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Increasing Nutrient Density of Food Crops through Soil Fertility Management and Cultivar SelectionProposal presented By Md J MeagyDepartment of Plant, Soil, and Insect Sciences University of Massachusetts Amherst Table of ContentsLiterature ReviewsResearch Justification ObjectivesExperiments Experiment 1:Experiment 2:Experiment 3:Experiment 4:Experiment 1:Introduction Materials and MethodsMaterials Methods Results DiscussionExperiment 2:Introduction Materials and MethodsMaterials Methods Results DiscussionExperiment 3: Introduction Materials and MethodsMaterials Methods Results DiscussionExperiment 3: Introduction Materials and MethodsMaterials Methods Results DiscussionBibliography Increasing Nutrient Density of Food Crops through Soil Fertility Management and Cultivar SelectionProposal presented By Md J MeagyDepartment of Plant, Soil, and Insect Sciences University of Massachusetts Amherst Increasing Nutrient Density of Food Crops through Soil Fertility Management and Cultivar SelectionA Disertation Proposal Presented ByMD J. MEAGYApproved as to style and content by: ____________________________________________________Allen V. Barker, Chair ____________________________________________________Geunhwa Jung, Member____________________________________________________Touria El Jaoual Eaton, Member Department of Plant, Soil, and Insect Sciences University of Massachusetts Amherst Increasing Nutrient Density of Food Crops through Soil Fertility Management and Cultivar SelectionAbstractLettuce is the most widely used leafy vegetable around the world. This experiment will be conducted on lettuce crop, organic and conventional fertility regimes, fertility management practices, assessing the relationship between fertility regimes and cultivars selection for increasing nutrient densities, and assessing the molecular similarities and variations among the cultivars. Experiments will be conducted in greenhouse and field sites. Organic and conventional fertilizer regimes will be used as treatment for the plant. Growth parameter such as height, fresh wt and dry wt will be reported for each experiment. Elemental analysis will be resulted accumulated by plants afterwards. Nutrients densities in plants might be increases through fertility and management practices.Increasing Nutrient Density of Food Crops through Soil Fertility Management and Cultivar SelectionBackgroundMalnutrition is a primary factor limiting human productivity in modern times, and deficiencies of certain elements including calcium, magnesium, potassium, phosphorus, zinc, copper, and others known as mineral nutrients, in diets of humans are a substantial nutritional problem throughout the World PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5EYXJudG9uLUhpbGw8L0F1dGhvcj48WWVhcj4yMDA1PC9Z
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ADDIN EN.CITE.DATA (Darnton-Hill et al., 2005; Kataki and Babu, 2002; Schaetzel and Sankar, 2000). Davis (2009) reports 5% to 40% declines in mineral contents of vegetables and fruits in the past 50 to 70 years in the United States. Reports from the United Kingdom indicated that foods were depleted by about 20% during this time ADDIN EN.CITE <EndNote><Cite><Author>Anonymous</Author><Year>2005</Year><RecNum>49</RecNum><record><rec-number>49</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">49</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Anonymous</author></authors></contributors><titles><title>Plants lose their value</title><secondary-title>Food Magazine 68 (Jan/Mar):1</secondary-title></titles><periodical><full-title>Food Magazine 68 (Jan/Mar):1</full-title></periodical><dates><year>2005</year></dates><urls></urls></record></Cite><Cite><Author>Anonymous</Author><Year>2006</Year><RecNum>50</RecNum><record><rec-number>50</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">50</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Anonymous</author></authors></contributors><titles><title>Meat and dairy: where have the minerals gone</title><secondary-title>Food Magazine 72 (Jan/Mar):10</secondary-title></titles><periodical><full-title>Food Magazine 72 (Jan/Mar):10</full-title></periodical><dates><year>2006</year></dates><urls></urls></record></Cite><Cite><Author>Mayer</Author><Year>1997</Year><RecNum>36</RecNum><record><rec-number>36</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">36</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Mayer, Anne Marie </author></authors></contributors><titles><title>Historical changes in the mineral content of fruits and vegetables</title><secondary-title>British Food Journal</secondary-title></titles><periodical><full-title>British Food Journal</full-title></periodical><pages>207</pages><volume>99</volume><number>6</number><section>211</section><dates><year>1997</year></dates><urls></urls></record></Cite></EndNote>(Anonymous, 2005; Anonymous, 2006; Mayer, 1997). Research on the diets of subjects in Philadelphia warranted a downward revision in the intakes of iron, magnesium, and vitamins ADDIN EN.CITE <EndNote><Cite><Author>Guenther</Author><Year>1994</Year><RecNum>51</RecNum><record><rec-number>51</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">51</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Guenther, P. M.</author><author>Perloff, B. P.</author><author>Vizioli, T. L.</author></authors></contributors><titles><title>Separating fact from artifact in changes in nutrient intake over time</title><secondary-title>Journal of the American Dietetic Association</secondary-title></titles><periodical><full-title>Journal of the American Dietetic Association</full-title></periodical><pages>270-275</pages><volume>94</volume><number>3</number><dates><year>1994</year></dates><urls></urls></record></Cite></EndNote>(Guenther et al., 1994). The result of a study concluded that many Americans are not meeting the current recommendations for calcium intake through diet alone or with supplements ADDIN EN.CITE <EndNote><Cite><Author>Ma</Author><Year>2007</Year><RecNum>34</RecNum><record><rec-number>34</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">34</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ma, Jun</author><author>Johns, Rachel A.</author><author>Stafford, Randall S.</author></authors></contributors><titles><title>Americans are not meeting current calcium recommendations</title><secondary-title>Am J Clin Nutr</secondary-title></titles><periodical><full-title>Am J Clin Nutr</full-title></periodical><pages>1361-1366</pages><volume>85</volume><number>5</number><dates><year>2007</year><pub-dates><date>May 1, 2007</date></pub-dates></dates><urls><related-urls><url>;(Ma et al., 2007). Davis (2009) suggests that the decline in mineral and protein content in fruits and vegetables is due partly to a dilution effect of high yields. Side-by-side comparisons of low- and high-yielding vegetables and grains showed negative correlations between yield of produce and concentrations of minerals. However, White et al. (2009) reported that with potato (Solanum spp. L.), a dilution effect of high yields on nutrient concentration was not observed universally and that soil fertility affected the mineral nutrients more than the dilution effect.Soil fertility problems associated with nutrient depletion by crop production are worldwide ADDIN EN.CITE <EndNote><Cite><Author>Tan</Author><Year>2005</Year><RecNum>54</RecNum><record><rec-number>54</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">54</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Tan, Z. X.</author><author>Lal, R.</author><author>Wiebe, K. D.</author></authors></contributors><titles><title>Global soil nutrient depletion and yield reduction</title><secondary-title>Journal of Sustainable Agriculture</secondary-title></titles><periodical><full-title>Journal of Sustainable Agriculture</full-title></periodical><pages>123-146</pages><volume>26</volume><number>1</number><dates><year>2005</year></dates><urls></urls></record></Cite></EndNote>(Tan et al., 2005). In the United States, potassium and phosphorus contents are being drawn down in soils at an increasing rate every year, and the depletion occurring for 40 years for potassium and for nearly 30 years for phosphorus ADDIN EN.CITE <EndNote><Cite><Author>Stewart</Author><Year>2004</Year><RecNum>55</RecNum><record><rec-number>55</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">55</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Stewart, W.M</author></authors></contributors><titles><title> Inorganic nutrient use in the United States: Past and present. Environmental Impact of Fertilizer on Soil and Water </title><secondary-title>ACS Symposium Series 872:151-160</secondary-title></titles><periodical><full-title>ACS Symposium Series 872:151-160</full-title></periodical><dates><year>2004</year></dates><urls></urls></record></Cite></EndNote>(Stewart, 2004). 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ADDIN EN.CITE.DATA (Ayoub, 1998; Dobermann et al., 1996; Lal and Singh, 1998; Nandwa and Bekunda, 1998). Elemental depletion of soils must be compensated for by fertilization for sustainable production of nutrient-sufficient foods ADDIN EN.CITE <EndNote><Cite><Author>Buol</Author><Year>1995</Year><RecNum>130</RecNum><record><rec-number>130</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">130</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Buol, S. W.</author></authors></contributors><titles><title>Sustainability of soil use</title><secondary-title>Annual Review of Ecology and Systematics</secondary-title></titles><periodical><full-title>Annual Review of Ecology and Systematics</full-title></periodical><pages>25-44</pages><volume>26</volume><number>1</number><dates><year>1995</year></dates><urls></urls></record></Cite></EndNote>(Buol, 1995).Organically grown fruits and vegetables might differ from conventionally grown produce due to differences in the types of fertilizers used in the two practices. Low availability of nutrients in organic fertilizers could limit mineral accumulation in plants relative to fertilization with chemical fertilizers with high nutrient availability, thereby making the chemical fertilizers a superior nutrient source. On the other hand, high availability of certain nutrients or failure to provide elements in chemical fertilizers could lead to nutrient imbalances in foods ADDIN EN.CITE <EndNote><Cite><Author>Lundegaardh</Author><Year>2003</Year><RecNum>35</RecNum><record><rec-number>35</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">35</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Lundegaardh, B.</author><author>Maartensson, A.</author></authors></contributors><titles><title>Organically Produced Plant Foods- Evidence of Health Benefits</title><secondary-title>Acta Agriculturae Scandinavica. Section B. Soil and Plant Science</secondary-title></titles><periodical><full-title>Acta Agriculturae Scandinavica. Section B. Soil and Plant Science</full-title></periodical><pages>3-15</pages><volume>53</volume><number>1</number><dates><year>2003</year></dates><urls></urls></record></Cite></EndNote>(Lundegaardh and Maartensson, 2003). A study noted that compost increased nutrient concentrations in soils but not always in plants ADDIN EN.CITE <EndNote><Cite><Author>Roe</Author><Year>1998</Year><RecNum>131</RecNum><record><rec-number>131</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">131</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Roe, N. E.</author></authors></contributors><titles><title>Compost utilization for vegetable and fruit crops</title><secondary-title>HortScience: a publication of the American Society for Horticultural Science (USA)</secondary-title></titles><periodical><full-title>HortScience: a publication of the American Society for Horticultural Science (USA)</full-title></periodical><dates><year>1998</year></dates><urls></urls></record></Cite></EndNote>(Roe, 1998). The result of another study reported no consistent differences in nitrogen, phosphorus, and potassium concentrations in several vegetables in crops fertilized with composts and crop residues or with synthetic mineral fertilizers ADDIN EN.CITE <EndNote><Cite><Author>Herencia</Author><Year>2007</Year><RecNum>24</RecNum><record><rec-number>24</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">24</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Herencia, J. F.</author><author>Ruiz-Porras, J. C.</author><author>Melero, S.</author><author>Garcia-Galavis, P. A.</author><author>Morillo, E.</author><author>Maqueda, C.</author></authors></contributors><titles><title>Comparison between Organic and Mineral Fertilization for Soil Fertility Levels, Crop Macronutrient Concentrations, and Yield</title><secondary-title>Agron J</secondary-title></titles><periodical><full-title>Agron J</full-title></periodical><pages>973-983</pages><volume>99</volume><number>4</number><dates><year>2007</year><pub-dates><date>June 5, 2007</date></pub-dates></dates><urls><related-urls><url>;(Herencia et al., 2007). A recently published study ADDIN EN.CITE <EndNote><Cite><Author>Benbrook</Author><Year>2008</Year><RecNum>6</RecNum><record><rec-number>6</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">6</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Benbrook, C.</author><author>Zhao, X.</author><author>Yá?ez, J. </author><author>Davies, N.</author><author>Andrews, P.</author></authors></contributors><titles><title>New evidence confirms the nutritional superiority of plant-based organic foods</title><secondary-title>The Organic Center, Organic Center, ;(Benbrook et al., 2008) reported that organically grown foods derived from plants were superior to those grown conventionally with respect to phosphorus, potassium, nitrates, several antioxidants, and vitamin C. An experiment result suggested that the high yields achieved through farming systems with high nitrogen fertilizer inputs led to a dilution of nutrient density in vegetables relative to organic systems with low nitrogen inputs ADDIN EN.CITE <EndNote><Cite><Author>Benbrook</Author><Year>2009</Year><RecNum>133</RecNum><record><rec-number>133</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">133</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Benbrook, C.</author><author>Scientist, C.</author></authors></contributors><titles><title>The Impacts of Yield on Nutritional Quality: Lessons from Organic Farming</title><secondary-title>HortScience</secondary-title></titles><periodical><full-title>HortScience</full-title></periodical><pages>12-14</pages><volume>44</volume><number>1</number><dates><year>2009</year></dates><urls></urls></record></Cite></EndNote>(Benbrook and Scientist, 2009). High nitrate concentrations in foods are considered as a factor adversely affecting human health ADDIN EN.CITE <EndNote><Cite><Author>Maynard</Author><Year>1976</Year><RecNum>38</RecNum><record><rec-number>38</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">38</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Maynard, D. N.</author><author>Barker, A. V.</author><author>Minotti, P. L.</author><author>Peck, N. H.</author></authors></contributors><titles><title>Nitrate accumulation in vegetables</title><secondary-title>Advances in Agronomy</secondary-title></titles><periodical><full-title>Advances in Agronomy</full-title></periodical><pages>71-118</pages><volume>28</volume><dates><year>1976</year></dates><urls></urls></record></Cite></EndNote>(Maynard et al., 1976). Organically grown vegetables may have less nitrate accumulation than conventionally fertilized vegetables ADDIN EN.CITE <EndNote><Cite><Author>Benbrook</Author><Year>2008</Year><RecNum>6</RecNum><record><rec-number>6</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">6</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Benbrook, C.