菠菜果实刺型基因的精细定位及候选基因分析

Spinach fruit (sown seeds) morphology can be classified into pricked and non-pricked form, which is a very obvious morphological trait. In spinach breeding, it is closely related to the purification of spinach germplasm, breeding of inbred lines and breeding of varieties; in spinach production, pricked spinach has also a great impact on seed processing and mechanized sowing. In recent years, numbers of molecular markers related to sex, disease resistance on spinach have been developed at home and abroad, while the research on mechanism of formation of pricked seed and non-pricked seed is very limited. Previous genetic analysis of project has revealed spinach fruit prickle was regulated by a single dominant gene. We have constructed the first high density genetic map and located the gene in the physical interval of 1.9 Mb in chr3 by SLAF-BSA method combined with marker screening of BC1 population. We intend to further expand the BC1 segregation population and finely locate the fruit prickle gene. Combined with spinach transcriptome sequencing, the genes related to flower organ development are selected as candidate genes, and functional verification is performed by Arabidopsis transgenic experiments. The gene-linked markers were developed for molecular marker-assisted selection breeding. The research results can lay a foundation for large-scale application of breeding, and also provide a theoretical basis for elucidating the molecular mechanism of producing pricked spinach, and have important scientific significance and application value for spinach molecular breeding.

菠菜的果实（生产用种子）有刺/无刺，是非常明显的形态性状，在菠菜育种中，其与菠菜优良种质纯化、自交系选育以及品种选育密切相关；在菠菜生产中，刺型菠菜对于种子加工和机械化播种又造成了很大影响。近年来，国内外开发了系列与菠菜性别、抗病等相关的分子标记，但对果实刺型的研究鲜有报道。项目组前期遗传研究发现菠菜果实刺型受单显性基因调控；构建了菠菜首张高密度遗传图谱，并通过SLAF-BSA的方法结合BC1群体的标记筛选将菠菜果实刺型基因定位在3号染色体1.9 Mb的物理区间内。项目组拟进一步扩大BC1分离群体，对刺型基因进行精细定位；结合菠菜转录组测序，筛选花器官发育相关的基因为候选基因，通过拟南芥转基因实验进行功能验证，开发基因连锁标记用于分子标记辅助选择育种。预期结果可为该基因在育种中的规模化应用奠定基础，同时也为阐明菠菜果实刺型调控的分子机制提供理论依据，对菠菜分子育种具有重要的科学意义和应用。

菠菜是我国重要的绿叶蔬菜，也是主要的卡脖子蔬菜作物之一。菠菜果实刺型直接决定了菠菜品种的竞争力。对菠菜果刺的研究不仅有助于阐明菠菜花器官发育研究具有重要的科学意义，而且对菠菜杂交育种均具有重要的应用价值。项目组通过在BC1群体和RILs群体中筛选重组交换单株，将菠菜果刺基因定位在参考基因组（Sp75_v1）3号染色体0.27 Mb的物理区间内。由于参考基因组Sp75_v1的不连续性，项目组重新组装了一版高质量的刺型雌株菠菜基因组Sp_XX_v1，利用ONT三代测序技术结合高密度遗传图、HiC数据对菠菜刺型基因组组装到978Mb，N50分别达到34Mb。利用菠菜RILs群体将菠菜刺型基因定位在新基因组360 kb的物理区间内，在此区间内存在5个候选基因，其中存在一个SpAP2基因，通过克隆测序发现，无刺的SpAP2基因在CDS区域存在55bp的缺失导致翻译的提前终止。并基于此InDel开发的分子标记可100%鉴定菠菜的刺型。对菠菜SpAP2基因的功能验证以及下游基因的调控网络分析仍在进行，为下一步菠菜果刺基因的确定及调控的机制研究奠定基础。