柑橘EST-SSR分子标记分析

对来源于甜橙（Citrus sinensis Osbeck）、枳壳（Poncirus trifoliata Raf．）和其他柑橘非冗余EST数据库的38124条单-基因（Unigene）序列进行了简单重复序列SSRs（Simple Sequence Repeat）搜索，所分析的柑橘非冗余核酸序列总长23．29Mb，从中获得了8218条SSR，其中包括单碱基重复4913条（59．8％），2碱基重复1419条（17．3％），3碱基重复1709条（20．8％），4碱基重复114条（1．39％），5碱基重复23条（0．28％），6碱基重复40条（0．49％）。大约每2．8kb长度的单-基因序列中即存在1个SSR，即平均4．6个单-基因中存在1个SSR。随碱基重复单元（motif）的不同，SSR的最大长度在40-105之间，全部重复序列的平均长度为20．9bp。各种SSR（1-，2-，3-，4-，5-，6-核苷酸重复）的发生频率在甜橙和枳壳间非常接近。其中单碱基重复序列是最丰富的重复单元，其次为3碱基重复。在所得的SSR的重复单元中，富含A碱基的重复单元的分布占据优势地位，出现的频率与密度均较高，而富含CG碱基的重复单元出现频率和密度较低。用25对EST-SSR引物对6个柑橘品种的多样性进行了PCR检测，结果表明，所有25对引物在6个柑橘品种间均扩增到多样性条带，证实通过柑橘EST数据库的发掘能够高效地筛选到基因水平的SSR标记。

Analysis of Microsatellites in Citrus Unigenes

Simple sequence repeats (SSRs) were investigated in the unigene sequences from expressed sequence tags (EST) of sweet orange (Citrus sinensis osbeck), trifoliate orange (Poncirus trifoliata Raf.) and other citrus species and cultivars. A total of 37 802 citrus unigene sequences corresponding to 23.29 Mb were searched, resulting in the identification of 8 218 SSRs. Among them there were 4 913 (59.8%) mono-, 1 419 (17.3%) di-, 1 709 (20.8%) tri-, 114 (1.39%) tetra-, 23 (0.28%) penta- and 40 (0.49%) hexa-nucleotide SSRs. The estimated frequency of SSRs was approximately 1/2.8 kb, which could be extrapolated to 1 SSR-containing unigene in 4.6 unigenes. The maximum length of the SSR ranged from 40 to 105 bp depending on the repeating numbers of the motif in the SSR. The overall average length of SSRs was 20.9 bp. The frequencies of different SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) were very similar between sweet orange and trifoliate orange. The mononucelotide repeats appeared to be the most abundant SSRs within sweet orange and trifoliate orange, followed by trimeric repeats. The adenine rich repeats such as A/T, AG, AT, AAG, AAAT, AAAG, AAAT, AAAAG, AAAAT etc. were predominant in each type of SSRs (mono-, di-, tri-, tetra-, and penta-), whereas the C/G, CG, CCG repeats were less abundant. Twenty-five primer pairs flanking EST-SSR loci were designed to detect the possible polymorphism of six citrus cultivars including sweet orange and trifoliate orange. The PCR result with all these 25 primer pairs revealed the existence of polymorphism within six citrus cultivars confirming that citrus EST database could be efficiently exploited for the development of gene-derived SSR markers.