</author><author>Zhao, X.</author><author>Yá?ez, J. </author><author>Davies, N.</author><author>Andrews, P.</author></authors></contributors><titles><title>New evidence confirms the nutritional superiority of plant-based organic foods</title><secondary-title>The Organic Center, Organic Center, ;(Benbrook et al., 2008). The result of a study reported that farm manure had a more favorable effect on certain plant constituents (nitrate and oxalic acid) than potassium nitrate ADDIN EN.CITE <EndNote><Cite><Author>Turan</Author><Year>2005</Year><RecNum>134</RecNum><record><rec-number>134</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">134</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>M. Turan</author><author>F. Sevimli</author></authors></contributors><titles><title>Influence of different nitrogen sources and levels on ion content of cabbage (Brassica oleracea var. capitata)</title><secondary-title>New Zealand Journal of Crop and Horticultural Science</secondary-title></titles><periodical><full-title>New Zealand Journal of Crop and Horticultural Science</full-title></periodical><pages>241-249</pages><volume>33</volume><number>3</number><dates><year>2005</year></dates><urls></urls></record></Cite></EndNote>(Turan and Sevimli, 2005). However, Maynard et al. (1976) noted that the amount of nitrogen fertilization regardless of source was the principal factor leading to nitrate accumulation in vegetables. A study noted that with fertilization for optimum yield, nitrate concentrations of vegetables were not different between organically and chemically fertilized vegetables ADDIN EN.CITE <EndNote><Cite><Author>Barker</Author><Year>1975</Year><RecNum>5</RecNum><record><rec-number>5</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">5</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Barker, A. V.</author></authors></contributors><titles><title>Organic vs. inorganic nutrition and horticultural crop quality</title><secondary-title>HortScience</secondary-title></titles><periodical><full-title>HortScience</full-title></periodical><pages>50-53</pages><volume>10</volume><number>1</number><dates><year>1975</year></dates><urls></urls></record></Cite></EndNote>(Barker, 1975).Regardless of soil conditions, fruits and vegetables, although high in vitamins, are typically low in mineral nutrients ADDIN EN.CITE <EndNote><Cite><Author>Elless</Author><Year>2000</Year><RecNum>18</RecNum><record><rec-number>18</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">18</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Elless, Mark P.</author><author>Blaylock, Michael J.</author><author>Huang, Jianwei W.</author><author>Gussman, Christopher D.</author></authors></contributors><titles><title>Plants as a natural source of concentrated mineral nutritional supplements</title><secondary-title>Food Chemistry</secondary-title></titles><periodical><full-title>Food Chemistry</full-title></periodical><pages>181-188</pages><volume>71</volume><number>2</number><keywords><keyword>Nutraceuticals</keyword><keyword>Brassica juncea</keyword><keyword>Bioavailability</keyword><keyword>Mineral supplements</keyword></keywords><dates><year>2000</year></dates><isbn>0308-8146</isbn><urls><related-urls><url>;(Elless et al., 2000). Except for potassium, the fruit-vegetable food group contributes less than 30% of the total dietary intake of mineral nutrients ADDIN EN.CITE <EndNote><Cite><Author>Levander</Author><Year>1990</Year><RecNum>31</RecNum><record><rec-number>31</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">31</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Levander, O. A.</author></authors></contributors><titles><title>Fruit and vegetable contributions to dietary mineral intake in human health and disease</title><secondary-title>HortScience</secondary-title></titles><periodical><full-title>HortScience</full-title></periodical><pages>1486-1488</pages><volume>25</volume><number>12</number><dates><year>1990</year></dates><urls></urls></record></Cite></EndNote>(Levander, 1990). To counter this problem, several attempts have been made to increase the mineral nutrient content of fruits and vegetables. These actions have included enrichment of foods in processing, but not much attention has been directed toward improvement of foods through enhanced soil fertility and cultivar selection, although these two factors appear to be principal agents affecting the nutrient density of fruits and vegetables. Methods of increasing the nutrient content of foods must be developed through improved practices in fertilization and in the development and selection of crop varieties that accumulate the nutrients in amounts that are adequate for intake in normal diets of humans. This research will involve studies with heritage and modern hybrids of several types of vegetable crops grown under differing regimes of fertilization. It is likely that heritage and modern hybrids will differ in susceptibility to diseases. . The high levels of horizontal or broad resistance to pathogens are often observed in heritage cultivars due to their adaptation over time to climate and soil conditions where they have grown in when they are compared to modern cultivars. The modern cultivars drop in horizontal or broad resistance to pathogens during breeding and under protection of pesticides. Soil fertility practices also are likely to affect the prevalence of diseases in the soil and plant susceptibility to diseases. Plant pathogenic diseases can be stimulated by excess levels of available nitrogen under favorable conditions.Justification The need for this project was raised by the food consumers and producers as well as by the scientific community. Literature on food composition demonstrates that the mineral nutrient density of vegetables has fallen in the past 50 years. This decline is associated with declines in soil fertility and with the genetics of plant cultivars that increase yield at higher rates than mineral nutrients increases. Research is needed to develop systems of food crop production that will supply adequate mineral nutrition to people directly. Use of nutrient-dense crops provide an opportunity for vegetable producers to diversify production and market, and to increase income and profitability as there appear to be a ready market for these crops. Research ObjectivesTo determine if the mineral nutrient densities of selected vegetable crops can be increased through cultivar selection.To determine if the nutrient densities of selected vegetable crops can be increased by elevating nutrient contents in the medium in which these plants grow.To determine if the nutrient densities of selected vegetable crops can be increased through soil fertility practices (e.g., organic vs conventional fertilizers; different fertilizer regimes), and To assess genetic similarities among selected cultivars within species, for genetic purity and diversity, using molecular markers. ResearchFour experiments will be set up for this project based on objectives. The brief discussions of the experiment are as follows:Experiment 1. Assessment of the relationship between nutrient density in crops and cultivar selection and nutrient availability in the greenhouse.This experiment will be designed to determine the mineral nutrient contents of lettuce through cultivar selection using different fertilizer regimes.The experiment will be conducted in the greenhouse to evaluate regimes of fertilization and cultivars for evaluation in fields. The same fertilizers suggested for the field evaluation will be used in this experiment. The greenhouse experiments will help to evaluate fertility regimes before and after application in the field. The lettuce crop will be grown in peat-based media (Canadian Growing Mix 1-PV, Conrad Fafard Inc, Agawam, MA) ADDIN EN.CITE <EndNote><Cite><Author>Boodley</Author><Year>1996</Year><RecNum>7</RecNum><record><rec-number>7</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">7</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>Boodley, J. W.</author><author>Newman, S. E.</author><author>Jackson, D. W.</author><author>Plaster, E.</author><author>Sheaffer, C. C.</author><author>Moncada, K. M.</author><author>Emmons, R.</author><author>Loehrlein, M.</author><author>Reiley, H. E.</author><author>Shry Jr, C. L.</author></authors></contributors><titles><title><style face="normal" font="default" size="100%">The commercial greenhouse, 3</style><style face="superscript" font="default" size="100%">rd </style><style face="normal" font="default" size="100%">edition</style></title></titles><dates><year>1996</year></dates><pub-location>Albany, NY</pub-location><publisher>Delmar Publishers</publisher><urls></urls></record></Cite></EndNote>(Boodley et al., 1996). The elemental nutrient composition will be determined in edible parts (fresh leaves) of lettuce. Because of their adaptability to greenhouse production, the lettuce crop to be evaluated using different lettuce cultivars. Each crop will be evaluated for their growth and nutrient contents. The experimental design of the experiment will be followed by complete random block design.Experiment 2. Assess the effect between mineral nutrient density in crops and cultivar selection by elevating nutrient contents in the medium in which the plants grow.This experiment will be assessed differences in the mineral densities of lettuce cultivar through increasing the nutrient contents in the medium. The differences of this experiment from the experiment one will be in elevating nutrient contents in the medium in which the plant grow. The experiment will be conducted in greenhouse and field to assess the response of lettuce using the increasing nutrient contents. The same fertilizers will be used in the field experiment. The crop will be grown in peat-based media (Canadian Growing Mix 1-PV, Conrad Fafard Inc, Agawam, MA) ADDIN EN.CITE <EndNote><Cite><Author>Boodley</Author><Year>1996</Year><RecNum>7</RecNum><record><rec-number>7</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">7</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>Boodley, J. W.</author><author>Newman, S. E.</author><author>Jackson, D. W.</author><author>Plaster, E.</author><author>Sheaffer, C. C.</author><author>Moncada, K. M.</author><author>Emmons, R.</author><author>Loehrlein, M.</author><author>Reiley, H. E.</author><author>Shry Jr, C. L.</author></authors></contributors><titles><title><style face="normal" font="default" size="100%">The commercial greenhouse, 3</style><style face="superscript" font="default" size="100%">rd </style><style face="normal" font="default" size="100%">edition</style></title></titles><dates><year>1996</year></dates><pub-location>Albany, NY</pub-location><publisher>Delmar Publishers</publisher><urls></urls></record></Cite></EndNote>(Boodley et al., 1996). The fertilizer regimes will be chemical fertilizer organic fertilizer with increasing nutrient contents. The increasing nutrient contents in the fertilizer regimes will be calcium based on nutrient demand for human dietary nutrient requirement. The nutrient contents will be measured in the edible parts (fresh leaves) of the plant. This crop will be evaluated for growth and nutrient densities in produce. The experimental design of the experiment will be followed by complete random block design.Experiment 3. Assess the effect of organic and conventional soil fertility practices on mineral accumulation in vegetables in field experimentation. Fertilization will be provided in the experiment using organic and conventional fertilizer regime. Two regimes of organic and one conventional fertilizer will be selected to provide nutrients. Organic fertilizer will come from the individual sources includes soybean meal for nitrogen, colloidal rock phosphate for the phosphorus, and mined potassium sulfate for the potassium. The compost will be obtained from the University Office of Waste Management, which produces compost from dining commons food waste and yard waste from the Amherst campus. Produce will be harvested and evaluated for yield and nutrient densities at edible plant parts (e.g. fresh leaves). In addition, whenever pathogen resulted damages or symptoms are observed under natural conditions (no artificial inoculations), accurate identification of the disease will be carried out in conjunction with Plant Disease Diagnostician and a vegetable pathologist (Dr. Robert Wicks, Plant, Soil, and Insect Sciences) at the University of Massachusetts Amherst. The disease severity per cultivar will be rated. The experimental design of the experiment will be followed by complete random block design.Experiment 4. Assessment of the genetic purity and genetic diversity among cultivars of lettuce using molecular markers in greenhouse experiment.This experiment is planned to determine the genetic purity and genetic diversity among cultivars of lettuce crops using molecular markers. The experiment will be conducted in the greenhouse using the same cultivars as used in the previous experiments. Standard fertilizer will be used to grow the cultivars. Fresh tissue will be sampled in the middle of the growth period for genomic analysis. Genomic DNA will be extracted from plant tissues according to the procedures used in ADDIN EN.CITE <EndNote><Cite><Author>Curley</Author><Year>2004</Year><RecNum>13</RecNum><record><rec-number>13</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">13</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Curley, J.</author><author>Jung, G.</author></authors></contributors><titles><title>RAPD-Based Genetic Relationships in Kentucky Bluegrass: Comparison of Cultivars, Interspecific Hybrids, and Plant Introductions</title><secondary-title>Crop Sci</secondary-title></titles><periodical><full-title>Crop Sci</full-title></periodical><pages>1299-1306</pages><volume>44</volume><number>4</number><dates><year>2004</year><pub-dates><date>July 1, 2004</date></pub-dates></dates><urls><related-urls><url> app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">26</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Simko, Ivan</author></authors></contributors><titles><title>Development of EST-SSR Markers for the Study of Population Structure in Lettuce (Lactuca sativa L.)</title><secondary-title>J Hered</secondary-title></titles><periodical><full-title>J Hered</full-title></periodical><pages>256-262</pages><volume>100</volume><dates><year>2008</year><pub-dates><date>September 15, 2008</date></pub-dates></dates><urls><related-urls><url>;(Curley and Jung, 2004; Simko, 2008) and ADDIN EN.CITE <EndNote><Cite><Author>Sim</Author><Year>2009</Year><RecNum>76</RecNum><record><rec-number>76</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">76</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Sim, S. C.</author><author>Yu, J. K.</author><author>Jo, Y.</author><author>Sorrells, M. E.</author><author>Jung, G.</author></authors></contributors><titles><title>Transferability of cereal EST-SSR markers to ryegrass</title><secondary-title>Genome</secondary-title></titles><periodical><full-title>Genome</full-title></periodical><pages>431-437</pages><volume>52</volume><number>5</number><dates><year>2009</year></dates><urls></urls></record></Cite></EndNote>(Sim et al., 2009). EST- and genomic-SSR markers of respective vegetables, these will be selected from the most recent papers PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5DYXZhZ25hcm88L0F1dGhvcj48WWVhcj4yMDA5PC9ZZWFy
