小麥三雌蕊基因Pis1連鎖的SRAP分子標(biāo)記開(kāi)發(fā)
發(fā)布時(shí)間：2018-12-07 15:39
【摘要】：采用SRAP分子標(biāo)記技術(shù)篩選與小麥三雌蕊基因Pis1連鎖的分子標(biāo)記。以小麥三雌蕊近等基因系CM28TP及其輪回親本CM28(川麥28)為親本,雜交獲得F1,F1自交得到F2群體,提取基因組DNA,采用分離群體分組分析法(Bulked Segregant Analysis,BSA)構(gòu)建三雌蕊性狀池和正常雌蕊池,利用44條SRAP正向引物和44條SRAP反向引物組成1936個(gè)SRAP引物組合,逐步進(jìn)行篩選,結(jié)果如下:1、為研究小麥三雌蕊性狀的遺傳規(guī)律,以CM28與CM28TP為親本,雜交獲得F1代312株,F1代自交得到1046株F2代群體。觀察F1代單株,結(jié)果顯示F1代全為三雌蕊性狀;F2代統(tǒng)計(jì)結(jié)果為793株表現(xiàn)三雌蕊性狀,253株為正常雌蕊。χ2檢驗(yàn)結(jié)果表明,F2代三雌蕊和正常雌蕊分離比符合3:1比例,驗(yàn)證了三雌蕊性狀由顯性單基因控制。2、本實(shí)驗(yàn)采用傳統(tǒng)的CTAB法和試劑盒法提取基因組DNA,試劑盒法包括植物基因組DNA提取試劑盒和多糖多酚植物基因組DNA提取試劑盒兩種,并進(jìn)行了比較,提取的結(jié)果表明:采用多糖多酚植物基因組DNA提取試劑盒提取的基因組DNA,純度、濃度、OD260/OD280和OD260/OD230值等均符合要求。瓊脂糖凝膠電泳檢測(cè)的基因組DNA電泳條帶,條帶清晰且較亮,沒(méi)有拖尾現(xiàn)象。3、選用44條SRAP正向引物和44條SRAP反向引物組成1936個(gè)SRAP引物組合,對(duì)兩個(gè)親本CM28和CM28TP進(jìn)行SRAP分析,篩選出在親本間具有差異的特異性引物組合共224對(duì)。用在親本間表現(xiàn)出多態(tài)性的224對(duì)引物組合分別對(duì)三雌蕊池、正常雌蕊池進(jìn)行擴(kuò)增,有32對(duì)引物組合的PCR擴(kuò)增產(chǎn)物在基因池間表現(xiàn)出差異性。用在三雌蕊池和正常雌蕊池間表現(xiàn)差異的32對(duì)引物組合,分別用構(gòu)建基因池的11株三雌蕊單株和11株正常雌蕊單株進(jìn)行PCR擴(kuò)增,其中me2-em36引物組合在8個(gè)三雌蕊上擴(kuò)增出一條約100bp的條帶,而6個(gè)正常雌蕊上并沒(méi)有擴(kuò)增出。me3-em23引物組合在9個(gè)正常雌蕊上擴(kuò)增出一條約200bp的條帶,而7個(gè)三雌蕊上并沒(méi)有擴(kuò)增出。me16-em21引物組合在8個(gè)正常雌蕊上擴(kuò)增出一條約50bp的條帶,而7個(gè)三雌蕊上并沒(méi)有擴(kuò)增出。me16-em25引物組合在8個(gè)三雌蕊上擴(kuò)增出一條約50bp的條帶,而7個(gè)正常雌蕊上并沒(méi)有擴(kuò)增出。me16-em26引物組合在10個(gè)正常雌蕊擴(kuò)增出一條約80bp的條帶,而9個(gè)三雌蕊上并沒(méi)有擴(kuò)增出。me43-em27引物組合在8個(gè)三雌蕊上擴(kuò)增出一條約80bp的條帶,而7個(gè)正常雌蕊上并沒(méi)有擴(kuò)增出。me44-em39引物組合在8個(gè)三雌蕊上擴(kuò)增出一條約80bp的條帶,而8個(gè)正常雌蕊上并沒(méi)有擴(kuò)增出。將這7對(duì)引物組合在218株F2群體中進(jìn)行驗(yàn)證,其中有三雌蕊159株,正常雌蕊59株。利用Mapmaker 3.0計(jì)算遺傳距離,結(jié)果只有me2-em36和me16-em26這兩對(duì)引物組合擴(kuò)增的條帶與Pis1基因連鎖,將這兩個(gè)標(biāo)記命名為m2e36,m16e26。m2e36與Pis1的遺傳距離為18.22cM,標(biāo)記m16e26與Pis1的連鎖距離為22.63cM。這兩個(gè)分子標(biāo)記分別位于Pis1基因的兩側(cè)。
[Abstract]:SRAP molecular marker technique was used to screen the molecular markers linked to the Pis1 gene of Tripistil gene in wheat. F _ 2 population was obtained by crossing with CM28TP and its recurrent parent CM28 (Chuanmai 28). Genomic DNA, was extracted from F _ 2 population by separating population grouping analysis (Bulked Segregant Analysis,). BSA) was used to construct a three-pistil trait pool and a normal pistil pool. 1936 SRAP primer combinations were composed of 44 SRAP forward primers and 44 SRAP reverse primers. The results were as follows: 1. Using CM28 and CM28TP as parents, the F _ 1 generation was crossed with 312 plants, and 1046 F _ 2 populations were obtained from F _ 1 generation. The single plant of F1 generation was observed, and the results showed that all F1 generations were three pistil characters. In F2 generation, 793 plants showed three pistil traits and 253 were normal pistil. 