大豆幼胚培养再生基因的发掘与功能标记开发

Cultivated soybean [Glycine max (L.) Merr.] has its origin from wild soybean [Glycine soja Sieb. et Zucc.] in China and has been a major crop worldwide, which contributes significantly to the global economy as an important source of dietary oil and protein. However, soybean regeneration capacity remains relatively low in comparison with other crops, such that it is still recognized as one of the recalcitrant crops for genetic engineering. To date, research has focused on factors such as the media components, culture conditions and explants types to develop and optimize methods for plant regeneration and transformation. However, the genetic basis of tissue culture responses has not been investigated in detail. Conventional linkage mapping can be an effective tool for identifying genes underlying natural variation. However, the genes identified by this method are restricted to the ones segregating in the cross under consideration. Association mapping is emerging as a powerful method to rapidly identify genome locations, but it suffers from the limitation that it generates false positives due to population structure. To this end, it has been proposed that association mapping should be carried out in conjunction with genetic linkage analysis to detect relevant genes. Meanwhile, high density soybean linkage map and the accomplishment of soybean genome sequencing make it impossible to search candidate genes in the soybean genomic level. In this study, we aim to combine linkage and association mapping to discover the genes or allelic loci and exploit the functional molecular markers for soybean tissue culture response. The prospective results will certainly help complete our knowledge of the genetic basis underlying soybean tissue culture responses, the establishment of efficient soybean regeneration and transformation system and the creation of soybean artificial seeds. Moreover, the information would lead soybean breeders not only to improve soybean tissue culture response, but also to combine these qualities with other valuable traits to achieve the integrated improvement of soybean agronomic traits.

大豆起源于我国，是世界上主要的粮食和油料作物，但也是难再生和转化的作物，这影响了大豆转基因育种的进程，制约着我国大豆产业的发展。目前，人们主要从建立再生和转化体系的角度出发，着重研究了影响大豆再生能力的各种因素，而对其再生过程中的遗传机理研究却很少。基于分离群体的连锁分析和基于自然群体的关联分析具有明显的互补性，二者结合可以提高QTL定位的精度和广度；同时高密度大豆遗传图谱的构建和大豆基因组序列测定的完成，为在大豆全基因组水平上寻找目的基因提供了可能。本研究拟分别利用科丰重组自交家系群体(NJRIKY)和由200份大豆品种组成的自然群体，对大豆再生能力相关性状进行连锁作图分析和关联分析，综合分析发掘与大豆再生性状相关的等位位点或目的基因，开发相应的功能性标记，以期进一步弄清大豆体细胞胚胎发生再生体系的遗传机制，为建立高效、稳定的大豆再生、遗传转化体系和人工大豆种子的创造奠定理论基础。

大豆的再生和遗传转化体系是进行转基因育种的重要基础，而再生培养能力的高低是决定转基因效率的关键。目前的研究主要集中在影响大豆再生能力的各种因素，而对大豆组织培养再生能力的遗传机制研究较少。因此，本研究首先利用科丰1号×南农1138-2衍生的184个重组自交家系群体（NJRIKY）对大豆幼胚培养再生能力性状进行QTL定位分析，选用大豆愈伤组织诱导率和体细胞胚诱导率作为反映大豆幼胚培养再生能力的评价指标，采用复合区间作图法(CIM)进行QTL分析，结果共检测到3个与出愈率有关的QTL，位于B2和D2连锁群上，可解释5.84%-16.60%的表型变异；检测到4个与体细胞胚诱导率有关的QTL，全部位于G连锁群上，对表型性状变异的解释率为7.79%-14.16%；同时选取145份具有代表性的地方品种资源（Chinese soybean landrace population，CSLRP）组成自然群体，对出愈率和出胚率进行表型数据分析。根据具有广泛遗传变异的地方品种群体CSLRP的连锁不平衡（linkage disequilibrium，LD）水平建立一套由遍布全基因组SNP衍生的SNPLDB（SNP linkage disequilibrium block，SNP连锁不平衡区段）标记，分析其在全基因组的分布特点。利用SNPLDB标记并通过“两步法”对资源群体的2个性状进行全基因组关联分析，进而分析该群体中2个性状的遗传结构，了解不同生态区材料遗传结构的差异，发掘优异等位变异，为大豆再生性状的改良提供了标记辅助选择的依据。