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ADDIN EN.CITE.DATA (Cavagnaro et al., 2009; Kong et al., 2006; Liu et al., 2007b; Liu et al., 2007c; Richards et al., 2004; Simko, 2008; Yi et al., 2006) will be screened against selected cultivars within the vegetable species and used for genetic similarities. PCRs will be conducted in conditions as described in the respective papers. DNA similarities among cultivars within each of species will be estimated. The experimental design of the experiment will be followed by complete random block design.Statistical analysisPlant heights, fresh weights, and dry weights data will be processed as analysis of variance using the SAS (SAS 9.1.3, SAS Institute Inc., SAS Campus Drive, Cary, NC 27513) PROC GLM procedure. Mean separation will be conducted by F test and Duncan’s New Multiple Range Test (P=0.05). Total N and elemental analysis data will be analyzed using SAS PROC GLM procedure. Polynomial comparison among cultivars will be performed by orthogonal polynomial test using PROC GLM and PROC IML procedure. .Assessment of the relationship between nutrient density in crops and cultivar selection and nutrient availability in the greenhouse.Introduction Mineral deficiency adversely affect a third of world’s population. Consequently, micronutrient deficiencies affect global health and limit human productivity in recent times, and deficiency of certain mineral nutrients like calcium, potassium, magnesium, phosphorus, zinc, iron, copper, manganese and others are a substantial nutritional problem in diets of human around the world ADDIN EN.CITE <EndNote><Cite><Author>Darnton-Hill</Author><Year>2005</Year><RecNum>1</RecNum><record><rec-number>1</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">1</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Darnton-Hill, Ian</author><author>Webb, Patrick</author><author>Harvey, Philip W. J.</author><author>Hunt, Joseph M.</author><author>Dalmiya, Nita</author><author>Chopra, Mickey</author><author>Ball, Madeleine J.</author><author>Bloem, Martin W.</author><author>de Benoist, Bruno</author></authors></contributors><titles><title>Micronutrient deficiencies and gender: social and economic costs</title><secondary-title>Am J Clin Nutr</secondary-title></titles><periodical><full-title>Am J Clin Nutr</full-title></periodical><pages>1198S-1205</pages><volume>81</volume><number>5</number><dates><year>2005</year><pub-dates><date>May 1, 2005</date></pub-dates></dates><urls><related-urls><url> app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">2</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>Kataki, P. K.</author><author>Babu, S. C.</author></authors></contributors><titles><title>Food systems for improved human nutrition: linking agriculture, nutrition, and productivity</title></titles><dates><year>2002</year></dates><pub-location>N.Y</pub-location><publisher>Food Products Press, Binghampton</publisher><urls></urls></record></Cite></EndNote>(Darnton-Hill et al., 2005; Kataki and Babu, 2002). Nutrient content in vegetables and fruits reported declines of 5% to 40% or more in some soils in the past 50 to 100 years in the United States ADDIN EN.CITE <EndNote><Cite><Author>Davis</Author><Year>2009</Year><RecNum>4</RecNum><record><rec-number>4</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">4</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>D. R. Davis</author></authors></contributors><titles><title>Declining Fruit and Vegetable Nutrient Composition: What Is the Evidence?</title><secondary-title>HortScience</secondary-title></titles><periodical><full-title>HortScience</full-title></periodical><pages>15-19</pages><volume>44</volume><number>1</number><dates><year>2009</year></dates><urls></urls></record></Cite></EndNote>(Davis, 2009). Reports indicated the in the United Kingdom foods contents decreased by about 20% during this time ADDIN EN.CITE <EndNote><Cite><Author>Anonymous</Author><Year>2005</Year><RecNum>49</RecNum><record><rec-number>49</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">49</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Anonymous</author></authors></contributors><titles><title>Plants lose their value</title><secondary-title>Food Magazine 68 (Jan/Mar):1</secondary-title></titles><periodical><full-title>Food Magazine 68 (Jan/Mar):1</full-title></periodical><dates><year>2005</year></dates><urls></urls></record></Cite><Cite><Author>Mayer</Author><Year>1997</Year><RecNum>36</RecNum><record><rec-number>36</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">36</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Mayer, Anne Marie </author></authors></contributors><titles><title>Historical changes in the mineral content of fruits and vegetables</title><secondary-title>British Food Journal</secondary-title></titles><periodical><full-title>British Food Journal</full-title></periodical><pages>207</pages><volume>99</volume><number>6</number><section>211</section><dates><year>1997</year></dates><urls></urls></record></Cite><Cite><Author>Anonymous</Author><Year>2006</Year><RecNum>50</RecNum><record><rec-number>50</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">50</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Anonymous</author></authors></contributors><titles><title>Meat and dairy: where have the minerals gone</title><secondary-title>Food Magazine 72 (Jan/Mar):10</secondary-title></titles><periodical><full-title>Food Magazine 72 (Jan/Mar):10</full-title></periodical><dates><year>2006</year></dates><urls></urls></record></Cite></EndNote>(Anonymous, 2005; Anonymous, 2006; Mayer, 1997). Study on the diets of subjects in Philadelphia, PA warranted a downward revision in the intakes of iron, magnesium, and vitamins ADDIN EN.CITE <EndNote><Cite><Author>Guenther</Author><Year>1994</Year><RecNum>51</RecNum><record><rec-number>51</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">51</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Guenther, P. M.</author><author>Perloff, B. P.</author><author>Vizioli, T. L.</author></authors></contributors><titles><title>Separating fact from artifact in changes in nutrient intake over time</title><secondary-title>Journal of the American Dietetic Association</secondary-title></titles><periodical><full-title>Journal of the American Dietetic Association</full-title></periodical><pages>270-275</pages><volume>94</volume><number>3</number><dates><year>1994</year></dates><urls></urls></record></Cite></EndNote>(Guenther et al., 1994). Another study concluded that many Americans, particularly men, social disadvantaged groups, and ethnic minorities are not meeting the current recommendations for calcium intake through diet alone or with supplements ADDIN EN.CITE <EndNote><Cite><Author>Ma</Author><Year>2007</Year><RecNum>34</RecNum><record><rec-number>34</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">34</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ma, Jun</author><author>Johns, Rachel A.</author><author>Stafford, Randall S.</author></authors></contributors><titles><title>Americans are not meeting current calcium recommendations</title><secondary-title>Am J Clin Nutr</secondary-title></titles><periodical><full-title>Am J Clin Nutr</full-title></periodical><pages>1361-1366</pages><volume>85</volume><number>5</number><dates><year>2007</year><pub-dates><date>May 1, 2007</date></pub-dates></dates><urls><related-urls><url>;(Ma et al., 2007).Davis (2009) suggests that the decline in mineral and protein content in fruits and vegetables is due partly to a dilution effect of high yields. Side-by-side comparisons of low- and high-yielding vegetables and grains showed negative correlations between yield of produce and concentrations of minerals. However, a dilution effect of high yield on nutrient concentration was not observed universally on potato (Solanum spp L.) and that soil fertility affected the mineral nutrients more than the dilution effect ADDIN EN.CITE <EndNote><Cite><Author>White</Author><Year>2009</Year><RecNum>52</RecNum><record><rec-number>52</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">52</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>White, P.J.</author><author>Bradshaw, E.E. </author><author> Dale, M.F.B. </author><author> Ramsa, G.</author><author>Hammond, J.P. </author><author> Broadley, M.R. </author></authors></contributors><titles><title>Relationships between yield and mineral concentrations in potato tubers</title><secondary-title>HortScience</secondary-title></titles><periodical><full-title>HortScience</full-title></periodical><pages>6-14</pages><volume>44</volume><number>1</number><dates><year>2009</year></dates><urls></urls></record></Cite></EndNote>(White et al., 2009).Micronutrient malnutrition is a growing concern all over the developing world having consequence a mental retardation, impairments of the immune system, and overall poor health ADDIN EN.CITE <EndNote><Cite><Author>Cakmak</Author><Year>2009</Year><RecNum>53</RecNum><record><rec-number>53</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">53</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Cakmak, I.</author></authors></contributors><titles><title>Enrichment of fertilizers with zinc: An excellent investment for humanity and crop production in India</title><secondary-title>Journal of Trace Elements in Medicine and Biology</secondary-title></titles><periodical><full-title>Journal of Trace Elements in Medicine and Biology</full-title></periodical><pages>281-289</pages><volume>23</volume><number>4</number><dates><year>2009</year></dates><urls></urls></record></Cite></EndNote>(Cakmak, 2009).Soil fertility problems associated with nutrient depletion by crop production are worldwide, and also nutrient depletion can be caused to insufficient fertilizers use and unbalanced fertilization ADDIN EN.CITE <EndNote><Cite><Author>Tan</Author><Year>2005</Year><RecNum>54</RecNum><record><rec-number>54</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">54</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Tan, Z. X.</author><author>Lal, R.</author><author>Wiebe, K. D.</author></authors></contributors><titles><title>Global soil nutrient depletion and yield reduction</title><secondary-title>Journal of Sustainable Agriculture</secondary-title></titles><periodical><full-title>Journal of Sustainable Agriculture</full-title></periodical><pages>123-146</pages><volume>26</volume><number>1</number><dates><year>2005</year></dates><urls></urls></record></Cite></EndNote>(Tan et al., 2005). In United States, potassium and phosphorus are being drawn in soils on a national basis at an increasing rate every year, and the depletion has occurred for 40 years for potassium and for nearly 30 years for phosphorus ADDIN EN.CITE <EndNote><Cite><Author>Stewart</Author><Year>2004</Year><RecNum>55</RecNum><record><rec-number>55</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">55</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Stewart, W.M</author></authors></contributors><titles><title> Inorganic nutrient use in the United States: Past and present. Environmental Impact of Fertilizer on Soil and Water </title><secondary-title>ACS Symposium Series 872:151-160</secondary-title></titles><periodical><full-title>ACS Symposium Series 872:151-160</full-title></periodical><dates><year>2004</year></dates><urls></urls></record></Cite></EndNote>(Stewart, 2004). A study suggest prediction of nitrogen uptake for sustainable nitrate pool of lettuce was depended on the water content ADDIN EN.CITE <EndNote><Cite><Author>Seginer</Author><Year>2004</Year><RecNum>127</RecNum><record><rec-number>127</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">127</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Seginer, I. D. O.</author><author>Bleyaert, Peter</author><author>Breugelmans, Maaike</author></authors></contributors><titles><title>Modelling Ontogenetic Changes of Nitrogen and Water Content in Lettuce</title><secondary-title>Ann Bot</secondary-title></titles><periodical><full-title>Ann Bot</full-title></periodical><pages>393-404</pages><volume>94</volume><number>3</number><dates><year>2004</year><pub-dates><date>September 1, 2004</date></pub-dates></dates><urls><related-urls><url>;(Seginer et al., 2004).The objective of the study will be to determine if the mineral nutrient densities of selected vegetable crops can be increase through cultivar selection.Materials and MethodsMaterialsEighteen lettuce (Lactuca sativa) cultivars will be used in this experiment. Nine varieties will be heritage and nine will be modern for this study. Three varieties (Table 1) of each group (Butterhead, Romaine/Cos, and Loose leaf) will be chosen in heritage and modern type of lettuce based on widely used and marketability. Then eighteen varieties (nine heritages and nine moderns) of both groups will be used for the treatment. Three fertilizer regimes will be used for this experiment.Methods Eighteen different cultivars of lettuce will be grown in the Bowditch greenhouse at University of Massachusetts Amherst. Seeds of those cultivars were collected from professional seed producers. Heritage type of seeds will be collected from certified organic seeds producer (Seed of Change Seeds Co., Spicer, MN 56288) and modern type of seeds will be collected from another successful seeds producer (Johnny Seeds Co., Winslow, ME 04901). All seeds of these cultivars will be planted in peat moss medium on December 18, 2009 in the greenhouse at 2-3 seeds/cube. . For best germination of seeds media temperature will be maintained of 68oF (20oC). After 32 days, lettuce seedlings will be transferred to plastic containers (6 inch diameter) in a peat-based medium (MetroMix 360, SunGro Horticulture, Vancouver, Canada).Three different combinations (regimes) of fertilizers will be chosen for nutrient sources of lettuce cultivars. With different cultivars different nutrient regimes will be tested if nutrient content increases through cultivars selection. Three treatments will be Hoagland #1 solution (complete), Jack’s Fertilizer (20-10-20) Peat-Lite (add CaSO4 to medium), and organic Pure blend Pro Grow (3-1.5-4) for this experiment. The purpose of the application of different fertilizer regimes is to investigate the nutrient content of the lettuce through cultivar selection. The lettuce will be grown in greenhouse for about 7-9 weeks from seedlings to maturation. Table 1. Varietal selection of each cultivarHeritage variety (H)Modern variety (M)Butterhead (C1):Buttercrunch (V1)Bronze Mignonette (V2)Tom thumb (V3)Romaine/Cos (C2):Coastal Star (V1)Cosmo-Savoy Leaf (V2)Forellenschluss (V3)Loose leaf (C3):Salad Bowl (V1)Simpson black-seeded (V2)Red deer tongue (V3)Butterhead (C1):Adriana (V1)Focea (V2)Australe (V3)Romaine/Cos (C2):Red rosie (V1)Claremont (V2)Winter density (V3)Loose leaf (C3):Tropicana (V1)Simpson elite (V2)Two star (V3)Each regime will carry a fertilizer treatment with different combination. The first fertilizer treatment contained of 210 ppm N, 31 ppm P, 234 ppm K, 200 ppm Ca, 48 ppm Mg, 64 ppm S, and micronutrient composite and Fe chelate 1 ppm from a Hoagland’s solution ADDIN EN.CITE <EndNote><Cite><Author>Hoagland</Author><Year>1950</Year><RecNum>43</RecNum><record><rec-number>43</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">43</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Hoagland, D. R.</author><author>Arnon, D. I.