中国农业科学 ›› 2015, Vol. 48 ›› Issue (6): 1052-1062.doi: 10.3864/j.issn.0578-1752.2015.06.02

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于RNA-seq的百萨偃麦草染色体特异分子标记开发与应用

李晨旭,刘志涛,庄丽芳,亓增军   

  1. 南京农业大学农学院/作物遗传与种质创新国家重点实验室/江苏省现代作物生产协同创新中心,南京210095
  • 收稿日期:2014-09-25 出版日期:2015-03-16 发布日期:2015-03-16
  • 通讯作者: 亓增军,E-mail:zjqi@njau.edu.cn
  • 作者简介:李晨旭,E-mail:2011101116@njau.edu.cn
  • 基金资助:
    国家自然科学基金(31170302)、高等学校创新引智计划(B08025)、江苏省高校优势学科建设工程资助项目

Development and Application of Specific Molecular Markers of Thinopyrum bessarabicum Löve Based on RNA-seq

LI Chen-xu, LIU Zhi-tao, ZHUANG Li-fang, QI Zeng-jun   

  1. College of Agronomy, Nanjing Agricultural University/National Key Laboratory of Crop Genetics and Germplasm Enhancement /JCIC-MCP, Nanjing 210095
  • Received:2014-09-25 Online:2015-03-16 Published:2015-03-16

摘要: 【目的】百萨偃麦草(Thinopyrum bessarabicum Löve, 2n = 2x = 14, JJ或EbEb)是小麦改良的重要亲缘物种,开发染色体特异分子标记对于加快其有利基因向小麦中的转移和应用有重要意义。【方法】利用百萨偃麦草分蘖期叶片RNA-seq获得的EST序列与节节麦D基因组序列进行比对,鉴定出4 957条没有相似性的序列作为筛选百萨偃麦草特异序列的基础序列。从这些基础序列中随机选择部分序列设计EST-PCR引物507对,通过在普通小麦中国春、百萨偃麦草和中国春-百萨偃麦草双二倍体中的扩增分析,筛选出百萨偃麦草基因组特异标记,然后在已经选育出的8个小麦-百萨偃麦草异染色体系中进行染色体定位,并探讨这些标记在小麦染色体工程中的应用潜力;根据谷类作物的共线性,以百萨偃麦草EST序列设计共线性引物100对,并比较这些引物的扩增和定位结果。【结果】在开发的507对引物中,204对(40.2%)在百萨偃麦草和中国春-百萨偃麦草双二倍体中具有特异扩增,多态率远高于利用小麦(12%)和百萨偃麦草(14%)EST设计的234对共线性引物产生的多态率,建立了高效开发小麦亲缘物种特异标记的新方法;利用8个中国春-百萨偃麦草异染色体系,共定位了198个百萨偃麦草特异标记,分别位于染色体1J(31)、2JS(15)、2JL(26)、3JS(20)、4JS(12)、4JL(12)、5J(27)、6JS(13)、6JL(22)和7JS(20),其中189个是根据百萨偃麦草转录组序列设计;利用定位于1J和6J的特异标记确定了4个易位系的染色体身份,其中1个涉及1J的大片段易位,2个涉及6JS的不同区段易位,1个为小片段中间插入易位;利用这些易位系,将30个1J和12个6J特异标记分别定位于2个物理区段。【结论】通过RNA-seq结合与小麦基因组序列比对可以获得小麦亲缘物种相对特异的EST序列并据此开发引物,建立了开发外源染色体特异标记的新方法,开发的标记可应用于小麦异易位系鉴定和缺失物理图谱的绘制。

关键词: 百萨偃麦草, 转录组, 分子标记, 易位系

Abstract: 【Objective】Development of adequate molecular markers covering the whole genome of an alien species is becoming more and more important in identifying the homoeology and structure of the alien chromosomes, developing small segmental translocations, tracing target genes and physical mapping. Thinopyrum bessarabicum Löve is an important alien species with multiple beneficial genes for wheat breeding, 8 wheat alien lines involving seven chromosomes (arm) of Th. bessarabicum have been developed in Cytogenetics Institute, Nanjing Agricultural University, China. Recently, many small segmental translocations involving different chromosomes of Th. bessarabicum have been induced via irradiation, gametocidal chromosomes and ph1b system. However, the current markers are not enough to identify these translocations. To develop more specific markers, a transcriptome of Th. bessarabicum leaves at tillering stage was sequenced and assembled.【Method】The sequences were then used to blast against the D genome sequences of Aegilops tauschii and 4957 “no hits found” sequences probably representing Th.bessarabicum specific were used for primers development.【Result】Of 507 pairs of primers, 204 produced specific amplicons in Th. bessarabicum with a polymorphism (40.2%) quite higher than 12% showed in 134 primers developed based on wheat ESTs and 14% showed in 100 primers developed based on the transcriptome sequences of Th. bessarabicum. Of them, 64 pairs of primers had no amplification in wheat indicating species specific. Using 8 wheat alien lines involving seven chromosomes or arms of Th. bessarabicum, total 198 including 189 markers developed based on the transcriptome sequences of Th.bessarabicum were chromosomally mapped, which distributed on 1J, 2JS, 2JL, 3JS, 4JS, 4JL, 5J, 6JS, 6JL and 7JS, respectively, with a number of 31, 15, 26, 20, 12, 12, 27, 13, 22 and 20. Using these markers, three wheat alien translocations involved chromosomes 1J and 6J preliminarily designated as T1JS·1JL-W, T6JS·W, T6JS-W·W and one intercalary translocation with two specific markers were identified. After analysis on these translocations, 7 markers were mapped on the distal part of 1JL and 23 on the left part of 1J. For 6JS, 8 markers were mapped on the distal region and 4 on the pericentric region of 6JS. 【Conclusion】Blast against wheat D genome sequences with the transcriptome sequences of Th. bessarabicum, a high efficient way to develop markers specific for alien species of both wheat and other crops was developed. These markers facilitate both the identification of wheat alien translocations and physical mapping.

Key words: Thinopyrum bessarabicum, transcriptome, molecular markers, translocations