大豆花叶病毒病抗性基因位点RN1的精细定位及相关基因挖掘与功能验证

Soybean mosaic virus (SMV) is a worldwide disease, which seriously impact on yield and commodities quality of soybean. N1 strain generally occur in Heilongjiang Province, thus, an urgent need was to identify its resistance loci (RN1) to improve the efficiency and accuracy of breeding varieties with resistance to SMV.. In this study, 30 resistance and 30 suscetible lines of F2:5 , derived from a cross between Hefeng25 and Dongnong93046, was used to construct resistance/ suscetible to N1 strain pool through bulked segregation analysis(BSA) method. DNA of resistance/susceptible to N1 strain pool, was respectively isolated, which was genome-wide sequenced through specific length amplified fragment(SLAF) sequencing method. A total of 51,927 SLAF tag were developed, which was comparised with reference genome of Williasms82 through BLastN. A total of 4,922 polymorphic SNP marker could be obtained. The strength of correlation between SNP marker with SMV resistance, was determined by expression abunce difference of SLAF tag between resistance and susceptibility to N1 strain pool. According to associated strength with SMV resistance, SNP marker was arranged in refference genome of Williams82, and then, 122 strong associated SNP marker and 3 candidated region, was defined. A total of 8,742 F2 line were developed from a cross between Hefeng25 and Dongnong93046, which was inoculated with N1 strain. After inoculation, about 2,000 recessive F2 line could be obtained, which was further analyzed to narrow the candidated region through strong associated SNP marker, according to stragegy of BSA combination with recessive group analysis(RCA).If cosegregated SNP markers weren't identified in candidated region, seriers of new SNP marker would be designed according to reference genome of Williams82. After completing fine mapping, targeted gene region could be obtained, which were sequenced through series of PCR products.. All canditated gene, located in target gene region, were predicted through database of www.phytozome.net and www.ncbi.nlm.nih.gov. In the annotation information, predicted gene had homology with typical resistance gene or had typical domain of resistance gene, which would be preferred candidate resistance genes to SMV N1 strain. Predicted genes with no valuable annotation information, were also used as a possible candidated genes for subsequent analysis. These canditated genes were analyzed through Real-time PCR to find real canditated gene..Candidated resistance gene was cloned from Dongnong93046 through RACE method. Function of candidated resistance gene was further analyzed through virus induced gene silencing(VIGS) and agrobacterium-mediated cotyledonary node transformation.

大豆花叶病毒病是一种世界性大豆病害,严重影响大豆产量和商品品质。N1株系是黑龙江省的优势株系，发生普遍。目前，迫切需要寻找紧密连锁的抗病分子标记和克隆抗病基因，以提高选育抗病品种效率和准确性，简化选育程序，加快培育抗病品种的进程。.本项目采用SLAF-seq(Specific length amplified fragment sequencing)技术对抗/感N1株系池进行全基因组测序分析，高通量获得多态性SNP标记，并定义出强关联SNP标记和抗病基因热点区；结合合丰25与东农93046杂交的F2代超大群体，精细定位RN1位点，PCR产物测序获得抗病基因目的区域DNA序列信息；通过基因注释信息和QPCR技术获得抗病候选基因；通过VIGS（Virus Induced Gene Silencing）和农杆菌介导转化技术鉴定候选基因功能；进而通过分子标记辅助选择和转基因技术创制抗病新种质。

大豆花叶病毒病是一种世界性大豆病害,目前尚无有效药剂防治SMV。若从根本上解决SMV对我国大豆生产的威胁，一个重要途径是利用现代测序技术开发全基因组SNP标记，对SMV抗病位点进行精细定位，挖掘抗病候选基因并验证其功能。进而通过与抗病毒紧密连锁的功能标记辅助选择，创制高度抗病新材料，实现抗病毒病顶层分子聚合育种设计，从而解除该病害对生产的威胁。本项目经过三年的研究，结果如下：1) 通过2个重组自交系的接种鉴定的遗传分析，表明说明东农93046对SMV N1株系的成株抗性受一对基因控制。从东农93046×合丰25的后代群体进行简化基因组测序，得到3个与大豆SMV 成株抗性相关的候选基因组区段，并获得25 个包含抗病基因保守结构域的候选基因。2)利用其对240株隐性感病单株进行基因分型，通过感病表型与抗病等位基因的重组情况，获得与抗病等位重组率为0的两个分子标记分别为SSR64和ST1，在该区间内存在10个编码基因，其中有3个基因有NBS-LRR结构域，推测其可能与抗病性有关。QRT-PCR结果表明水杨酸处理的叶片中Rg2-4, Rg2-5, Rg2-9, Rg2-10在待测样本间存在差异表达。3)利用BPMV（豌豆荚斑驳病毒）基因沉默载体系统对4个候选基因进行功能初步鉴定，在Rg2-5及Rg2-10的沉默植株中检测到SMV N1株系病毒外壳蛋白基因，而在Rg2-4及Rg2-9中检测不到该基因的表达，这表明Rg2-5及Rg2-10为抗病基因，子叶节法转化大豆植株，获得转Rg2-5基因阳性植株15株，获得转Rg2-10基因阳性植株7株。 在转Rg2-10 和Rg2-5基因植株中未检测到未检测到病毒蛋白外壳基因转录产物，说明这两个基因参与抗病反应。通过遗传转化获得转Rg2-10基因和Rg2-5基因的大豆植株15株；分子标记辅助选择和获得抗大豆花叶病毒病新材料25份。4）相关研究结果发表SCI论文和中文核心期刊论文各3篇，申请专利1项，专利授权1项，培养研究生3名。.通过本项目研发的分子标记，对花叶病毒病抗性基因进行精细定位，可作为前景选择的手段，克服大豆品种中抗性基因难以准确鉴别和检测的困难，进而进行图位克隆，利用分子辅助育种技术加速了抗花叶病毒病的大豆新品种培育进程。