粗山羊草II型杂种致死基因NetM7-262的精细定位

Aegilops tauschii (Coss.) Schmal, as the D genome donor of common wheat, harboring abundant useful genes related to disease resistance, stress tolerance, quality and so on, plays an important role in wheat improvement. However, hybrid necrosis has been observed in many interspecific hybrids from crosses between tetraploid wheat and Ae.Tauschii，which hinders the effective transfer of excellent genes from Ae. tauschii to wheat. In our previous study,A triploid F1 population derived from the distant hybridization between tetraploid wheat and F1 (Ae. tauschii Ku2074×Ae. tauschii M7-262) was developed to map the necrosis gene using microsatellite markers. Phenotype identification and genetic analysis showed that hybrid necrosis effect was controlled by a single gene, located on chromosome 2DS, named as NetM7-262. In furtherstudy, the structure variation of NetM7-262linked region will be analyzed by comparing the 10×Genomics data with the genome of Ae. tauschii and Chinese spring. New molecular markers will be developed in the region with good colinearity. Recombinants in F2 segregating population are going to be screened using closely linked markers and subsequently hybridized with Langdon. Phenotypes of triploid F1will be identified after embryo rescue and vernalization treatment, and NetM7-262 will be fine-mapped. Above all, the current study will provide foundation for hybrid necrosis gene cloning and effective utilization of Ae. tauschii in wheat breeding.

粗山羊草作为小麦D基因组供体，遗传变异丰富，具有抗病、抗逆、优质等性状特点，是小麦遗传改良的重要基因资源。但部分粗山羊草种质与四倍体小麦的杂种F1致死，阻碍了优异基因从粗山羊草向小麦的有效转移。我们前期利用携带II型致死基因的亲本M7-262和正常亲本Ku2074的F1与四倍体小麦Langdon杂交，表型鉴定和遗传分析表明，致死效应由单基因控制，定位在2D染色体短臂，命名为NetM7-262。在此基础上，本研究拟借助10×Genomics测序粗山羊草亲本，与已测序粗山羊草、中国春基因组比较，分析目标基因区段结构变异，在共线性良好的区间开发分子标记，扩大F2分离群体，根据前期定位结果利用两侧紧密连锁标记筛选重要重组体，重组体再与Langdon杂交，通过幼胚拯救和春化处理，鉴定表型，实现对NetM7-262的精细定位，为致死基因克隆及粗山羊草优异基因的有效利用奠定基础。

本项目利用粗山羊草种质与Langdong杂交，成功获得85份人工合成六倍体小麦。并对其表型进行鉴定，发现15份粗山羊种质携带致死基因。利用不携带致死基因的粗山羊草种质Ku2074与携带致死基因的种质M7-262组合获得F2分离群体15000个单株，结合小麦、粗山羊草基因组信息开发了紧密连锁的新的分子标记，将致死基因NetM7-262精细定位在以AL8/78基因组为参照的2D染色体的约270kb的物理区间。.通过三代测序技术对亲本M7-262进行测序，最终确定将致死区间定位在物理区间333Kb区间内。对M7-262与Langdong的杂种后代，即人工合成六倍体小麦DHM7-262分不同时间段低温处理，通过转录组测序分析，最终在定位目标区间内获得4个表达基因，均响应温度变化。结合粗山羊种质重测序数据进行单倍型分析，发现候选基因4起始密码子上游4595bp有两个相连的SNPs，具有三种单倍型，单倍型Ⅱ表现杂种致死，单倍型Ⅰ和与单倍型Ⅲ表现为正常表型，候选基因4为NetM7-262重要候选基因。通过农杆菌介导的遗传转化方法将该候选基因转化小麦科农199，获得15株转基因植株，并均成功表达目的基因，为进一步验证该候选基因的功能奠定了基础。.通过筛选到关键SNP位点开发的位点特异引物，可以有效筛选粗山羊草种质中杂种致死型，为在小麦遗传改良中，对粗山羊草种质资源的选择及杂交后代中致死基因的淘汰提供了有效筛选标记。本项目利用粗山羊草与四倍体小麦Langdon杂交获得的85份人工合成六倍体小麦为拓宽小麦遗传基础提供了丰富的种质资源。