</author></authors></contributors><titles><title>The water culture method for growing plants without soil</title><secondary-title>California Agricultural Experiment Station Circular</secondary-title></titles><periodical><full-title>California Agricultural Experiment Station Circular</full-title></periodical><pages>1-32</pages><volume>347</volume><dates><year>1950</year></dates><urls></urls></record></Cite></EndNote>(Hoagland and Arnon, 1950). The second fertilizer treatment carried of using 3% N, 1.5% P, 4% K with trace minerals 1.0% Ca and 0.5% Mg from a 3-1.5-4 formula organic fertilizer (Pure Blend Pro Grow, Botanicare, Tempe, AZ) and with this fertilizer 4 ml 0.5 M MgSO4/liter will be added for Mg and S. The third fertilizer treatment consisted of 20 % N, 10 % P, 20 % K, 1.5 % Mg, 0.02 % B, 0.01 % Cu, 0.1 Fe, 0.05 % Mn, 0.01 % Mo, and 0.05 % Zn from a Jack’s 20-10-10 Peat-Lite commercial fertilizers (Jack’s Professional Fertilizers, J.R. Peters, Inc., Allentown, PA 18106) and with this fertilizer I g CaSO4/pot will be mixed in media for Ca, and additionally 4 ml 0.5 M MgSO4/liter will be added for Mg and S. The treatment of fertilizer regime will be applied one time in one day interval at the amount 100 ml/plant for 1st 10 days after transplanted of lettuce seedlings. Then amount of treatment of each regime then will be increased at the rate 200 ml/plant for one day interval and continued at maturity. The fertilizer regimes will be applied to a plant in every two days.Media pH values will be measured in a water extract after shaking for 15 m ADDIN EN.CITE <EndNote><Cite><Author>Hesse</Author><Year>1971</Year><RecNum>46</RecNum><record><rec-number>46</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">46</key></foreign-keys><ref-type name="Generic">13</ref-type><contributors><authors><author>Hesse, P. R.</author></authors></contributors><titles><title>A Textbook of Soil Chemical Analysis, Chemical Pub</title></titles><dates><year>1971</year></dates><publisher>Co</publisher><urls></urls></record></Cite></EndNote>(Hesse, 1971) using a pH meter (CRISON micro pH 2002). Media EC values will be determined in a media/water extract after shaking for 15 m (Hesse, 1971) using a conductivity meter (CRISON micro pH 2002).At harvest, growth parameters including plant height, plant size and color, leaf size or whole heads, leaf color, number of leaf, and fresh weights of leaf will be recorded. Oven-dry weights of plant leaf and stem will be recorded afterwards.Portions of dry plant sample of leaves will be ground to pass a 30-mesh screen and sub sampled and stored in coin envelops for elemental analysis of N, P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr accumulated by lettuce leaves. Kjeldhal N will be determined by acid-digestion and posterior determination by auto analyzer (BRAN+LUEBBE, method G-188-97, BRAN+LUEBBE, Norderstedt, Germany). Pulverized samples (0.5 g) will be ashed at 500oC for 12 h in a muffle furnace and the ash will be dissolved in 10% HCl solution prepared in deionized H2O. For P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr, the ash extracts will be analyzed by inductive coupled plasma (ICP) spectrophotometric analysis ADDIN EN.CITE <EndNote><Cite><Author>Jones Jr</Author><Year>1991</Year><RecNum>27</RecNum><record><rec-number>27</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">27</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Jones Jr, J. B.</author><author>Wolf, B.</author><author>Mills, H. A.</author></authors></contributors><titles><title>Plant Analysis Handbook. Micro-Macro Publ</title><secondary-title>Inc., Athens, GA</secondary-title></titles><periodical><full-title>Inc., Athens, GA</full-title></periodical><dates><year>1991</year></dates><urls></urls></record></Cite><Cite><Author>Kalra</Author><Year>1998</Year><RecNum>28</RecNum><record><rec-number>28</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">28</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>Kalra, Y. P.</author></authors></contributors><titles><title>Handbook of reference methods for plant analysis</title></titles><dates><year>1998</year></dates><pub-location>Boca Raton</pub-location><publisher>CRC Press</publisher><urls></urls></record></Cite></EndNote>(Jones Jr et al., 1991; Kalra, 1998) at UMass Soil and Plant Tissue Laboratory. Lettuce growth parameters will be used for evaluating the consequences of different fertilizer regimes and their combination on lettuce growth and nutrient management through cultivars selection.Pest will be managed by using certified organic or conventional herbicides, fungicides or insecticides as required to bring about control. During the growing season, appearance of diseases symptoms and occurrences will be carefully monitored and diseases severity will be evaluated for each vegetables at a scale of 0-9 scale indicating 0 being no visible symptoms and 9 being severe symptoms.Summary Assess the effect between mineral nutrient density in crops and cultivar selection by elevating nutrient contents in the medium in which the plants grow.IntroductionMineral elements are required for human body to maintain good health. Plants are the main sources for these elements. Fresh fruits and vegetables are high in vitamins but are aften low in essential minerals ADDIN EN.CITE <EndNote><Cite><Author>Ashmead</Author><Year>1982</Year><RecNum>90</RecNum><record><rec-number>90</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">90</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>Ashmead, H. </author></authors></contributors><titles><title> Chelated mineral nutrition in plants. In D. W. Ashmead, Animals and man (pp. xi±xv). Springfield, IL: C.C. Thomas.</title></titles><dates><year>1982</year></dates><urls></urls></record></Cite></EndNote>(Ashmead, 1982). Plants like Brassica juncea cultivated in hydrophonics condition contains high levels of nutritionally important minerals such as Cr, Fe, Mn, Se, and Zn ADDIN EN.CITE <EndNote><Cite><Author>Elless</Author><Year>2000</Year><RecNum>18</RecNum><record><rec-number>18</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">18</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Elless, Mark P.</author><author>Blaylock, Michael J.</author><author>Huang, Jianwei W.</author><author>Gussman, Christopher D.</author></authors></contributors><titles><title>Plants as a natural source of concentrated mineral nutritional supplements</title><secondary-title>Food Chemistry</secondary-title></titles><periodical><full-title>Food Chemistry</full-title></periodical><pages>181-188</pages><volume>71</volume><number>2</number><keywords><keyword>Nutraceuticals</keyword><keyword>Brassica juncea</keyword><keyword>Bioavailability</keyword><keyword>Mineral supplements</keyword></keywords><dates><year>2000</year></dates><isbn>0308-8146</isbn><urls><related-urls><url>;(Elless et al., 2000). Critical functions in human body are mainly conducted by minerals. Calcium, phosphorus, and magnesium are required for constructing and maintaining bones ADDIN EN.CITE <EndNote><Cite><Author>Anonymous</Author><Year>1989</Year><RecNum>91</RecNum><record><rec-number>91</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">91</key></foreign-keys><ref-type name="Book Section">5</ref-type><contributors><authors><author>Anonymous</author></authors></contributors><titles><title>Committee on Diet and Health. Food and Nutrition Board. National Research Council. Minerals. </title><secondary-title>In: Diet and health: implecations for reducing chronic disease risk</secondary-title></titles><pages>347-366</pages><dates><year>1989</year></dates><pub-location>Washington, DC</pub-location><publisher>National Academy Press</publisher><urls></urls></record></Cite></EndNote>(Anonymous, 1989). Diet with enough calcium helps in maintaining healthy bones and reduces risk of osteoporosis ADDIN EN.CITE <EndNote><Cite><Author>Anonymous</Author><Year>2000</Year><RecNum>92</RecNum><record><rec-number>92</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">92</key></foreign-keys><ref-type name="Book Section">5</ref-type><contributors><authors><author>Anonymous</author></authors></contributors><titles><title>U.S. Department of Health and Human Services. Healthy People 2010. 2nd edition. Objectives for Improving Health (Part B: Focus Areas 15–28). Vol. II. Objective 19–11.</title></titles><dates><year>2000</year></dates><pub-location>Washington, DC: U.S. Government Printing Office</pub-location><urls></urls></record></Cite></EndNote>(Anonymous, 2000). In addition, calcium is required in blood clotting ADDIN EN.CITE <EndNote><Cite><Author>Karll</Author><Year>2000</Year><RecNum>93</RecNum><record><rec-number>93</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">93</key></foreign-keys><ref-type name="Book Section">5</ref-type><contributors><authors><author>E. Karll</author></authors><secondary-authors><author>Singh, M. A. F</author></secondary-authors></contributors><titles><title>Calcium and vitamin D</title><secondary-title>Exercise, nutrition, and the older woman: wellness for women over fifty</secondary-title></titles><pages>173-181</pages><dates><year>2000</year></dates><pub-location>Washington, DC</pub-location><publisher>CRC Press.</publisher><urls></urls></record></Cite><Cite><Author>Krause</Author><Year>1984</Year><RecNum>94</RecNum><record><rec-number>94</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">94</key></foreign-keys><ref-type name="Book Section">5</ref-type><contributors><authors><author>M. V. Krause </author><author>L. K. Mahan </author></authors><secondary-authors><author>Krause </author><author>Mahan </author></secondary-authors></contributors><titles><title>Minerals</title><secondary-title>Food, nutrition, and diet therapy. 7th edition</secondary-title></titles><pages>144–80</pages><dates><year>1984</year></dates><pub-location>Philadelphia, PA</pub-location><publisher>W. B. Saunders Company</publisher><urls></urls></record></Cite></EndNote>(Karll, 2000; Krause and Mahan, 1984), and phosphorus and magnesium are essential in energy metabolism. Iron is a crucial part of hemoglobin and myoglobin ADDIN EN.CITE <EndNote><Cite><Author>Anonymous</Author><Year>1989</Year><RecNum>91</RecNum><record><rec-number>91</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">91</key></foreign-keys><ref-type name="Book Section">5</ref-type><contributors><authors><author>Anonymous</author></authors></contributors><titles><title>Committee on Diet and Health. Food and Nutrition Board. National Research Council. Minerals. </title><secondary-title>In: Diet and health: implecations for reducing chronic disease risk</secondary-title></titles><pages>347-366</pages><dates><year>1989</year></dates><pub-location>Washington, DC</pub-location><publisher>National Academy Press</publisher><urls></urls></record></Cite><Cite><Author>Krause</Author><Year>1984</Year><RecNum>94</RecNum><record><rec-number>94</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">94</key></foreign-keys><ref-type name="Book Section">5</ref-type><contributors><authors><author>M. V. Krause </author><author>L. K. Mahan </author></authors><secondary-authors><author>Krause </author><author>Mahan </author></secondary-authors></contributors><titles><title>Minerals</title><secondary-title>Food, nutrition, and diet therapy. 7th edition</secondary-title></titles><pages>144–80</pages><dates><year>1984</year></dates><pub-location>Philadelphia, PA</pub-location><publisher>W. B. Saunders Company</publisher><urls></urls></record></Cite></EndNote>(Anonymous, 1989; Krause and Mahan, 1984), and is required for carrying oxygen in the blood and muscles ADDIN EN.CITE <EndNote><Cite><Author>Krause</Author><Year>1984</Year><RecNum>94</RecNum><record><rec-number>94</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">94</key></foreign-keys><ref-type name="Book Section">5</ref-type><contributors><authors><author>M. V. Krause </author><author>L. K. Mahan </author></authors><secondary-authors><author>Krause </author><author>Mahan </author></secondary-authors></contributors><titles><title>Minerals</title><secondary-title>Food, nutrition, and diet therapy. 7th edition</secondary-title></titles><pages>144–80</pages><dates><year>1984</year></dates><pub-location>Philadelphia, PA</pub-location><publisher>W. B. Saunders Company</publisher><urls></urls></record></Cite></EndNote>(Krause and Mahan, 1984). Anemia is caused by iron deficiency ADDIN EN.CITE <EndNote><Cite><Author>Krause</Author><Year>1984</Year><RecNum>94</RecNum><record><rec-number>94</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">94</key></foreign-keys><ref-type name="Book Section">5</ref-type><contributors><authors><author>M. V. Krause </author><author>L. K. Mahan </author></authors><secondary-authors><author>Krause </author><author>Mahan </author></secondary-authors></contributors><titles><title>Minerals</title><secondary-title>Food, nutrition, and diet therapy. 7th edition</secondary-title></titles><pages>144–80</pages><dates><year>1984</year></dates><pub-location>Philadelphia, PA</pub-location><publisher>W. B. Saunders Company</publisher><urls></urls></record></Cite></EndNote>(Krause and Mahan, 1984). Deficiency of trace elements like zinc, copper, and selenium has a positive correlation with risk of cancer ADDIN EN.CITE <EndNote><Cite><Author>Chan</Author><Year>1998</Year><RecNum>95</RecNum><record><rec-number>95</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">95</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Chan, S.</author><author>Gerson, B.</author><author>Subramaniam, S.</author></authors></contributors><titles><title>The role of copper, molybdenum, selenium, and zinc in nutrition and health</title><secondary-title>Clinics in laboratory medicine</secondary-title></titles><periodical><full-title>Clinics in laboratory medicine</full-title></periodical><pages>673</pages><volume>18</volume><number>4</number><dates><year>1998</year></dates><urls></urls></record></Cite><Cite><Author>Abdulla</Author><Year>2000</Year><RecNum>96</RecNum><record><rec-number>96</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">96</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Abdulla, M.</author><author>Gruber, P.</author></authors></contributors><titles><title>Role of diet modification in cancer prevention</title><secondary-title>Biofactors</secondary-title></titles><periodical><full-title>Biofactors</full-title></periodical><pages>45-51</pages><volume>12</volume><number>1</number><dates><year>2000</year></dates><urls></urls></record></Cite></EndNote>(Abdulla and Gruber, 2000; Chan et al., 1998). A number of chronic cardiovascular diseases are shown to be correlated with magnesium and selenium ADDIN EN.CITE <EndNote><Cite><Author>Brown</Author><Year>2007</Year><RecNum>97</RecNum><record><rec-number>97</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">97</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Brown, K. M.</author><author>Arthur, J. R.</author></authors></contributors><titles><title>Selenium, selenoproteins and human health: a review</title><secondary-title>Public Health Nutrition</secondary-title></titles><periodical><full-title>Public Health Nutrition</full-title></periodical><pages>593-599</pages><volume>4</volume><number>2b</number><dates><year>2007</year></dates><urls></urls></record></Cite><Cite><Author>Fox</Author><Year>2001</Year><RecNum>98</RecNum><record><rec-number>98</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">98</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Fox, C.</author><author>Ramsoomair, D.</author><author>Carter, C.