Restriction site amplified polymorphism (RSAP) was used, for the first time, to analyze the genetic structure and diversity of four, mainly cultivated, varieties of the brown alga, Saccharina japonica. Eighty-eight sa...Restriction site amplified polymorphism (RSAP) was used, for the first time, to analyze the genetic structure and diversity of four, mainly cultivated, varieties of the brown alga, Saccharina japonica. Eighty-eight samples from varieties "Rongfu", "Fujian", "Ailunwan" and "Shengchanzhong" were used for the genetic analyses. One hundred and ninety-eight bands were obtained using eight combinations of primers. One hundred and ninety-one (96.46%) were polymorphic bands. Nei's genetic diversity was 0.360, and the coefficient of genetic differentiation was 0.357. No inbreeding-type recession was found in the four brown alga varieties and the results of the "Ailunwan" variety using samples from 2 years showed that the variety was becoming less diverse during the selection inherent in the breeding program. Genetic diversity and cluster analyses results were consistent with these genetic relationships. The results show the RSAP method is suitable for genetic analysis. Continuous inbreeding and selection could reduce the genetic diversity effectively; therefore periodical supervision is required.展开更多
【目的】对限制性位点扩增多态性（Restriction site amplified polymorphism，RsAP）标记技术进行改进，并优化了其反应体系。【方法】遵循引物设计原则，使限制性位点序列位于中间，在其3’端增加3个选择性碱基，5’端设计为10～12个...【目的】对限制性位点扩增多态性（Restriction site amplified polymorphism，RsAP）标记技术进行改进，并优化了其反应体系。【方法】遵循引物设计原则，使限制性位点序列位于中间，在其3’端增加3个选择性碱基，5’端设计为10～12个碱基的随机序列，设计了14条长为19bp的限制性位点扩增多态性（RSAP）新引物。以大白菜、紫菜薹自交系及其F1、F2的DNA为模板，对新引物的反应体系进行了优化，并对其重复性和适用性进行了检验。【结果】新设计引物PCR扩增的前5个循环退火温度为35℃，后35个循环为52℃；在25μL优化反应体系中，模板DNA用量为2．0μL（20．0ng／μL）、Mg^3＋ 3．0μL（25mmol／L）、Taq酶（5U／μL）1．5U、dNTPs2．0μL（2．5mmol／L）、引物各0．6μL（10μmol／L）；新引物扩增出的谱带较原引物更多，多态性更好；只改变选择性碱基，新设计引物就能扩增出新的谱带；新设计引物的重复性、稳定性好，在荞麦和小麦品系，以及紫菜薹、大白菜及其F1、F2中，新引物均能扩增出清晰谱带。【结论】新引物适用性和重复性好，可广泛应用于其他作物的基因组DNA分析。展开更多
基金Supported by the National Special Research Fund for Non-Profit Sector(No.200805075)the Shandong Foundation for Development of Scienceand Technology, China (No.2007GG10005018)+2 种基金the Genetically Modified Organism Technology Major Project (No.2009ZX08009-100B)the National High Technology Research and Development Program of China(863 Program) (Nos.2010AA10A401,2012AA10A406)the National Agricultural Transformation (No.2010GB23600666)
文摘Restriction site amplified polymorphism (RSAP) was used, for the first time, to analyze the genetic structure and diversity of four, mainly cultivated, varieties of the brown alga, Saccharina japonica. Eighty-eight samples from varieties "Rongfu", "Fujian", "Ailunwan" and "Shengchanzhong" were used for the genetic analyses. One hundred and ninety-eight bands were obtained using eight combinations of primers. One hundred and ninety-one (96.46%) were polymorphic bands. Nei's genetic diversity was 0.360, and the coefficient of genetic differentiation was 0.357. No inbreeding-type recession was found in the four brown alga varieties and the results of the "Ailunwan" variety using samples from 2 years showed that the variety was becoming less diverse during the selection inherent in the breeding program. Genetic diversity and cluster analyses results were consistent with these genetic relationships. The results show the RSAP method is suitable for genetic analysis. Continuous inbreeding and selection could reduce the genetic diversity effectively; therefore periodical supervision is required.
文摘【目的】对限制性位点扩增多态性（Restriction site amplified polymorphism，RsAP）标记技术进行改进，并优化了其反应体系。【方法】遵循引物设计原则，使限制性位点序列位于中间，在其3’端增加3个选择性碱基，5’端设计为10～12个碱基的随机序列，设计了14条长为19bp的限制性位点扩增多态性（RSAP）新引物。以大白菜、紫菜薹自交系及其F1、F2的DNA为模板，对新引物的反应体系进行了优化，并对其重复性和适用性进行了检验。【结果】新设计引物PCR扩增的前5个循环退火温度为35℃，后35个循环为52℃；在25μL优化反应体系中，模板DNA用量为2．0μL（20．0ng／μL）、Mg^3＋ 3．0μL（25mmol／L）、Taq酶（5U／μL）1．5U、dNTPs2．0μL（2．5mmol／L）、引物各0．6μL（10μmol／L）；新引物扩增出的谱带较原引物更多，多态性更好；只改变选择性碱基，新设计引物就能扩增出新的谱带；新设计引物的重复性、稳定性好，在荞麦和小麦品系，以及紫菜薹、大白菜及其F1、F2中，新引物均能扩增出清晰谱带。【结论】新引物适用性和重复性好，可广泛应用于其他作物的基因组DNA分析。
