SLAF-seq BSA定位水稻黄叶转绿基因grc2的效果研究  

Effects on Mapping the Gene of Green-revertible Chlorina 2(grc2) Using SLAF-seq BSA in Rice(Oryza sativa)

作　　者：谭炎宁[1] 余东[1,2] 盛夏冰 康伟伟[2] 李哲理 段美娟 袁定阳[1,3] TAN Yan-Ning;YU Dong;SHENG Xia-Bing;KANG Wei-Wei;LI Zhe-Li;DUAN Mei-Juan;YUAN Ding-Yang(State Key Laboratory of Hybrid Rice,Hunan Hybrid Rice Research Center,Changsha 410125,China;College of Biosciences and Biotechnology,Hunan Agricultural University,Changsha 410128,China;College of Agronomy,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南杂交水稻研究中心杂交水稻国家重点实验室,长沙410125 [2]湖南农业大学生物科技学院,长沙410128 [3]湖南农业大学农学院,长沙410128

出　　处：《农业生物技术学报》2020年第3期381-388,共8页Journal of Agricultural Biotechnology

基　　金：湖南农业科技创新资金项目(2017XC09);湖南省自然科学基金项目(2019JJ40206);湖南杂交水稻研究中心科研创新基金(YB201907)。

摘　　要：将传统分群分析法(bulk segregant analysis, BSA)与全基因组特异性位点扩增片段测序手段(specific-locus amplified fragment sequencing, SLAF-seq)深度整合的SLAF-seq BSA技术为快速定位质量性状基因提供了可能。然而,对这一技术的定位准确性程度却少有报道。本研究以水稻(Oryza sativa)黄化转绿基因grc2 (green-revertible chlorina, grc2)为对象,分析了SLAF-seq BSA定位结果与传统图位定位结果的差异。首先,通过简化基因组测序获得了在’grc2’突变体和正常叶色水稻’Y58S’之间表现出单核苷酸多态性的5 302个SLAF标记;然后,利用Y58S/grc2的F2群体中的100个野生型单株和100个突变型单株构建了两个极端混池,再通过自动化计算各SLAF标记在极端混池中的父母本基因型频率,筛选到了符合在0.01水平满足父本(’grc2’)、母本(’Y58S’)基因型在野生型混池中为33∶66且在突变型混池中为99∶1的关联标记共13个,这些标记集中分布在Chr06前臂的0.98~1.21 Mb与1.57~1.99 Mb两个相邻区域。经连锁分析发现,第1区域的关联标记AM1、AM6与grc2基因的遗传距离仅为0.625和0 cM;将关联标记比对至日本晴参考基因组后,发现第1关联区域恰好覆盖了grc2基因的传统定位区间,表明SLAF-seq BSA的定位结果具有参考价值。本研究结果为支撑SLAF-seq BSA能快速关联质量性状基因提供了证据。SLAF-seq BSA, integrated traditional bulk segregant analysis(BSA) with specific-locus amplified fragment sequencing(SLAF-seq), makes it possible to quickly locate qualitative trait genes. However, little is known about the reliability of its mapping results. In this study, the green-revertible chlorina gene(grc2) in rice(Oryza sativa) was selected as a target for comparing the mapping results provided with map-based mapping and SLAF-seq BSA. Firstly, 5302 SLAF markers were identified which showed single nucleotide polymorphism between ’grc2’ and ’Y58 S’(a cultivar presenting normally green leaves) by SLAF-seq. Later,the wild-type bulk and the mutant bulk were established, each consisting of 100 plants from a F2 population of’Y58 S’/’grc2’. By automatically calculating the genotype frequency in two pools, 13 SLAF markers were found associated with grc2 which accorded with an expected separation ratio of 33∶66 for paternal/maternal in wild-type bulk and 99∶1 in mutant bulk at 0.01 level. Interestingly, all of the associated markers were distributed on two neighbour regions of 0.98~1.21 Mb and 1.57~1.99 Mb on Chromosome 6 referenced as ’93-11’(an indica cultivar). Furthermore, two markers named AM1 and AM6 on the first region were confirmed linkaged with grc2, just exhibiting a genetic distance of 0.625 cM and 0 cM respectively. While referenced as’ Nipponbare’(a japonica cultivar), it was observed that the first region just covered the mapping area with traditional method, indicating the result of SLAF-seq BSA is for consulting. Therefore, the results presented herein supported SLAF-seq BSA would be a useful tool for rapidly obtaining the region associated with qualitative trait gene.

关 键 词：基因定位 质量性状基因 特异性位点扩增片段测序(SLAF-seq) 分群分析法(BSA) 水稻 

分 类 号：S336[农业科学—作物遗传育种]