利用已经公布的杉木444条EST序列和未公布的杉木基因组文库中1 142条基因序列,进行引物开发效率的比较。去冗余后,利用MISA 搜索SSR 位点,分别得到109个和39个含有SSR的 位点。杉木EST序列中SSR分布密度为964.58个/Mbp,基因组中平均每Mbp出现1 037.24个SSR。在两个独立来源的数据库序列中,六核苷酸重复均为最多的重复类型,且AT-rich的重复类型占较大比例。AGC/CTG是杉木EST序列和基因组库中最多的三碱基重复,通过Primer 3.0分别设计出SSR引物95对和37对。为考察设计引物在杉木不同种源(群体)中的有效性,取12个种源(个体)的优良个体, 利用随机抽取的10个EST-SSR和8个gSSR(基因组SSR)进行引物筛选,结果表明:EST-SSR和gSSR各有4对引物在12个种源(个体)中表现出明显的多态性,多态率分别为40%和50%。8对多态性的SSR引物共扩增出 25 个多态性等位位点,平均每个引物产生 3.125 个多态性等位位点,平均有效的等位位点为2.399 5,PIC平均值为0.519 1; Hot平均为0.307 4。其中gSSR标记在检测群体间存在较大的分化,4个gSSR比4个EST-SSR扩增出更多的等位位点数、平均等位位点数,以及更大的PIC值。
Abstract
In order to develop SSR primers of Cunninghamia lanceolata(Lamb.)Hook based on EST-SSR and genomic SSR, 292 non-repeated Chinese fir EST-sequences were assembled by removing low-quality and redundant fragments depend upon 444 ESTs sequences from NCBI Public database, and 143 genome-sequences were assembled by removing redundant fragments in 1 142 genome sequences. All those non-redundant sequences of Cunninghamia lanceolata(Lamb.)Hook was searched for mono-to hexa-nucleotide simple sequence repeats(SSR or microsatellite)with software MISA, and the type,size and frequency of these SSRs were determined. Totally, There were 109 SSRs to be picked out among EST-sequences, the distribution density was 964.58 SSR/Mbp; and 39 SSRs were found in genome sequences, accounting for the higher density of 1 037.24 SSR/Mbp. In both Genomic and EST sequence library, the Hexanucleotides repeats, especially AT-rich repeat, was the most repeated type, and the AGC/CTG was the most trinucleotides repeats, The 37 and 95 pairs of SSR primers were designed based on EST library and Genomic by Primer 3.0, respectively. Ten EST-SSR primers and eight pairs of gSSR primers were selected randomly to perform PCR amplification with 12 individuals. There were four pairs of primers showed polymorphism both in EST-SSR and gSSR, with polymorphic rate of 40% and 50%, respectively. Twenty-five polymorphic alleles were amplified by 8 SSR primer pairs, which showed an average 3.125 polymorphism alleles for each primer, the average effective alleles(Na)2.399 5, average PIC 0.519 1, average Hot 0.307 4. There were more polymorphism alleles from gSSR markers than EST-SSR among groups. More allelic loci were amplified with four pairs of primer from gSSR than the four pairs of primer from EST-SSR, and the former had higher PIC value also.
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基金
收稿日期:2013-07-31 修回日期:2013-10-10
基金项目:国家林业公益性行业科研专项项目(201004049); 国家自然科学基金重点项目(30930077); 江苏高校优势学科建设工程资助项目(PAPD)
第一作者:徐阳,博士生。*通信作者:施季森,教授。E-mail:jshi@njfu.edu.cn。
引文格式:徐阳,陈金慧,李亚,等. 杉木EST-SSR与基因组SSR引物开发[J]. 南京林业大学学报:自然科学版,2014,38(1):9-14.
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