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作者	单位
杨东升，李冉，宝格日乐，王海伟，卢倩倩，郝水源，刘建华	（1.河套学院/硬质小麦技术创新中心/河套地区绿色农产品安全生产与预警控制实验室，内蒙古巴彦淖尔 015000； 2.巴彦淖尔市临河区农牧和科技局，内蒙古巴彦淖尔 015000）

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中文摘要:

冰草是小麦抗逆遗传改良的重要近缘植物，开发冰草基因组分子标记对于加快冰草的遗传育种效率和利用具有重要意义。本研究基于转录组测序（RNA-seq）技术，对2个冰草材料的幼嫩根、茎、叶片及结实期的花穗、种子进行生物信息学分析，开发高多态性的EST-SSR标记并进行验证。结果表明,共获得23 491 793条高质量序列，其碱其长7.03 Gb，GC含量56.44%，Q30为93.04%。共获得33 993条Unigene，总长度为29 309 958 bp。将所获Unigene与9大功能数据库进行比对，共有25 614条Unigene获得功能注释，其中Nr数据库共注释24 557条，GO数据库注释到66 193条Unigene，按功能分为3个大类和42个亚类；共14 412条Unigene注释到KEGG数据库的136条代谢通路；有11 321条Unigene在KOG数据库获得注释，归属为25个功能类别；共预测到大于100 bp的CDS有7 288条，SSR位点2 443个，其中三碱基重复1 439个，占总数的58.90%；获得EST-SSR多态性引物42对，从中随机选择10对在冰草的30个F₂代分离单株进行有效性验证，平均多态性比率为48.33%。以上结果说明利用RNA-seq技术大量开发的EST-SSR引物，可高效地应用于四倍体冰草产量、品质性状相关的标记开发、优异新种质指纹图谱构建及精准分子标记辅助育种。

英文摘要:

Agropyron is an important relative plant for genetic improvement of stress resistance in wheat. The development of molecular markers is of great significance to accelerate the efficiency of genetic breeding and innovative utilization of wheatgrass. In this study, based on transcriptome sequencing technology, the young roots, stems, leaves, spikes at grain-filling stage, and seeds of two wheatgrass accessions have been bioinformatics analyzed, and highly polymorphic EST-SSR markers were developed and verified. The results showed that a total of 23 491 793 high-quality sequences were obtained, with a base length of 7.03 Gb, the GC content of 56.44%, and the Q30 content of 93.04%. 33 993 Unigenes were obtained, with a total length of 29 309 958 bp. A total of 25 614 Unigene annotations were obtained by comparing Unigene with nine functional databases, including 24 557 annotations in the Nr database and 66 193 annotations in the GO database, which were divided into 3 categories and 42 subcategories according to their functions. A total of 14 412 Unigene pathways were annotated into 136 metabolic pathways in the KEGG database. A total of 11 321 Unigenes were annotated in the KOG database, categorized into 25 functional categories. A total of 7 288 CDSs with 2 443 SSR sites greater than 100 bp were predicted, of which 1 439 were tribase repeats, accounting for 58.90% of the total. A total of 42 pairs of primers with EST-SSR polymorphisms were obtained, and 10 pairs were randomly selected for validation using 30 F₂ individuals. The average polymorphism ratio was 48.33%. The results indicated that the EST-SSR primers, which were developed in large quantities by RNA-seq technology, can be efficiently applied to the development of markers related to yield and quality traits of tetraploid wheatgrass, the construction of excellent new germplasm fingerprints, and precise molecular marker-assisted breeding.

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