蠂 2 test showed that the segregation ratio of three pistils and normal pistil of F2 was 3:1, which confirmed that the trait of three pistil was controlled by dominant single gene. In this experiment, the traditional CTAB method and kit method were used to extract genomic DNA, from plants, including plant genomic DNA extraction kit and polysaccharide polyphenol plant genomic DNA extraction kit, and compared with each other. The results showed that the purity, concentration, OD260/OD280 and OD260/OD230 value of genomic DNA, extracted from polysaccharides polyphenol plant genomic DNA extraction kit met the requirements. The genomic DNA bands detected by agarose gel electrophoresis were clear and bright, and there was no trailing phenomenon. 3. 44 SRAP forward primers and 44 SRAP reverse primers were selected to form 1936 SRAP primer combinations. SRAP analysis was performed on two parent CM28 and CM28TP. A total of 224 pairs of specific primer combinations with different parents were screened. Two hundred and twenty-four pairs of primer combinations were used to amplify the three pistil cisterns and the normal pistil cisterns, and 32 pairs of primer combinations were used to amplify the PCR products among the gene pools. Using 32 pairs of primer combinations which showed the difference between the three pistil cisterns and the normal pistil cisterns, the PCR amplification was carried out by using 11 single plants of three pistil and 11 single plants of normal pistil, respectively. The me2-em36 primer combinations amplified a treaty 100bp band on 8 pistil, but not on 6 normal pistil. Me3-em23 primer combination amplified a treaty 200bp band on 9 normal pistil. Me16-em21 primer combinations amplified a treaty 50bp band on 8 normal pistil. Me16-em25 primer combinations amplified a treaty 50bp band on 8 triecium. Me16-em26 primer combinations amplified a treaty 80bp band in 10 normal pistil. The me43-em27 primer combination amplified a treaty 80bp band on 8 triecium. Me44-em39 primer combinations amplified a treaty 80bp band on 8 triecium, but not on 8 normal pistil. The 7 pairs of primer combinations were tested in 218 F2 populations. There were 159 gynoecium and 59 normal pistil. The genetic distance was calculated by Mapmaker 3.0. Only the bands amplified by the combination of me2-em36 and me16-em26 were linked to the Pis1 gene. The genetic distance between m2e36 m16e26.m2e36 and Pis1 was 18.22 cm. The linkage distance between marker m16e26 and Pis1 was 22.63 cm. These two molecular markers are located on both sides of the Pis1 gene, respectively.