</author></authors></contributors><titles><title>Magnesium: its proven and potential clinical significance</title><secondary-title>Southern Medical Journal</secondary-title></titles><periodical><full-title>Southern Medical Journal</full-title></periodical><pages>1195-1201</pages><volume>94</volume><number>12</number><dates><year>2001</year></dates><urls></urls></record></Cite></EndNote>(Brown and Arthur, 2007; Fox et al., 2001), and diabetes with zinc ADDIN EN.CITE <EndNote><Cite><Author>Thompson</Author><Year>1995</Year><RecNum>99</RecNum><record><rec-number>99</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">99</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Thompson, K. H.</author><author>Godin, D. V.</author></authors></contributors><titles><title>Micronutrients and antioxidants in the progression of diabetes</title><secondary-title>Nutrition Research</secondary-title></titles><periodical><full-title>Nutrition Research</full-title></periodical><pages>1377-1410</pages><volume>15</volume><number>9</number><keywords><keyword>Diabetes Mellitus</keyword><keyword>Micronutrient</keyword><keyword>Zinc</keyword><keyword>Ascorbic Acid</keyword><keyword>Vitamin E</keyword><keyword>Antioxidant</keyword></keywords><dates><year>1995</year></dates><isbn>0271-5317</isbn><urls><related-urls><url>;(Thompson and Godin, 1995). The results of the study suggest that it is possible to improve the dietary intake of local dwelling elders by increasing consumption of fruits, vegetables and calcium-rich foods ADDIN EN.CITE <EndNote><Cite><Author>Bernstein</Author><Year>2002</Year><RecNum>102</RecNum><record><rec-number>102</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">102</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Bernstein, Melissa A.</author><author>Nelson, Miriam E.</author><author>Tucker, Katherine L.</author><author>Layne, Jennifer</author><author>Johnson, Elizabeth</author><author>Nuernberger, Andrea</author><author>Castaneda, Carmen</author><author>Judge, James O.</author><author>Buchner, David</author><author>Singh, Maria Fiatarone</author></authors></contributors><titles><title>A Home-Based Nutrition Intervention to Increase Consumption of Fruits, Vegetables, and Calcium-Rich Foods in Community Dwelling Elders</title><secondary-title>Journal of the American Dietetic Association</secondary-title></titles><periodical><full-title>Journal of the American Dietetic Association</full-title></periodical><pages>1421-1427</pages><volume>102</volume><number>10</number><dates><year>2002</year></dates><isbn>0002-8223</isbn><urls><related-urls><url>;(Bernstein et al., 2002). A study in vitro revealed that vegetables such as kale, celery, collard, Chinese cabbage, and soybean sprout contained high levels of dialyzable calcium (20-39%) compared to milk powder, which contains 25 mg Ca/100 g ADDIN EN.CITE <EndNote><Cite><Author>Kamchan</Author><RecNum>103</RecNum><record><rec-number>103</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">103</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Kamchan, Achiraya</author><author>Puwastien, Prapasri</author><author>Sirichakwal, P. P. Prapaisri P.</author><author>Kongkachuichai, Ratchanee</author></authors></contributors><titles><title>In vitro calcium bioavailability of vegetables, legumes and seeds</title><secondary-title>Journal of Food Composition and Analysis</secondary-title></titles><periodical><full-title>Journal of Food Composition and Analysis</full-title></periodical><pages>311-320</pages><volume>17</volume><number>3-4</number><keywords><keyword>Calcium bioavailability</keyword><keyword>Vegetables</keyword><keyword>Seeds and legumes</keyword><keyword>Thailand</keyword></keywords><dates><pub-dates><date>2004/8//</date></pub-dates></dates><isbn>0889-1575</isbn><urls><related-urls><url>;(Kamchan et al.). A study on individuals age 65 and older reported that calcium is required for skeletal maintenance and that supplemental intakes to a total in the range of 1300-1700 mg Ca/day stop age-related bone loss and reduce fracture risk ADDIN EN.CITE <EndNote><Cite><Author>Heaney</Author><Year>2001</Year><RecNum>104</RecNum><record><rec-number>104</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">104</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Heaney, Robert P.</author></authors></contributors><titles><title>Calcium Needs of the Elderly to Reduce Fracture Risk</title><secondary-title>J Am Coll Nutr</secondary-title></titles><periodical><full-title>J Am Coll Nutr</full-title></periodical><pages>192S-197</pages><volume>20</volume><number>2</number><dates><year>2001</year><pub-dates><date>April 1, 2001</date></pub-dates></dates><urls><related-urls><url>;(Heaney, 2001).Magnesium is the fourth most abundant cation in the body. It is believed that more than 300 enzymatic reactions are involved with magnesium. The recommended dietary intakes (RDI) for magnesium are about 30 mg/day for adults. Though magnesium deficiency is rare in healthy people, but diabetes, kidney diseases, hypertension, and coronary artery diseases may induce magnesium depletion ADDIN EN.CITE <EndNote><Cite><Author>Wahlqvist</Author><Year>2002</Year><RecNum>105</RecNum><record><rec-number>105</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">105</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Wahlqvist, M. L.</author><author>Darmadi-Blackberry, I.</author></authors></contributors><titles><title>Magnesium in human health</title><secondary-title>Current Therapeutics</secondary-title></titles><periodical><full-title>Current Therapeutics</full-title></periodical><pages>35</pages><volume>43</volume><number>6</number><dates><year>2002</year></dates><urls></urls></record></Cite></EndNote>(Wahlqvist and Darmadi-Blackberry, 2002). Green vegetables, some legumes (beans and peas), nuts and seeds, and whole and unrefined grains are good sources of magnesium ADDIN EN.CITE <EndNote><Cite><Author>USD-ARS</Author><Year>2003</Year><RecNum>106</RecNum><record><rec-number>106</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">106</key></foreign-keys><ref-type name="Electronic Article">43</ref-type><contributors><authors><author>USD-ARS</author></authors></contributors><titles><title>U.S. Department of Agriculture, Agricultural Research Service. USDA National Nutrient Database for Standard Reference, Release 16. Nutrient Data Laboratory Home Page, . </title></titles><dates><year>2003</year></dates><urls></urls></record></Cite></EndNote>(USD-ARS, 2003). Eating lot of green vegetables, whole grains and legumes will help meet your dietary need for magnesium ADDIN EN.CITE <EndNote><Cite><Author>USD-ARS</Author><Year>2003</Year><RecNum>106</RecNum><record><rec-number>106</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">106</key></foreign-keys><ref-type name="Electronic Article">43</ref-type><contributors><authors><author>USD-ARS</author></authors></contributors><titles><title>U.S. Department of Agriculture, Agricultural Research Service. USDA National Nutrient Database for Standard Reference, Release 16. Nutrient Data Laboratory Home Page, . </title></titles><dates><year>2003</year></dates><urls></urls></record></Cite></EndNote>(USD-ARS, 2003).Dark green leafy vegetables are good sources of iron, especially sixty percent of non-heme irons are found in fruits, vegetables, grain, and nut. Recommended dietary intakes of iron are 10 mg/day for a child, 12 mg/day for a man, and 15 mg/day for a woman ADDIN EN.CITE <EndNote><Cite><Author>Baynes</Author><Year>1990</Year><RecNum>107</RecNum><record><rec-number>107</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">107</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Baynes, R. D.</author><author>Bothwell, T. H.</author></authors></contributors><titles><title>Iron Deficiency</title><secondary-title>Annual Review of Nutrition</secondary-title></titles><periodical><full-title>Annual Review of Nutrition</full-title></periodical><pages>133-148</pages><volume>10</volume><number>1</number><dates><year>1990</year></dates><urls><related-urls><url>;(Baynes and Bothwell, 1990). An experiment conducted on butter head lettuce reported that plants grown with high Fe levels (10 mg/L) had notable tip burn incidence but no bolting was observed ADDIN EN.CITE <EndNote><Cite><Author>Chow</Author><Year>1999</Year><RecNum>108</RecNum><record><rec-number>108</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">108</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Chow, K. K.</author><author>Meng-Wei, L.</author><author>Choo, H. L.</author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Effects of Nutrient Composition on Butterhead Lettuce (</style><style face="italic" font="default" size="100%">Lactuca sativa</style><style face="normal" font="default" size="100%"> L. cv. </style><style face="italic" font="default" size="100%">Panama</style><style face="normal" font="default" size="100%">) Grown in Deep Flow Technique for the Tropics</style></title><secondary-title>Acta Horticulturae</secondary-title></titles><periodical><full-title>ACTA HORTICULTURAE</full-title></periodical><pages>135-146</pages><dates><year>1999</year></dates><urls></urls></record></Cite></EndNote>(Chow et al., 1999). The results also indicated that high Fe could be beneficial in producing desired head size. In accordance with another experiment revealed tip burn increased with nitrogen supply and was related to an increase in head size ADDIN EN.CITE <EndNote><Cite><Author>Brumm</Author><Year>1992</Year><RecNum>109</RecNum><record><rec-number>109</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">109</key></foreign-keys><ref-type name="Conference Proceedings">10</ref-type><contributors><authors><author>Brumm, I.</author><author>Schenk, M.</author></authors></contributors><titles><title>Influence of Nitrogen Supply on the Occurence of Calcium Deficiency in Field Grown Lettuce</title></titles><pages>125-136</pages><dates><year>1992</year></dates><publisher>ISHS</publisher><urls></urls></record></Cite></EndNote>(Brumm and Schenk, 1992).Fresh leafy vegetables are major sources of minerals for recommended dietary intakes in the recent times. Lettuce has been an important vegetable crop with high market value, and its nutritional characteristics have been studied throughout the world ADDIN EN.CITE <EndNote><Cite><Author>Ashkar</Author><Year>1970</Year><RecNum>110</RecNum><record><rec-number>110</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">110</key></foreign-keys><ref-type name="Thesis">32</ref-type><contributors><authors><author>Ashkar, S. A.</author><author>Ries, S. K.</author></authors></contributors><titles><title>Lettuce tipburn as related to nutrient imbalance and nitrogen composition</title></titles><dates><year>1970</year></dates><publisher>Michigan State University. Dept. of Horticulture</publisher><urls></urls></record></Cite><Cite><Author>Chow</Author><Year>1999</Year><RecNum>108</RecNum><record><rec-number>108</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">108</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Chow, K. K.</author><author>Meng-Wei, L.</author><author>Choo, H. L.</author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Effects of Nutrient Composition on Butterhead Lettuce (</style><style face="italic" font="default" size="100%">Lactuca sativa</style><style face="normal" font="default" size="100%"> L. cv. </style><style face="italic" font="default" size="100%">Panama</style><style face="normal" font="default" size="100%">) Grown in Deep Flow Technique for the Tropics</style></title><secondary-title>Acta Horticulturae</secondary-title></titles><periodical><full-title>ACTA HORTICULTURAE</full-title></periodical><pages>135-146</pages><dates><year>1999</year></dates><urls></urls></record></Cite></EndNote>(Ashkar and Ries, 1970; Chow et al., 1999). The objective of the experiment will be to determine if the nutrient densities of selected vegetable crops can be increased through increasing the nutrient contents in the medium in which the plants grow.Materials and MethodsMaterialsSix lettuce cultivars will be used for this experiment. The six cultivars will be taken from the eighteen cultivars based on nutritional requirement and growth observes in the experiment above. Three cultivars of each group will be selected as modern and heritage. Two fertilizer regimes with elevating concentration will be selected for this study. One fertilizer regime will be chemical fertilizer, and another regime will be organic fertilizer with varying nutrient concentration in solution. Methods The seedlings of lettuce cultivars will be grown in the Bowditch greenhouse at UMass Amherst. Seeds of those cultivars will be collected from the professional seeds growers. Heritage type of seeds will be collected from certified organic seeds producer (Seed of Change Seeds Co., Spicer, MN 56288) and modern type of seeds were collected from another successful seeds producer (Johnny Seeds Co., Winslow, ME 04901). All seeds will be planted in peat moss medium for raising seedlings. For best germination rate, the optimum temperature will be maintained of 68oF (20oC). After four weeks from seeding, same sized seedlings will be transplanted to the 6?? round pot filled with peat moss media (MetroMix 360, SunGro Horticulture, Vancouver, Canada).Two different fertilizer regimes will be selected for nutrient sources of lettuce cultivars. The elevating fertilizer regimes will be tested if the nutrient densities of the lettuce crop increases through cultivar selection and nutrient management. Two fertilizer regime will be Jack’s 20-10-10 Peat-Lite commercial fertilizers (Jack’s Professional Fertilizers, J.R. Peters, Inc., Allentown, PA) and organic fertilizer (Pure Blend Pro Grow 3-1.5-4, Botanicare, Tempe, AZ) with varying concentration of Ca for this experiment. The goal of the application of different fertilizer regimes with varying concentration is to determine the nutrient densities of the lettuce through cultivar selection and nutrient management. The lettuces will be grown in the greenhouse for about 4-5 weeks from transplanting to marketable size.Each regime of fertilizer will be divided into three treatment groups based on varying internal nutrient concentration. The first fertilizer regime will be contained N, P, K, and micronutrient composite from a Jack’s 20-10-10 Peat-Lite commercial fertilizers with varying Ca (200, 300, 400 ppm) as CaCl2. Another fertilizer regime will be obtained from organic fertilizer with varying amount of Ca (200, 300, 400 ppm) as CaCl2. Three treatments of each group of fertilizer regime will be tested for this study. Therefore six treatments will be selected in two groups of fertilizer regimes. The treatment will be applied in one day interval at 100 ml/plant for first week then at 200 ml/plant until harvest. At harvest, growth parameters including plant height, plant size and color, and fresh weights of plant will be recorded. Oven-dry weights of plant will be recorded afterwards.Portions of dry plant sample of leaves will be ground to pass a 30-mesh screen and sub sampled and stored in plastic containers for elemental analysis of N, P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr accumulated by lettuce leaves. Kjeldhal N will be determined by acid-digestion and posterior determination by auto analyzer (BRAN+LUEBBE, method G-188-97, BRAN+LUEBBE, Norderstedt, Germany). Pulverized samples (0.5 g) will be ashed at 500oC for 12 h in a muffle furnace and the ash will be dissolved in 10% HCl solution prepared in deionized H2O. For P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr, the ash extracts will be analyzed by inductive coupled plasma (ICP) spectrophotometric analysis ADDIN EN.CITE <EndNote><Cite><Author>Jones Jr</Author><Year>1991</Year><RecNum>27</RecNum><record><rec-number>27</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">27</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Jones Jr, J. B.