基于EST-SSR标记的花楸树和少叶花楸杂交F_(1)代群体的杂种鉴定及遗传关系分析

Hybrid identification and genetic relationship analysis of F_(1)hybrid population of Sorbus pohuashanensis and Sorbus hupehensis var.paucijuga based on EST-SSR marker

以花楸树〔Sorbus pohuashanensis(Hance)Hedl.〕为母本、少叶花楸〔S.hupehensis var.paucijuga(D.K.Zang et P.C.Huang)L.T.Lu〕为父本进行种间杂交,利用筛选出的9对扩增结果稳定且具多态性的EST-SSR引物,对杂交F_(1)代群体进行杂种鉴定,并对亲本和杂交F_(1)代群体的基因分离规律以及遗传多样性和遗传关系进行分析。结果显示:共获得184株杂交F_(1)代,其中175株在9个EST-SSR位点上均扩增出双亲条带,说明这些单株为真杂种,杂种率达95.1%;另9株在3~7个位点上呈现异常基因型,即缺失双亲条带或出现新条带。9对EST-SSR引物中,仅sorsd29在亲本中的扩增结果为aa×bb型(纯合互补型),其他8对引物在亲本中的扩增结果为aa×bc型(父本型)、ab×cd型(双亲互补型)或ab×ac型(杂合互补型),因而,在175株真杂种中,仅sorsd29位点未出现基因分离,另8个位点均出现了基因分离,经χ^(2)检验,F_(1)代分离比与期望分离比均无显著(P>0.05)差异。用9对EST-SSR引物从175株F_(1)代真杂种中共扩增出28个等位基因,观测等位基因数、有效等位基因数、多态性信息含量、Shannon s信息指数、观测杂合度和期望杂合度的均值分别为3.1、2.9、0.566、1.076、0.920和0.640。基于Nei s遗传距离,采用UPGMA法构建系统聚类图,结果显示:在Nei s遗传距离0.28处,少叶花楸单独成组Ⅰ,花楸树和175株F_(1)代单株聚为组Ⅱ;在Nei s遗传距离0.24处,组Ⅱ可进一步分为2个亚组,其中81株F_(1)代单株聚为亚组Ⅱ_(1),另94株F_(1)代单株与花楸树聚为亚组Ⅱ_(2)。综合分析结果表明:采用EST-SSR分子标记法可对花楸树和少叶花楸的杂交F_(1)代群体进行有效的杂种鉴定;获得的杂交F_(1)代单株的基因分离情况符合孟德尔分离定律,且与母本花楸树有较近的遗传关系;总体上看,杂交F_(1)代群体中175株真杂种的遗传多样性较为丰富。

The interspecific hybridization was conducted by using Sorbus pohuashanensis(Hance)Hedl.as female parent and S.hupehensis var.paucijuga(D.K.Zang et P.C.Huang)L.T.Lu as male parent,the F_(1)hybrid population was identified by using nine pairs of EST-SSR primers with stable amplification results and polymorphism,and the gene segregation rule,genetic diversity,and genetic relationship of parents and F_(1)hybrid population were analyzed.The results show that 184 F_(1)hybrid individuals are obtained,in which the parental bands are all amplified in 175 individuals at nine EST-SSR loci,indicating that these individuals are real hybrids,and the hybrid rate reaches 95.1%;the other nine individuals have abnormal genotypes at 3-7 loci,either missing parental bands or appearing new bands.Among nine pairs of EST-SSR primers,only the amplification result of sorsd29 in parents is aa×bb type(homozygous and complementary type),and the amplification results of the other eight pairs of primers in parents are aa×bc type(male parent type),ab×cd type(parental complementary type),or ab×ac type(heterozygous and complementary type),therefore,among 175 real hybrid individuals,only sorsd29 locus doesn t show gene segregation,while the other eight loci all show gene segregation,and there is no significant(P>0.05)difference between the segregation ratio of F_(1)generation and expected segregation ratio according toχ2 test.In total,28 alleles are amplified from 175 F_(1)real hybrid individuals by using nine pairs of EST-SSR primers,and the means of number of observed alleles,number of effective alleles,polymorphism information content,Shannon s information index,observed heterozygosity,and expected heterozygosity are 3.1,2.9,0.566,1.076,0.920,and 0.640,respectively.The UPGMA method was used to construct systematic clustering diagram based on Nei s genetic distance,and the result shows that at the Nei s genetic distance of 0.28,S.hupehensis var.paucijuga is groupⅠindividually and S.pohuashanensis and 175 F_(1)individuals are clustered into groupⅡ;at the Nei s genetic distance of 0.24,groupⅡcan be further divided into two subgroups,in which 81 F_(1)individuals are clustered into subgroupⅡ1,and the other 94 F_(1)individuals and S.pohuashanensis are clustered into subgroupⅡ2.The comprehensive analysis result shows that an effective hybrid identification can be conducted for F_(1)hybrid population of S.pohuashanensis and S.hupehensis var.paucijuga by using the EST-SSR molecular marker method;the gene segregation situations of F_(1)hybrid individuals are all in accord with Mendel s law of segregation,and they have relatively close genetic relationships with the female parent S.pohuashanensis.In general,the 175 real hybrid individuals of F_(1)hybrid population have relatively rich genetic diversities.