新疆野生偃麦草种质耐盐性及相关分子标记研究

Xinjiang is a main distribution of Elytrigria repens，abundant variation existed among different accession for salt resistance.However,at present, the salt resistant mechnism of E. repens was unclear, and absent of simple, effective, usable marker for salt resistance identifing, thus restrained breeding process of E. repens. Hence based on past research, with 50 accessions of Elytrigria repens as test material,salt resistance during germination and seedling period were identified with gray correlation analysis method.Based on searching EST about salt tesistance in Genebank to design EST-SSR primers, along with EST-SSR primers from Triticum aestvium,PCR reaction was carried out, and polymorphic primers were selected out. Molecular markers correlation with salt resistance were obtained by the correlation analysis and bulk segregation analysis,the PCR was carried out based on salt-resistant gene pool and sensitive-resistant gene pool,allelic variation bands were explored.Based on the above studies,full-length cDNA correlatin with salt resistance of E. repens was cloned with bioinformatics methods.As made the base for understanding salt-resistant molecular mechanism of E.repens and assistant breeding.

新疆是偃麦草资源的主要分布区，已有研究发现其在耐盐性方面存在丰富的遗传变异。但目前其抗盐机制不清，无简便、高效、可用的抗盐筛选标记，限制其育种进程。因此本研究拟在已有研究基础上，以50份偃麦草材料为对象，采用灰色关联分析法鉴定其芽苗期及苗期耐盐性；在Genebank中搜索与抗盐有关EST序列，设计EST-SSR引物，结合小麦EST-SSR引物对供试材料进行PCR扩增，筛选多态性引物。通过关联分析结合分群分析法（BSA）获取抗盐相关标记，并对抗盐及敏盐基因池进行扩增，获得等位差异条带。以此为基础，利用生物信息学方法，克隆偃麦草抗盐基因，为进一步了解偃麦草耐盐分子机制、辅助偃麦草耐盐育种奠定基础。

针对新疆偃麦草耐盐机制不清，缺乏高效、可用的耐盐分子标记，限制其育种的问题。以47份偃麦草居群为供试材料，采用NaCl胁迫进行芽苗期和幼苗期耐盐鉴定；利用Genebank中偃麦草属序列结合小麦EST-SSR引物进行高效、多态偃麦草EST-SSR筛选；通过关联分析法结合BSA法获取偃麦草耐盐相关标记，对耐盐及敏盐基因池进行PCR扩增，获得等位差异条带，经电子克隆获得偃麦草耐盐基因。结果表明：（1）偃麦草种子萌发期适宜的NaCl鉴定浓度为1.6%-2.0%，S50%为0.53-2.22%，E04、E08、E23、E11耐盐较强；E27、E35、E37、E38为敏盐材料；在苗期，绿叶率可做耐盐鉴定指标，S50%为9.9-23.6g/L。其中E01、E02、E09、E19耐盐较强，E17、E46、E48、E50不耐盐。（2）获得在偃麦草中能稳定扩增、多态性高的EST-SSR引物101对，平均PIC值为0.57，有较高应用价值。（3）采用BSA法获得与偃麦草耐盐相关的EST-SSR引物2对，利用Tassel 3.0软件的GLM和MLM对1.0%NaCl下的耐盐生理指标数据库与101对EST-SSR构建的分子标记数据库进行关联分析，GLM程序检测到18个SSR位点与耐盐性状关联，MLM检测到12个，两个程序均检测到的关联位点7个，与枯黄率、相对电导率、生长速率、叶绿素含量、生物量相关联，解释率为15.53%-26.68%；（4）利用偃麦草耐盐标记在耐盐及敏盐池中进行扩增，得差显片段25条，其中16条成功测序；经NCBI数据库进行序列比对，5条具高同源序列，其中21KXM4与节节麦GDSL酯酶/脂肪酶EXL1一致性达93%，经电子克隆，获得1个全长1337bp的序列，其ORF长度为375bp，编码124个氨基酸，可能与偃麦草逆境GDSL脂肪酶表达有关。