</author><author>Wolf, B.</author><author>Mills, H. A.</author></authors></contributors><titles><title>Plant Analysis Handbook. Micro-Macro Publ</title><secondary-title>Inc., Athens, GA</secondary-title></titles><periodical><full-title>Inc., Athens, GA</full-title></periodical><dates><year>1991</year></dates><urls></urls></record></Cite><Cite><Author>Kalra</Author><Year>1998</Year><RecNum>28</RecNum><record><rec-number>28</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">28</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>Kalra, Y. P.</author></authors></contributors><titles><title>Handbook of reference methods for plant analysis</title></titles><dates><year>1998</year></dates><pub-location>Boca Raton</pub-location><publisher>CRC Press</publisher><urls></urls></record></Cite></EndNote>(Jones Jr et al., 1991; Kalra, 1998) at UMass Soil and Plant Tissue Laboratory. Lettuce growth parameters will be used for evaluating the consequences of different fertilizer regimes and their combination on lettuce growth and nutrient management through cultivars selection.Pest will be managed by using certified organic or conventional herbicides, fungicides or insecticides as required to bring about control. During the growing season, appearance of diseases symptoms and occurrences will be carefully monitored and diseases severity will be evaluated for each vegetables at a scale of 0-9 scale indicating 0 being no visible symptoms and 9 being severe symptoms.Assess the effect of organic and conventional soil fertility practices on mineral accumulation in vegetables in field experiment.IntroductionOrganic farming differs from conventional farming mainly tillage methods, crop rotation, fertilizer applications, and pest control methods. Organically grown vegetables have greater market values than conventional farms. Synthetic chemical fertilizers and pesticides uses in intensive agriculture may cause serious impact on public health and the environment ADDIN EN.CITE <EndNote><Cite><Author>Pimentel</Author><Year>2005</Year><RecNum>113</RecNum><record><rec-number>113</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">113</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Pimentel, David</author><author>Hepperly, Paul</author><author>Hanson, James</author><author>Douds, David</author><author>Seidel, Rita</author></authors></contributors><titles><title>Environmental, Energetic, and Economic Comparisons of Organic and Conventional Farming Systems</title><secondary-title>BioScience</secondary-title></titles><periodical><full-title>BioScience</full-title></periodical><pages>573-582</pages><volume>55</volume><number>7</number><dates><year>2005</year></dates><urls><related-urls><url>(2005)055[0573:EEAECO]2.0.CO;2</electronic-resource-num></record></Cite></EndNote>(Pimentel et al., 2005). Integrated pest and nutrient management systems and certified organic agriculture may reduce reliance on agrochemical inputs as well as make agriculture environmentally and economically sound ADDIN EN.CITE <EndNote><Cite><Author>Pimentel</Author><Year>2005</Year><RecNum>113</RecNum><record><rec-number>113</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">113</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Pimentel, David</author><author>Hepperly, Paul</author><author>Hanson, James</author><author>Douds, David</author><author>Seidel, Rita</author></authors></contributors><titles><title>Environmental, Energetic, and Economic Comparisons of Organic and Conventional Farming Systems</title><secondary-title>BioScience</secondary-title></titles><periodical><full-title>BioScience</full-title></periodical><pages>573-582</pages><volume>55</volume><number>7</number><dates><year>2005</year></dates><urls><related-urls><url>(2005)055[0573:EEAECO]2.0.CO;2</electronic-resource-num></record></Cite></EndNote>(Pimentel et al., 2005).Applications of compost in vegetable crop production systems have been shown better yield and fruit quality responses a compared with those obtained from standard vegetable crop commercial practices ADDIN EN.CITE <EndNote><Cite><Author>Stoffella</Author><Year>2001</Year><RecNum>111</RecNum><record><rec-number>111</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">111</key></foreign-keys><ref-type name="Conference Proceedings">10</ref-type><contributors><authors><author>Stoffella, P. J.</author><author>Ozores-Hampton, M.</author><author>Roe, N. E.</author><author>Li, Y.</author><author>Obreza, T. A.</author></authors></contributors><titles><title>Compost Utilization in Vegetable Crop Production Systems</title></titles><pages>125-128</pages><dates><year>2001</year></dates><publisher>ISHS</publisher><urls></urls></record></Cite></EndNote>(Stoffella et al., 2001). The impact of organic production practices on soil quality revealed that there was 22% more organic carbon (1257 kg/ha) and 20% more total N (970 kg/ha) on organic farm than conventional farm ADDIN EN.CITE <EndNote><Cite><Author>Liebig</Author><Year>1999</Year><RecNum>112</RecNum><record><rec-number>112</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">112</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Liebig, M. A.</author><author>Doran, J. W.</author><author>Usda, A. R. S.</author></authors></contributors><titles><title>Impact of organic production practices on soil quality indicators</title></titles><dates><year>1999</year></dates><urls></urls></record></Cite></EndNote>(Liebig et al., 1999). The findings of the study also reported that soils of organic farms had soil pH closer to neutral, lower bulk density, and higher available water-holding capacity, microbial biomass C and N, and soil respiration compared with conventional farms ADDIN EN.CITE <EndNote><Cite><Author>Liebig</Author><Year>1999</Year><RecNum>112</RecNum><record><rec-number>112</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">112</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Liebig, M. A.</author><author>Doran, J. W.</author><author>Usda, A. R. S.</author></authors></contributors><titles><title>Impact of organic production practices on soil quality indicators</title></titles><dates><year>1999</year></dates><urls></urls></record></Cite></EndNote>(Liebig et al., 1999). Ultimately, organic farming resulted better in maintaining soil productivity and reducing soil erosion than conventional farming ADDIN EN.CITE <EndNote><Cite><Author>Reganold</Author><Year>1987</Year><RecNum>114</RecNum><record><rec-number>114</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">114</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Reganold, J. P.</author><author>Elliott, L. F.</author><author>Unger, Y. L.</author></authors></contributors><titles><title>Long-term effects of organic and conventional farming on soil erosion</title><secondary-title>Nature</secondary-title></titles><periodical><full-title>Nature</full-title></periodical><pages>370-372</pages><volume>330</volume><number>6146</number><dates><year>1987</year></dates><urls></urls></record></Cite></EndNote>(Reganold et al., 1987). It has been believed that by applying good organic composts supplemented with lower amount of chemical fertilizers, high crop production and maintenance, or even increasing soil fertility can be achieved.The objective of the study will be to determine if the nutrient densities of selected vegetable crops can be increased through soil fertility practices (e.g., organic vs conventional fertilizers; different fertilizer used).Materials and MethodsMaterialsEighteen different lettuce cultivars will be used in this study. Nine heritage and nine modern cultivars will be used for this experiment. Two regimes of organic fertilizer and one conventional fertilizer regime will be used for this study. One kind of organic fertilizer regime will be obtained from individual nutrients sources providing nitrogen, phosphorus, and potassium requirements of crops; and other kind of organic fertilizer regime will be obtained from compost source providing those nutrients. Fertilizer for individual nutrients will be included soybean meal for nitrogen, colloidal rock phosphate for the phosphorus, mined potassium sulfate for the potassium. These materials are used commonly by organic growers in the region. The compost will be collected from the University of Massachusetts Amherst office of waste management, which produce compost from dinning commons food waste and yard waste from the Amherst campus. Conventional fertilizer will be obtained from a commercial fertilizers (10-10-10 all purpose) providing nutrient requirements of crops. Methods The experiment will be conducted at UMass field experiment station. Eighteen different cultivars of lettuce will be grown in the field at UMass Amherst field experiment station. Seeds of those cultivars will be collected from the professional seed producers. Heritage type of seeds will be collected from certified organic seeds producer (Seed of Change Seeds Co., Spicer, MN) and modern type of seeds will be collected from another successful seeds producer (Johnny Seeds Co., Winslow, ME). Both types of seed will be planted in mixed soil (soil with peat moss 1:1) at Bowditch greenhouse. For best germination of seeds soil temperature will be maintained of 68oF (20oC). At 4 weeks of age tomato seedlings will be transplanted in UMass field experiment station. Therefore, three different regimes of fertilizer will be chosen for this experiment. Two will be organic fertilizer regimes and one convention fertilizer regime. One group of organic regime will be obtained from some individual sources mentioned above, another group of organic regime will be obtained from compost sources collected from UMass Amherst office of waste management, and a conventional fertilizer regime will be obtained from a 10-10-10 all purpose commercial fertilizer. The compost will be analyzed for its nutrients contents before application. The reason for the use of different fertility regimes is to find out the higher nutrient densities in plants, growth and soil fertility management practices. The fertilizer will be applied based on the nutrient requirement of the crops according to the management guide ADDIN EN.CITE <EndNote><Cite><Author>Rosen</Author><Year>2005</Year><RecNum>27</RecNum><record><rec-number>27</rec-number><foreign-keys><key app="EN" db-id="sp2tda0pfee2wae0e0qpztw995rx0900ddv5">27</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Rosen, C. J.</author><author>Eliason, R.</author></authors></contributors><titles><title>Nutrient management for commercial fruit and vegetable crops in Minnesota</title><secondary-title>Univ. Minn. Extension Serv. BU-05886</secondary-title></titles><dates><year>2005</year></dates><urls></urls></record></Cite></EndNote>(Rosen and Eliason, 2005). The organic fertilizer regime will be applied at the rate of 20 ton/acre during soil preparation in one time as basal dose. The conventional fertilizer regime will be applied at the rate of 100 Ib/acre before transplant in one time as basal dose. In addition, 100 Ib CaSO4 /acre will be added during land preparation for calcium.Soil samples will be taken after plant harvest for N, P, K, Mg, and Ca measurement to determine the effect of the treatment on soil nutrient availability. A soil testing method proposed by Morgan will be used to extract the soil available nutrients ADDIN EN.CITE <EndNote><Cite><Author>Morgan</Author><Year>1941</Year><RecNum>79</RecNum><record><rec-number>79</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">79</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>M. F. Morgan</author></authors></contributors><titles><title>Chemical Soil Diagnosis by the Universal Soil Testing System</title><secondary-title>Bulletin 450 (ARevision of Bulletin 392), Connecticul Agricultural Experiment Station, New Haven.</secondary-title></titles><periodical><full-title>Bulletin 450 (ARevision of Bulletin 392), Connecticul Agricultural Experiment Station, New Haven.</full-title></periodical><dates><year>1941</year></dates><urls></urls></record></Cite></EndNote>(Morgan, 1941). Nitrogen will be assessed distillation and titration of NH3 in solution ADDIN EN.CITE <EndNote><Cite><Author>Bradstreet</Author><Year>1965</Year><RecNum>80</RecNum><record><rec-number>80</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">80</key></foreign-keys><ref-type name="Generic">13</ref-type><contributors><authors><author>Bradstreet, R. B.</author></authors></contributors><titles><title>The Kjeldahl method for organic nitrogen (1965) London</title></titles><dates><year>1965</year></dates><publisher>UK: Academic Press</publisher><urls></urls></record></Cite></EndNote>(Bradstreet, 1965); P will be determined by colorimetry (blue molybdophosphoric acid method) ADDIN EN.CITE <EndNote><Cite><Author>Olsen</Author><Year>1965</Year><RecNum>81</RecNum><record><rec-number>81</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">81</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>S. R. Olsen</author><author>L. A. Dean</author></authors></contributors><titles><title> Phosphorus. pp. 0351049. In: C. A. Black (ed.), Methods of soil analysis. Part 2. Agronomy 9.</title><secondary-title>American Society of Agronomy, Madison. WI</secondary-title></titles><periodical><full-title>American Society of Agronomy, Madison. WI</full-title></periodical><volume>9</volume><dates><year>1965</year></dates><urls></urls></record></Cite></EndNote>(Olsen and Dean, 1965); Ca and Mg will be determined by atomic absorption spectrometry; and K by atomic emission spectrometry ADDIN EN.CITE <EndNote><Cite><Author>Thomas</Author><Year>1967</Year><RecNum>83</RecNum><record><rec-number>83</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">83</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Thomas, R. L.</author><author>Sheard, R. W.</author><author>Moyer, J. R.</author></authors></contributors><titles><title>Comparison of conventional and automated procedures for nitrogen, phosphorus, and potassium analysis of plant material using a single digestion</title><secondary-title>Agronomy Journal</secondary-title></titles><periodical><full-title>Agronomy Journal</full-title></periodical><pages>240</pages><volume>59</volume><number>3</number><dates><year>1967</year></dates><urls></urls></record></Cite><Cite><Author>Lierop</Author><Year>1976</Year><RecNum>84</RecNum><record><rec-number>84</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">84</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Lierop, W.</author></authors></contributors><titles><title>Digestion procedures for simultaneous automated determination of NH4, P, K, Ca and Mg in plant material</title><secondary-title>Canadian Journal of Soil Science (Canada)</secondary-title></titles><periodical><full-title>Canadian Journal of Soil Science (Canada)</full-title></periodical><dates><year>1976</year></dates><urls></urls></record></Cite></EndNote>(Lierop, 1976; Thomas et al., 1967). At harvest, growth indices including plant height, plant size, leaf color, leaf sizes, and fresh weights of whole plant will be recorded. Oven dry weights of whole plants will be recorded afterwards.Dry leaves sample will be grounded to pass a 30-mesh screen for tissue nutrient analysis of N, P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr. Kjeldhal N will be determined by acid-digestion and posterior determination by auto analyzer (Bran+luebbe method G-188-97, Bran+luebbe, Norderstedt, Germany). Pulverized samples (0.5 g) will be ashed at 500oC for 12 h in a muffle furnace and the ash will be dissolved in 10% HCl solution prepared in deionized H2O. For P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr, the ash extracts will be analyzed by inductive coupled plasma (ICP) spectrophotometric analysis ADDIN EN.CITE <EndNote><Cite><Author>Jones Jr</Author><Year>1991</Year><RecNum>27</RecNum><record><rec-number>27</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">27</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Jones Jr, J. B.