桉树EST-SSR标记开发及其遗传图谱构建.pdf

摘要从基因库中下载桉属EST序列1 5606条,经序列拼接,发现重叠群和单一 EST序列共1568个含有简单重复序列(ssg),,以二核苷酸重复分布最为广泛。共设计了eSSR引物891对,利用尾叶桉×细叶桉作图群体的Pl尾叶桉进行PCR条件优化,其中683对引物PCR特异性较好。对扩增产物小于500bp的引物,用Pl和/或父本P2细叶桉测序,获得含SSR 的标记共1 85个,其中AG/%,CCG/%,基本与SSR在桉属EST中的分布一致。有1 3条测序成功但无SSR。测序结果中发现存在SSR重复单元和次数的变异。将源EST与测序结果比对,33条测序序列中有内含子。比对结果中,同源性80%以上的有1 27条,60%以下的仅12条。同源性较高,因此开发的标记可信度较高,可进一步利用。 85个引物中,有53个在作图群体的l 32个子代中有分离,结合前期作图的EST-CAPS标记(Cleaved amplified polymorphie sequence,切割扩增多态性序列),构建了桉树EST标记遗传图谱。尾叶桉图谱含eSSR标记l 8个,图距总长 636eM;细叶桉图谱含eSSR标记27个,总长123 ;整合图谱含eSSR标记 33个,。经比较,eSSR标记最终在尾叶桉和细叶桉的同源连锁群中有9个同源标记, 存在共线性的仪有4个。eSSR标记在两个树种图谱上的排序与整合图谱基本一致,未发牛重排,同源标记的功能有待于进一步研究确定。关键词:尾叶桉、细叶桉、eSSR、遗传图谱 Development ofEST-SSR markers and their application to C0nStrUCtlon Ol ic maPS in邑ucalyptus ●■1 Abstract Inthis study,1 5606 ESTs were obtained from Genebank analysis,a total of 1,568 bi—to hexa-type SSR—containing contigs and singlets were SSR per kb of EST sequence was found on average,and di-nucleotide motifs appeared tobethemost abundant designed 89 pairs of eSSR primers and used P ofthemapping groups to optimize the conditions ofPCR. Amplified fragments shorter than 500 bp were used forsequencing to detect reliability of SSR sequences,using P and/or 85 pairs were essfully sequenced within the AG/CT repeat accounting for %G/ CGG about %.which was similar to the SSR distirbution in ESTs for 3were sequenced essfully but without SSR results also revealed the variation inSSR repeat number and units. The sequencing results pared with original ESTs through alignment analysis,and there were introns 27 sequences had ahigh homology more than 80%while only 2 below 60%.We found higher homology with these mined eSSR markers were higher credible and could beused further. Finally,53 ofthese 85 pairs ofprimers showed separation in1