【作者】 王宇；

【导师】 徐立安；

【作者基本信息】 南京林业大学 ， 遗传学， 2009， 硕士

【摘要】 从基因库中下载桉属EST序列15606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(SSR),平均每2.9kb含有一个SSR,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P1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P1和/或父本P2细叶桉测序,获得含SSR的标记共185个,其中AG/CT重复约占43.01%,CCG/CGG约占12.37%,基本与SSR在桉属EST中的分布一致。有13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127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。185个引物中,有53个在作图群体的132个子代中有分离,结合前期作图的EST-CAPS标记(Cleaved amplified polymorphic 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18个,图距总长636cM;细叶桉图谱含eSSR标记27个,总长1231.6cM;整合图谱含eSSR标记33个,总长1560.2cM。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,存在共线性的仅有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,未发生重排,同源标记的功能有待于进一步研究确定。更多还原

【Abstract】 In this study,15606 ESTs were obtained from Genebank of Eucalyptus. After analysis,a total of 1,568 bi- to hexa-type SSR-containing contigs and singlets were identified.One SSR per 2.9 kb of EST sequence was found on average, and di-nucleotide motifs appeared to be the most abundant type.We designed 891 pairs of eSSR primers and used P1 of the mapping groups for E. urophylla×E. tereticornis to optimize the conditions of PCR.Amplified fragments shorter than 500 bp were used for sequencing to detect reliability of SSR sequences,using P1 and/or P2.185 pairs were successfully sequenced within SSR,while the AG / CT repeat accounting for 43.01% and CCG / CGG about 12.37%,which was similar to the SSR distirbution in ESTs for Eucalyptus.13 were sequenced successfully but without SSR contained. Sequencing results also revealed the variation in SSR repeat number and units.The sequencing results were compared with original ESTs through alignment analysis,and there were introns in 33 sequences.127 sequences had a high homology more than 80% while only 12 below 60%.We found higher homology with them.Therefore these mined eSSR markers were higher credible and could be used further.Finally,53 of these 185 pairs of primers showed separation in 132 sibs.These 53 eSSR markers,together with pre-mined EST-CAPS markers,were used to construct the parental and consensus maps for Eucalyptus.The genetic linkage map for E. urophylla had a total map distance of 636cM,and with 18 eSSR markers included. The map for E. tereticornis had a total map distance of 1231.6cM,and with 27 eSSR markers included.The consensus map had a total map distance of 1560.2cM,and with 33 eSSR markers included.By comparison,there were 9 homologous eSSR markers existed in the homologous linkage groups between E. urophylla and E. tereticornis,and only 4 had good colinearity.The order of eSSR markers had no variety between the parental maps and the consensus map,and also had no rearrangement in chromosome. The function of homologous markers should be further studied to determine.更多还原