</author><author>Wolf, B.</author><author>Mills, H. A.</author></authors></contributors><titles><title>Plant Analysis Handbook. Micro-Macro Publ</title><secondary-title>Inc., Athens, GA</secondary-title></titles><periodical><full-title>Inc., Athens, GA</full-title></periodical><dates><year>1991</year></dates><urls></urls></record></Cite><Cite><Author>Kalra</Author><Year>1998</Year><RecNum>28</RecNum><record><rec-number>28</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">28</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>Kalra, Y. P.</author></authors></contributors><titles><title>Handbook of reference methods for plant analysis</title></titles><dates><year>1998</year></dates><pub-location>Boca Raton</pub-location><publisher>CRC Press</publisher><urls></urls></record></Cite></EndNote>(Jones Jr et al., 1991; Kalra, 1998) at UMass Soil and Plant Tissue Laboratory. Lettuce growth parameters will be used for evaluating the consequences of different fertilizer regimes and their treatment on nutrient management through cultivars selection and soil fertility. Pest and disease symptoms will be managed by using certified organic herbicides and fungicides or insecticides to bring about control. The severity of diseases will be evaluated using a scale of 0-9 indicating 0 being no visible symptoms and 9 being severe symptoms.Assessment of the genetic purity and genetic diversity among cultivars of lettuce using molecular markers in the greenhouse experimentation.IntroductionMolecular markers are having uses as an effective tool for efficient selection of desired agronomic traits. Molecular markers can facilitate tomato breeding by means of marker assisted selection (MAS) to improve agronomical important characteristics such as yield, fruit quality, and disease resistance. Molecular markers such as RFLP (Restriction fragment length polymorphism) ADDIN EN.CITE <EndNote><Cite><Author>Ooijen</Author><Year>1994</Year><RecNum>115</RecNum><record><rec-number>115</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">115</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ooijen, J. W.</author><author>Sandbrink, J. M.</author><author>Vrielink, M.</author><author>Verkerk, R.</author><author>Zabel, P.</author><author>Lindhout, P.</author></authors></contributors><titles><title>An RFLP linkage map of Lycopersicon peruvianum</title><secondary-title>TAG Theoretical and Applied Genetics</secondary-title></titles><periodical><full-title>TAG Theoretical and Applied Genetics</full-title></periodical><pages>1007-1013</pages><volume>89</volume><number>7</number><dates><year>1994</year></dates><urls><related-urls><url> app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">116</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Sandbrink, J. M.</author><author>Ooijen, J. W.</author><author>Purimahua, C. C.</author><author>Vrielink, M.</author><author>Verkerk, R.</author><author>Zabel, P.</author><author>Lindhout, P.</author></authors></contributors><titles><title>Localization of genes for bacterial canker resistance in Lycopersicon peruvianum using RFLPs</title><secondary-title>TAG Theoretical and Applied Genetics</secondary-title></titles><periodical><full-title>TAG Theoretical and Applied Genetics</full-title></periodical><pages>444-450</pages><volume>90</volume><number>3</number><dates><year>1995</year></dates><urls><related-urls><url>;(Ooijen et al., 1994; Sandbrink et al., 1995), RAPD (Random amplification of polymorphic DNA) ADDIN EN.CITE <EndNote><Cite><Author>Stevens</Author><Year>1995</Year><RecNum>117</RecNum><record><rec-number>117</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">117</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Stevens, M. R.</author><author>Lamb, E. M.</author><author>Rhoads, D. D.</author></authors></contributors><titles><title>Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomatoes using RAPD and RFLP analyses</title><secondary-title>TAG Theoretical and Applied Genetics</secondary-title></titles><periodical><full-title>TAG Theoretical and Applied Genetics</full-title></periodical><pages>451-456</pages><volume>90</volume><number>3</number><dates><year>1995</year></dates><urls><related-urls><url> app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">118</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Qian, W.</author><author>Ge, S.</author><author>Hong, D. Y.</author></authors></contributors><titles><title>Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers</title><secondary-title>TAG Theoretical and Applied Genetics</secondary-title></titles><periodical><full-title>TAG Theoretical and Applied Genetics</full-title></periodical><pages>440-449</pages><volume>102</volume><number>2</number><dates><year>2001</year></dates><urls></urls></record></Cite></EndNote>(Qian et al., 2001; Stevens et al., 1995), ISSR (Inter simple sequence repeat) ADDIN EN.CITE <EndNote><Cite><Author>Zietkiewicz</Author><Year>1994</Year><RecNum>119</RecNum><record><rec-number>119</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">119</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Zietkiewicz, E.</author><author>Rafalski, A.</author><author>Labuda, D.</author></authors></contributors><titles><title>Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification</title><secondary-title>Genomics</secondary-title></titles><periodical><full-title>Genomics</full-title></periodical><pages>176-183</pages><volume>20</volume><number>2</number><dates><year>1994</year></dates><urls></urls></record></Cite><Cite><Author>Joshi</Author><Year>2000</Year><RecNum>120</RecNum><record><rec-number>120</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">120</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Joshi, S. P.</author><author>Gupta, V. S.</author><author>Aggarwal, R. K.</author><author>Ranjekar, P. K.</author><author>Brar, D. S.</author></authors></contributors><titles><title>Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus Oryza</title><secondary-title>TAG Theoretical and Applied Genetics</secondary-title></titles><periodical><full-title>TAG Theoretical and Applied Genetics</full-title></periodical><pages>1311-1320</pages><volume>100</volume><number>8</number><dates><year>2000</year></dates><urls></urls></record></Cite></EndNote>(Joshi et al., 2000; Zietkiewicz et al., 1994), AFLP ( Amplified fragment length polymorphism) ADDIN EN.CITE <EndNote><Cite><Author>Vos</Author><Year>1995</Year><RecNum>121</RecNum><record><rec-number>121</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">121</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Vos, P.</author><author>Hogers, R.</author><author>Bleeker, M.</author><author>Reijans, M.</author><author>Lee, T.</author><author>Hornes, M.</author><author>Friters, A.</author><author>Pot, J.</author><author>Paleman, J.</author><author>Kuiper, M.</author></authors></contributors><titles><title>AFLP: a new technique for DNA fingerprinting</title><secondary-title>Nucleic acids research</secondary-title></titles><periodical><full-title>Nucleic acids research</full-title></periodical><pages>4407</pages><volume>23</volume><number>21</number><dates><year>1995</year></dates><urls></urls></record></Cite></EndNote>(Vos et al., 1995), and Microsatellite polymorphism ADDIN EN.CITE <EndNote><Cite><Author>Panaud</Author><Year>1996</Year><RecNum>122</RecNum><record><rec-number>122</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">122</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Panaud, O.</author><author>Chen, X.</author><author>McCouch, S. R.</author></authors></contributors><titles><title>Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.)</title><secondary-title>Molecular and General Genetics MGG</secondary-title></titles><periodical><full-title>Molecular and General Genetics MGG</full-title></periodical><pages>597-607</pages><volume>252</volume><number>5</number><dates><year>1996</year></dates><urls></urls></record></Cite></EndNote>(Panaud et al., 1996) have been developed in tomato and other crops since last decade. However, RFLP uses breeding purposes is limited because it requires the use of radioactivity and is laborious. RAPD, ISSR and AFLP markers either identify only dominant alleles or are sensitive to PCR amplification conditions.Microsatellites or Simple Sequence Repeats (SSRs) are short (mostly 2-4 bp) tandem repeats of DNA sequences. The variation or polymorphism of SSRs are a result of polymerase slippage during DNA replication ADDIN EN.CITE <EndNote><Cite><Author>Levinson</Author><Year>1987</Year><RecNum>123</RecNum><record><rec-number>123</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">123</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Levinson, G.</author><author>Gutman, G. A.</author></authors></contributors><titles><title>Slipped-strand mispairing: a major mechanism for DNA sequence evolution</title><secondary-title>Molecular biology and evolution</secondary-title></titles><periodical><full-title>Molecular biology and evolution</full-title></periodical><pages>203</pages><volume>4</volume><number>3</number><dates><year>1987</year></dates><urls></urls></record></Cite></EndNote>(Levinson and Gutman, 1987). SSR markers are becoming the preferred molecular markers in crop breeding because of their properties of genetic co-dominance, high reproducibility and multi allelic variation. They are the most practical markers for genomic mapping, variety identification and marker-assisted selection. In lettuce, some microsatellite markers have been used ADDIN EN.CITE <EndNote><Cite><Author>Areshchenkova</Author><Year>1999</Year><RecNum>124</RecNum><record><rec-number>124</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">124</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Areshchenkova, T.</author><author>Ganal, M. W.</author></authors></contributors><titles><title>Long tomato microsatellites are predominantly associated with centromeric regions</title><secondary-title>Genome</secondary-title></titles><periodical><full-title>Genome</full-title></periodical><pages>536-544</pages><volume>42</volume><number>3</number><dates><year>1999</year></dates><urls></urls></record></Cite><Cite><Author>Smulders</Author><Year>1997</Year><RecNum>126</RecNum><record><rec-number>126</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">126</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Smulders, M. J. M.</author><author>Bredemeijer, G.</author><author>Rus-Kortekaas, W.</author><author>Arens, P.</author><author>Vosman, B.</author></authors></contributors><titles><title>Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species</title><secondary-title>TAG Theoretical and Applied Genetics</secondary-title></titles><periodical><full-title>TAG Theoretical and Applied Genetics</full-title></periodical><pages>264-272</pages><volume>94</volume><number>2</number><dates><year>1997</year></dates><urls></urls></record></Cite></EndNote>(Areshchenkova and Ganal, 1999; Smulders et al., 1997), but the number of SSR markers are available for molecular breeding is still few and only a limited number of SSR markers have been mapped to the tomato genome ADDIN EN.CITE <EndNote><Cite><Author>Areshchenkova</Author><Year>1999</Year><RecNum>124</RecNum><record><rec-number>124</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">124</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Areshchenkova, T.</author><author>Ganal, M. W.</author></authors></contributors><titles><title>Long tomato microsatellites are predominantly associated with centromeric regions</title><secondary-title>Genome</secondary-title></titles><periodical><full-title>Genome</full-title></periodical><pages>536-544</pages><volume>42</volume><number>3</number><dates><year>1999</year></dates><urls></urls></record></Cite><Cite><Author>Broun</Author><Year>1996</Year><RecNum>125</RecNum><record><rec-number>125</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">125</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Broun, P.</author><author>Tanksley, S. D.</author></authors></contributors><titles><title>Characterization and genetic mapping of simple repeat sequences in the tomato genome</title><secondary-title>Molecular and General Genetics MGG</secondary-title></titles><periodical><full-title>Molecular and General Genetics MGG</full-title></periodical><pages>39-49</pages><volume>250</volume><number>1</number><dates><year>1996</year></dates><urls></urls></record></Cite></EndNote>(Areshchenkova and Ganal, 1999; Broun and Tanksley, 1996).The objectives of the present study will be to assess the genetic similarities, variation, and purity among Lactuca sativa accessions in the USDA collection; and to develop and characterize the more SSRs markers for lettuce.Materials and MethodsMaterials Plant materialEighteen lettuce cultivars will be used for this experiment. Nine will be modern and nine will be heritage lettuce cultivars. The reason for choosing these plant cultivars for assessing the similarities and variation among cultivars are using in the previous experiment. All cultivars and its parental lines will be collected from the U.S. National Plant Germplasm System (NPGS) at North Central Region Plant Introduction Station in Ames, IA, USA; and heritage cultivars line will be collected from a commercial source (Seed of Change Seeds Co. Inc, Spicer, MN). All purposes commercial fertilizer of peters professional 20-20-20 TE formula will be used to grow lettuce. The rate of the fertilizer will be used at1 g/L, and additional CaSO4 will be added in the media at 1g/pot for Ca.Methods Genomic DNA IsolationThe heritage and modern seeds of the cultivars will be grown in the greenhouse with a day temperature of 24 ± 3oC and a night temperature of 18 ± 3oC in the Bowditch greenhouse at University of Massachusetts Amherst. One-month-old seedlings will be transplanted to the pots where individual of each cultivar will randomly be selected and numbered. Regular irrigation, fertilization, staking and crop protection measures will be adopted and purity visual evaluation will be conducted based on the main important morphological characters throughout the growth period. Total genomic DNA of the 18 lines will be isolated from young leaves following the method described by ADDIN EN.CITE <EndNote><Cite><Author>Liu</Author><Year>2003</Year><RecNum>85</RecNum><record><rec-number>85</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">85</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Liu,</author><author>L. Liu</author><author>Guo,</author><author>W. Guo</author><author>Zhu,</author><author>X. Zhu</author><author>Zhang,</author><author>T. Zhang</author></authors></contributors><titles><title>Inheritance and fine mapping of fertility restoration for cytoplasmic male sterility in Gossypium hirsutum L</title><secondary-title>TAG Theoretical and Applied Genetics</secondary-title></titles><periodical><full-title>TAG Theoretical and Applied Genetics</full-title></periodical><pages>461-469</pages><volume>106</volume><number>3</number><dates><year>2003</year></dates><urls><related-urls><url> app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">137</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Yu, K.