黄瓜黑色果刺基因染色体定位及候选基因分析

Molecular Mapping and Candidate Gene Analysis of Black Fruit Spine Gene in Cucumber(Cucumis sativus L.)

【目的】黄瓜作为重要的果菜类蔬菜,果实品质一直是黄瓜育种研究的重点。果实品质包括内在品质和外观品质,其中外观品质对黄瓜的商品性具有重要影响。果刺颜色作为黄瓜重要的品质性状之一,对其进行遗传分析和基因定位将有助于了解果刺颜色遗传的分子机理,为黄瓜果实性状改良提供理论依据和技术支撑,同时也可为刺色基因的精细定位及克隆奠定基础。【方法】研究利用黄瓜白色果刺自交系GY14(P1)和黑色果刺自交系NC76(P2)为亲本构建遗传群体,进行黄瓜果刺颜色的遗传分析。以F2分离群体为试材,应用分离群体分组分析法(BSA)和2 112对SSR引物进行SSR分析,结合9 930黄瓜全基因组序列信息和100份核心种质重测序结果开发新标记,对初定位区域进行标记加密。采用JoinMap 4.0作图软件和MapInspect软件构建连锁群并完成连锁群与染色体的对应,实现黄瓜黑色果刺性状的基因定位。运用生物信息学,在定位区域进行候选基因分析。利用包含156个株系的重组自交系(RILs)群体,对黑色果刺基因紧密连锁的两侧翼分子标记进行验证,确定标记用于分子标记辅助选择(MAS)育种的准确性。【结果】研究表明,黄瓜自交系NC76的黑色果刺符合质量性状遗传特点,由显性单基因B控制,黑色对白色为显性。初步定位筛选获得了与B基因连锁的8对SSR引物,将B定位于黄瓜4号染色体(Chr.4)上,最近的连锁标记为SSR22231,遗传距离为10.8 cM;根据初定位区域的序列信息,设计合成了新的SSR引物212对和Indel引物25对,利用这些引物对双亲和F2群体DNA进行分析,最终构建了一个包含14个SSR标记和1个InDel标记的分子标记连锁群,获得与B连锁的两侧翼标记SSRB-181和SSRB-130,遗传距离分别为2.0 cM和1.6 cM,该区段物理距离为422.1 kb,有60个预测候选基因,推测Csa4G003095和Csa4G001690是与黑色果刺形成相关性较大的候选基因。与B紧密连锁的两侧翼标记用于MAS育种的准确率分别为96.8%和96.2%,其中SSRB-181对黑色果刺植株鉴定的准确率达到100%,将在MAS育种发挥重要作用。【结论】黄瓜自交系NC76的黑色果刺性状由显性单基因控制,该基因位于Chr.4上422.1 kb范围内,两侧翼标记为SSRB-181和SSRB-130,遗传距离为3.6 cM。本研究结果为黑色果刺基因的精细定位和克隆及MAS育种奠定了良好基础。

[Objective]Cucumber （Cucumis sativus L.） is an important fruit vegetables. Fruit quality is always getting more attention in cucumber breeding program. Fruit quality includes inner quality and exterior quality, and fruit exterior quality of cucumber has important influences on its commodification. Spine color is one of the important fruit quality traits in cucumber. The clarification of the inheritance and identification of molecular markers for the fruit spine color gene will provide a theoretical basis for breeding of fruit quality and lay a foundation for fine mapping and gene cloning. [Method] Cucumber inbred lines GY14 with white fruit spines and NC76 with black fruit spines were used as the experiment materials for genetic analysis and gene mapping for black fruit spine in this study. Bulked segregation analysis （BSA） was performed in the F2 population using 2112 SSR markers. The sequence and re-sequencing information of 9930 and 100 core germplasms were used to develop new SSR and Indel markers in the primary mapping region of the black spine color gene （B）. JoinMap 4.0 and MapInspect software were employed to construct a linkage map for the B gene with SSR markers. Bio-informatics was adopted to predict candidate genes in the genomic region harboring the B gene. A set of 156 recombinant inbred lines （RILs） were used to test the veracity for marker-assisted selection （MAS） of flanking molecular markers linked to the B gene.[Result]Genetic analysis showed that the trait of black fruit spine in NC76 was qualitative, and a single dominant nuclear gene （B） controlled this trait. Black was dominant to white. In the primary genetic mapping of the B gene, eight SSR markers were screened to be linked with the black fruit spine color locus. The B gene was mapped on the chromosome 4 （Chr.4） of cucumber. The closest linked marker SSR22231 was 10.8 cM away from B. A total of 212 pairs of new SSR primer and 25 pairs of Indel primer were developed based on the sequence information in the primary mapping region of B. Fourteen SSR markers and one Indel marker were identified to be linked with the B gene after analysis of the F2 mapping population using these new developed molecular markers. The two closest flanking markers SSRB-181 and SSRB-130 were 2.0 and 1.6 cM away from B, respectively. The physical distance between SSRB-181 and SSRB-130 was 422.1Kb containing 60 predicted genes. Csa4G003095 and Csa4G001690 were the most possible candidate genes for the black fruit spine trait. The probability of the two flanking SSR markers to predict the spine color for marker-assisted selection （MAS） breeding was 96.8%and 96.2%, respectively. And the accuracy rate for SSRB-181 to predict the black spine color in MAS breeding was 100%.[Conclusion]The black fruit spine trait of NC76 is controlled by one dominant nuclear gene （B）. It is located on the Chr.4 of cucumber delimited in a physical distance of 422.1 Kb. The two closest flanking markers SSRB-181 and SSRB-130 could be used in the MAS breeding program. The results in this study will be of great benefit to fine mapping and gene cloning for the B gene and lay a good foundation for cucumber MAS breeding.