基于SRK基因序列分析的甘蓝自交不亲和系单倍型鉴定及验证

摘要

根据甘蓝自交不亲和性雌蕊决定因子SRK基因保守序列设计简并引物,克隆23份甘蓝自交不亲和系SRK基因并测序.利用NCBI数据库进行克隆序列的Blast比较以及序列之间的相似性分析,初步确定23份甘蓝自交不亲和系分属于SRK3、SRK7、SRK8、SRK16、SRK28、SRK36、SRK45、SRK46、SRK58、SRK68等10种S单倍型.其中有5份甘蓝自交不亲和系属于SRK36单倍型,占参试自交不亲和系的22%;SRK7、SRK16和SRK58单倍型均只有1份甘蓝自交不亲和系.进一步对不同自交不亲和系之间花期杂交后花粉萌发及花粉管伸长情况进行荧光显微观察,并对20个杂交组合的花期杂交亲和指数进行调查分析,结果表明根据SRK基因序列所确定的相同S单倍型自交不亲和系之间花期授粉表现为不亲和,花粉在柱头上萌发受阻,花期亲和指数低;而不同S单倍型自交不亲和系之间花期授粉表现高度亲和,花粉管伸入雌蕊正常,花期亲和指数高.表明基于SRK基因序列分析确定甘蓝自交不亲和系所属单倍型是一种简单而可靠的快速鉴定方法,有利于育种时有针对性地设计甘蓝自交不亲和系杂交组合配制,从而提高育种效率.%In the present study,based on the self-incompatibility pistil determinant SRK genes conserved sequence,a pair degenerate primer was designed to clone the SRK gene sequence in 23 self-incompatible lines and sequenced.SRK gene sequences from 23 cabbage self-incompatible lines were performed alignments analysis to NCBI database,total 10 S-haplotypes,SRK3,SRK7,SRK8,RK16,SRK28,SRK36,SRK45,SRK46, SRK58 and SRK68 were found.Among them,5 self-incompatible lines were belonged to SRK 36 S-haplotypes, accounting for 22% of all self-incompatible lines.However,only one S-haplotype self-incompatible line was detected in SRK7,SRK16 and SRK58.Moreover,pollen germination on stigma and pollen tube elongation in pistil were investigated using fluorescence microscopy after pollinated between different self-incompatible lines at flowering stage.The results indicated that pollen germinated well on the stigma and pollen tube grew well in pistil of those self-incompatible lines belonging to different S-haplotypes,whereas the pollen did not germinate on the stigma of those self-incompatible lines belonging to same S-haplotypes.The compatibility index of 20 cross-combinations of self-incompatible lines at flowering stage was also investigated.The crosses between self-incompatible lines with same S-haplotypes were highly incompatible after pollinated at flowering stage,however, those crosses between self-incompatible lines with different S-haplotypes were highly compatible after pollinated at flowering stage.All the results showed that haplotypes identification of cabbage self-incompatible lines based on the SRK gene sequence analysis was a simple,reliable and fast method for accurate cabbage cross-combination design,and was helpful to improve the efficiency of cabbage heterosis breeding.