</author><author>Pauls, K. P.</author></authors></contributors><titles><title>Optimization of DNA-extraction and PCR procedures for random amplified polymorphic DNA (RAPD) analysis in plants</title><secondary-title>PCR technology: current innovations. CRC press, Boca Raton, Florida</secondary-title></titles><periodical><full-title>PCR technology: current innovations. CRC press, Boca Raton, Florida</full-title></periodical><pages>193–200</pages><dates><year>1994</year></dates><urls></urls></record></Cite></EndNote>(Liu et al., 2003; Yu and Pauls, 1994) with some modifications. Leaves will be collected and immediately frozen in liquid nitrogen and stored at -70°C until use. For each sample, four fresh leaf obtained by punching leaves with the cap of a 1.5-ml Eppendorf tube, will putinto 400 μl of DNA extraction buffer (200 mM Tris–HCl, pH 7.4, 250 mM of NaCl, 25 mM of EDTA, pH 8.0, 0.5% SDS) and homogenized with a plastic pestle (Mandel Scientific Company Ltd.). Then 400 μl of 24:1 chloroform/isopropyl alcohol will be added to the homogenized solution, vortexed and left at room temperature for 30 min. The homogenate will spun in a micro centrifuge at a speed of 10,500 rpm for 2 min and 350 μl of the supernatant were transferred into a new Eppendorf tube. For DNA precipitation, an equal volume (350 μl) of isopropanol will be added to the tube that will left at room temperature for 5 min and then spun at 11,000 rpm for 5 min. Then, the DNA pellet will be air-dried at room temperature for 30 to 60 min before it will be dissolved in 200 μl of water at 4 °C overnight. The supernatant will be collected after micro centrifugation at 1,300 rpm for 2 min, yielding about 25 ng/μl of DNA.Search of DNA sequences and primer designA list of about 1,000 asteraceae microsatellites (the majority were L. sativa) showing the GenBank database accession numbers with their motifs, and the number of repeats will kindly provided by Andreas Matern, Cornell University, Ithaca, New York. The entire DNA sequence for each accession is searched, retrieved from the GenBank database and verified for the presence of SSRs. If the SSR is not at, or very close to, either the 5′ or 3′ end, the sequence is collected. Prior to primer design, all the saved DNA sequences will be analyzed using the program DNASIS (Hitachi America Ltd., San Bruno, Cal.) for homologous sequences. Each sequence will be compared with the rest of the DNA sequences. If homologous sequences are found, only one unique sequence will kept for primer design while the rest of the homologous DNA sequences will be eliminated because of their redundancy. PCR primers (forward and reverse) flanking the repeat sequence will be designed using the computer program GENE RUNNER (Hastings Software, Inc., N.Y.). The core parameters used in the primer design include the following: (1) the primer length is between 18 bp and 25 bp, (2) the percentage of GC is between 35% and 60%, (3) the Tm of the primers is over 40 °C which will be calculated using Tm = 59.9 + 0.41 (%G+C) – (675/primer length) based on the standard PCR conditions at a salt concentration of 50 mM ADDIN EN.CITE <EndNote><Cite><Author>Sharrocks</Author><Year>1994</Year><RecNum>138</RecNum><record><rec-number>138</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">138</key></foreign-keys><ref-type name="Book Section">5</ref-type><contributors><authors><author>A.D. Sharrocks </author></authors><secondary-authors><author>T. Weissensteiner</author><author>H. G. Griffin</author><author> A. M. Griffin</author></secondary-authors></contributors><titles><title>The design of primers for PCR</title><secondary-title>PCR technology: current innovations
</secondary-title></titles><pages>5-11</pages><dates><year>1994</year></dates><publisher>CRC press Inc.</publisher><urls></urls></record></Cite></EndNote>(Sharrocks, 1994), and (4) the predicted PCR products range from 100 to 350 bp in length with a preference of between 100 bp and 250 bp. In addition, the primer internal structures, such as hairpin loops, possible primer dimers, length of single base pair run at the 3′ end and the number of short repeats (such as CT, GA etc.) will also be taken into consideration. When two or more SSRs are located in the same DNA sequence but are at different sites, two flanking primers are designed separately for each of the SSRs. All designed oligonucleotides will be synthesized commercially by Sigmagenosys, Incorporated.SSR primers will be obtained from Operon Technologies Inc. (Alameda, CA) and tested. Single DNA molecular marker system, SSR, will be used to test seed genetic purity. SSR primers will be obtained from OperonTechnologies Inc., SSR-PCR will be performed in PTC-100 thermocycler (MJ Research Inc., USA) according to the protocol of ADDIN EN.CITE <EndNote><Cite><Author>Williams</Author><Year>1990</Year><RecNum>86</RecNum><record><rec-number>86</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">86</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Williams, John G. K.</author><author>Kubelik, Anne R.</author><author>Livak, Kenneth J.</author><author>Rafalski, J. Antoni</author><author>Tingey, Scott V.</author></authors></contributors><titles><title>DNA polymorphisms amplified by arbitrary primers are useful as genetic markers</title><secondary-title>Nucl. Acids Res.</secondary-title></titles><periodical><full-title>Nucl. Acids Res.</full-title></periodical><pages>6531-6535</pages><volume>18</volume><number>22</number><dates><year>1990</year><pub-dates><date>November 25, 1990</date></pub-dates></dates><urls><related-urls><url>;(Williams et al., 1990) and ADDIN EN.CITE <EndNote><Cite><Author>Zietkiewicz</Author><Year>1994</Year><RecNum>87</RecNum><record><rec-number>87</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">87</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Zietkiewicz, Ewa</author><author>Rafalski, Antoni</author><author>Labuda, Damian</author></authors></contributors><titles><title>Genome Fingerprinting by Simple Sequence Repeat (SSR)-Anchored Polymerase Chain Reaction Amplification</title><secondary-title>Genomics</secondary-title></titles><periodical><full-title>Genomics</full-title></periodical><pages>176-183</pages><volume>20</volume><number>2</number><dates><year>1994</year></dates><isbn>0888-7543</isbn><urls><related-urls><url>;(Zietkiewicz et al., 1994) respectively. The SSR amplification products will be separated and detected according to the reported methods ADDIN EN.CITE <EndNote><Cite><Author>Liu</Author><Year>2007</Year><RecNum>88</RecNum><record><rec-number>88</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">88</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Liu, G.</author><author>Liu, L.</author><author>Gong, Y.</author><author>Wang, Y.</author><author>Yu, F.</author><author>Shen, H.</author><author>Gui, W.</author></authors></contributors><titles><title>Seed genetic purity testing of F1 hybrid cabbage (Brassica oleracea var. capitata) with molecular marker analysis</title><secondary-title>Seed Science and Technology</secondary-title></titles><periodical><full-title>Seed Science and Technology</full-title></periodical><pages>477-486</pages><volume>35</volume><dates><year>2007</year></dates><urls><related-urls><url>;(Liu et al., 2007a). The oligonucleotide primers for SSR analysis will be synthesized by Invitrogen Biotech (Shanghai) Co., China, according to the reported sequences of microsatellite markers ADDIN EN.CITE <EndNote><Cite><Author>He</Author><Year>2003</Year><RecNum>89</RecNum><record><rec-number>89</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">89</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>He,</author><author>C. He</author><author>Poysa,</author><author>V. Poysa</author><author>Yu,</author><author>K. Yu</author></authors></contributors><titles><title>Development and characterization of simple sequence repeat (SSR) markers and their use in determining relationships among Lycopersicon esculentum cultivars</title><secondary-title>TAG Theoretical and Applied Genetics</secondary-title></titles><periodical><full-title>TAG Theoretical and Applied Genetics</full-title></periodical><pages>363-373</pages><volume>106</volume><number>2</number><dates><year>2003</year></dates><urls><related-urls><url>;(He et al., 2003). The SSR-PCR reactions will be performed and products will be detected according to the reported protocol ADDIN EN.CITE <EndNote><Cite><Author>Liu</Author><Year>2007</Year><RecNum>88</RecNum><record><rec-number>88</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">88</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Liu, G.</author><author>Liu, L.</author><author>Gong, Y.</author><author>Wang, Y.</author><author>Yu, F.</author><author>Shen, H.</author><author>Gui, W.</author></authors></contributors><titles><title>Seed genetic purity testing of F1 hybrid cabbage (Brassica oleracea var. capitata) with molecular marker analysis</title><secondary-title>Seed Science and Technology</secondary-title></titles><periodical><full-title>Seed Science and Technology</full-title></periodical><pages>477-486</pages><volume>35</volume><dates><year>2007</year></dates><urls><related-urls><url>;(Liu et al., 2007a) . PCR amplification and product electrophoresisPCR reactions will be performed in 96-well plates using either the Perkin Elmer GeneAmp PCR system 9600 (PE Biosystems) or the TECHNE Genius themal cycler (Techne Ltd., U.K.) with the same amplification program. Each 10-μl reaction mixture contained about 25 ng of lettuce genomic DNA, 0.3 μM of forward and reverse primers, 300 μM of each dNTP, 1 μl of 10 × PCR buffer containing 100 mM of Tris–HCl, pH 8.3, 500 mM of KCl, and 1 unit of Taq DNA polymerase. The PCR amplification conditions will be programmed as one cycle of denaturation at 94 °C for 2 min, followedby 35-cycles amplification with a 25 s denaturing at 94 °C, a 25 s annealing at the Tm (Tm varies for the individual primers) and a 25 s extension at 68 °C. After PCR amplication, the products will be mixed with 3 μl of stop buffer (97% deionized formamide, 0.3% each bromophenol blue and xylene cyanol FF and 10 mM of EDTA, pH 8.0) and then denatured at 94 °C for 5 min in a PCR machine. Four micro litres of each denatured PCR product will be used for fragment separation on a DNA sequencing gel (6% polyacrylamide, 8 M urea and 1 × TBE buffer) running at a constant power of 55 W for 2–2.5 h, using an S2 sequencing-gel apparatus (GIBCO BRL). A 1-kb-plus DNA size marker will also be loaded along with the samples for each run to estimate the fragment sizes of the separated DNA fragments. After each run, the gel was placed in 10% glacial acetic-acid fixation solution for 20 min with gentle shaking, silver-stained for 30 min and then immediately developed in a 3% sodium carbonate solution according to the DNA silver-staining kit (Promega).Nomenclature of SSR markersThe nomenclature of the SSR markers will be based on the method described by ADDIN EN.CITE <EndNote><Cite><Author>Yu</Author><Year>2000</Year><RecNum>139</RecNum><record><rec-number>139</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">139</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Yu, K.</author><author>Park, S. J.</author><author>Poysa, V.</author><author>Gepts, P.</author></authors></contributors><titles><title>Integration of simple sequence repeat (SSR) markers into a molecular linkage map of common bean (Phaseolus vulgaris L.)</title><secondary-title>J Hered</secondary-title></titles><periodical><full-title>J Hered</full-title></periodical><pages>429-434</pages><volume>91</volume><number>6</number><dates><year>2000</year><pub-dates><date>November 1, 2000</date></pub-dates></dates><urls><related-urls><url>;(Yu et al., 2000). The SSR name will be prefixed with LS standing for L. sativa, followed by the repeat motif in lowercase and a number starting from 001 for each distinct repeat motif. For example, LSaat001 and LSaat002 re-present, respectively, the SSR markers at two different loci with the same repeat motif “aat”. For the imperfect or compound repeats, such as (AAG)3T(TGA)7, only the motif with the highest repeat number, in this case TGA, is used. When two or more different repeats such as the SSR locus (CT)12(GATA)12(AT)2(AC)10 have the same number of repeats, the repeat motif at the 5′ end is used. Thus, the SSR name for (CT)12(GATA)12(AT)2(AC)10 is designated as LSct rather than LSgata. This SSR nomenclature system can be applied to any newly developed microsatellites and provides a simple way to track SSR loci for use in a breeding program.Genetic analysisAll 18 genotypes from different geographic origins will be used to screen the SSR primers for PCR amplification and product-length polymorphism. For primers that produced the expected fragments after PCR reactions, the number of alleles will be recorded and the polymorphism information content (PIC) of an SSR locus will be calculated as described by ADDIN EN.CITE <EndNote><Cite><Author>Saal</Author><Year>1999</Year><RecNum>140</RecNum><record><rec-number>140</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">140</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Saal, B.</author><author>Wricke, G.</author></authors></contributors><titles><title>Development of simple sequence repeat markers in rye (Secale cereale L.)</title><secondary-title>Genome</secondary-title></titles><periodical><full-title>Genome</full-title></periodical><pages>964-972</pages><volume>42</volume><number>5</number><dates><year>1999</year></dates><urls></urls></record></Cite></EndNote>(Saal and Wricke, 1999):PIC= 1-i=1Kpi2where pi is the frequency of the ith allele out of the total number of alleles at an SSR locus, and k is the total number of different alleles for that locus. For phylogenetic analysis, only the data for the polymorphic SSR loci are entered for all DNA samples, and a “1” or “0” is used if an allele is present or absent for a genotype, respectively. The data will be analyzed using the computer program TREECON (Van de Peer and De Wachter 1994). The genetic-distance estimation will be based on the method described by ADDIN EN.CITE <EndNote><Cite><Author>Nei</Author><Year>1979</Year><RecNum>141</RecNum><record><rec-number>141</rec-number><foreign-keys><key app="EN" db-id="xfdrx00a7dfdrlepd9dpv5ahtdexa05vpv29">141</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Nei, M.</author><author>Li, W. H.</author></authors></contributors><titles><title>Mathematical model for studying genetic variation in terms of restriction endonucleases</title><secondary-title>Proceedings of the National Academy of Sciences of the United States of America</secondary-title></titles><periodical><full-title>Proceedings of the National Academy of Sciences of the United States of America</full-title></periodical><pages>5269-5273</pages><volume>76</volume><number>10</number><dates><year>1979</year><pub-dates><date>October 1979</date></pub-dates></dates><urls><related-urls><url>;(Nei and Li, 1979). All 18 different lettuce genotypes will be clustered based on the estimated genetic distance, and the phylogenetic tree topology was inferred with the clustering method of the Unweighted Pair Group Method Using Arithmetic Average (UPGMA).Summary Literature Cited ADDIN EN.REFLIST Abdulla, M.and P. Gruber. 2000. Role of diet modification in cancer prevention. Biofactors. 12: 45-51.Anonymous. 1989. 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