基于陆地棉茎尖转录组序列genic-SSR标记的开发与评价

Development and Evaluation of genic-SSR Markers Based on Shoot Tip Transcriptome Information of Gossypium hirsutum L.

利用软件MISA和SSR Locator对陆地棉（Gossypium hirsutum L.）Coker312茎尖转录组测序得到的73515条Unigene序列进行分析,查找到4507个SSR位点,分布于4039条转录本序列中,SSR位点出现频率为5.5%,非编码区的SSR位点要显著高于编码区（2853/1654）。SSR重复基元中,三核苷酸重复基元占主导地位（51.03%）,且AAG/CTT（20.08%）类型最多;其次是二核苷酸重复基元（28.76%）,主要为AG/CT（55.47%）。利用引物批量设计软件共开发1569对SSR引物,在陆地棉TM-1、海岛棉3-79（G.barbadense L.）、阿非利加棉（G.herbaceum L.）、雷蒙德氏棉（G.raimondii Ulbrich.）4个棉种中进行初步评价,其中1117对引物能在4个棉种间扩增出稳定的条带,扩增率为71.2%。选择650对能稳定扩增出条带的引物,在涉及5个棉种（增加了亚洲棉（G.arboreum L.））的13份材料中进一步筛选,83对引物能在13份材料中扩增出特异性条带,PIC变幅在0.121~0.648之间,平均值为0.422,其中80对引物能在7份陆地棉材料中扩增出特异性条带,平均PIC值为0.336;挑选5对在本实验室作图亲本鲁棉研22和鲁原343间有明显多态的引物进行连锁分析,其中4对成功连锁到本实验室构建的遗传图谱上。本研究丰富了棉属genic-SSR的数量,为棉属遗传作图、性状关联分析提供了更多引物选择。

A total of 4507 SSRs distributed in 4039 transcript sequences were identified from 73515 unigenes in Gossypium hirsutum shoot tip transcriptome. The frequency of unigene sequences containing SSR was 5. 5% and the number of SSRs was significantly higher in non-coding regions than that in coding regions（ 2853 /1654）. The SSR repeat motifs dominated by trinucleotide repeats（ 51. 03%）,among which AAG / CTT（ 20. 08%） accounted for the highest proportion. Dinucleotide repeats motifs（ 28. 76%） were the next,and AG / CT（ 55. 47%） were the dominant type. Using primers batch design software,1569 pairs of genic-SSR primers were developed. Preliminary evaluation was conducted on four Gossypium species,TM-1（ Gossypium hirsutum L.）,3-79（ G. barbadense L.）,G. herbaceum L. var. africanum and G. raimondii Ulbrich. A total of 1117 primer pairs amplified stable bands and the amplification rate was 71. 2%. Six hundred and fifty pairs of primers were randomly selected and further screened in 13 accessions of five cotton species（ plus G. arboreum L.）. Eighty-three of these primers could amplify special bands,the PIC value ranged from 0. 121 to 0. 648,with an average of 0. 422. Eighty pairs of primers displayed polymorphisms within G. hirsutum,and the average PIC value was 0. 336. Five among the 83 genic-SSR primer pairs which distinctly revealed polymorphism between the laboratory mapping parents of Lumianyan22 and Luyuan343 were selected and carried on linkage analysis,four of the SSRs were successfully integrated into intraspecific genetic linkage map previously constructed in our lab. The results enriched the quantity of cotton genus genic-SSR for mapping and association analysis and related research on cotton species.