【學(xué)位授予單位】：西華師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943.2
本文編號(hào)：2367409
[Abstract]:SRAP molecular marker technique was used to screen the molecular markers linked to the Pis1 gene of Tripistil gene in wheat. F _ 2 population was obtained by crossing with CM28TP and its recurrent parent CM28 (Chuanmai 28). Genomic DNA, was extracted from F _ 2 population by separating population grouping analysis (Bulked Segregant Analysis,). BSA) was used to construct a three-pistil trait pool and a normal pistil pool. 1936 SRAP primer combinations were composed of 44 SRAP forward primers and 44 SRAP reverse primers. The results were as follows: 1. Using CM28 and CM28TP as parents, the F _ 1 generation was crossed with 312 plants, and 1046 F _ 2 populations were obtained from F _ 1 generation. The single plant of F1 generation was observed, and the results showed that all F1 generations were three pistil characters. In F2 generation, 793 plants showed three pistil traits and 253 were normal pistil. 蠂 2 test showed that the segregation ratio of three pistils and normal pistil of F2 was 3:1, which confirmed that the trait of three pistil was controlled by dominant single gene. In this experiment, the traditional CTAB method and kit method were used to extract genomic DNA, from plants, including plant genomic DNA extraction kit and polysaccharide polyphenol plant genomic DNA extraction kit, and compared with each other. The results showed that the purity, concentration, OD260/OD280 and OD260/OD230 value of genomic DNA, extracted from polysaccharides polyphenol plant genomic DNA extraction kit met the requirements. The genomic DNA bands detected by agarose gel electrophoresis were clear and bright, and there was no trailing phenomenon. 3. 44 SRAP forward primers and 44 SRAP reverse primers were selected to form 1936 SRAP primer combinations. SRAP analysis was performed on two parent CM28 and CM28TP. A total of 224 pairs of specific primer combinations with different parents were screened. Two hundred and twenty-four pairs of primer combinations were used to amplify the three pistil cisterns and the normal pistil cisterns, and 32 pairs of primer combinations were used to amplify the PCR products among the gene pools. Using 32 pairs of primer combinations which showed the difference between the three pistil cisterns and the normal pistil cisterns, the PCR amplification was carried out by using 11 single plants of three pistil and 11 single plants of normal pistil, respectively. The me2-em36 primer combinations amplified a treaty 100bp band on 8 pistil, but not on 6 normal pistil. Me3-em23 primer combination amplified a treaty 200bp band on 9 normal pistil. Me16-em21 primer combinations amplified a treaty 50bp band on 8 normal pistil. Me16-em25 primer combinations amplified a treaty 50bp band on 8 triecium. Me16-em26 primer combinations amplified a treaty 80bp band in 10 normal pistil. The me43-em27 primer combination amplified a treaty 80bp band on 8 triecium. Me44-em39 primer combinations amplified a treaty 80bp band on 8 triecium, but not on 8 normal pistil. The 7 pairs of primer combinations were tested in 218 F2 populations. There were 159 gynoecium and 59 normal pistil. The genetic distance was calculated by Mapmaker 3.0. Only the bands amplified by the combination of me2-em36 and me16-em26 were linked to the Pis1 gene. The genetic distance between m2e36 m16e26.m2e36 and Pis1 was 18.22 cm. The linkage distance between marker m16e26 and Pis1 was 22.63 cm. These two molecular markers are located on both sides of the Pis1 gene, respectively.
【學(xué)位授予單位】：西華師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943.2
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