‘满天红’ב红香酥’杂种鉴定及遗传变异Genic-SSR分析

Identification of the hybrids and analysis of genetic variation of a pear progeny derived from crossing between ‘Mantianhong' and 'Hongxiangsu' by Genic-SSR

【目的】对授粉时未做去雄处理的‘满天红’ב红香酥’组合进行杂种鉴定和遗传变异分析,排除非双亲杂种单株,为利用该组合进行连锁分析奠定基础。【方法】用9对从转录组数据开发的在双亲间表现多态性的genic-SSR引物和1对自交不亲和基因检测引物对‘满天红’ב红香酥’组合所有单株进行杂种鉴定、标记偏分离分析和群体聚类分析。【结果】348个杂交单株中,有339株同时拥有母本和父本来源的特征片段,真正为‘满天红’和‘红香酥’的杂种,其余9株在部分位点未见父本条带,排除为双亲杂种的可能。卡方检验发现6个位点符合孟德尔分离定律,4个出现偏分离现象。双亲和339个真正杂种后代经遗传聚类分析分为3组:一组包含77个后代,与‘满天红’聚在一起;另一组包含183个后代,与‘红香酥’聚到一起。另有79个后代,单独聚在一起。【结论】利用Genic-SSR分析成功地对‘满天红’ב红香酥’组合梨杂种后代进行了鉴定,表明外来花粉污染是造成杂交群体中非双亲杂种污染的主要原因,产生自交后代污染的概率较低,聚类分析表明S-RNAse和部分SSR位点在该杂交群体中发生偏分离,半数以上的单株与父本‘红香酥’聚为一类。

【Objective】Segregation population is usually used for localizing genes of specific agronomic traits and developing molecular markers in highly heterozygous and perennial fruit trees. Excluding the individuals of non-parental hybrids from a progeny derived from a certain crossing combination is necessary for insuring the accuracy of linkage analysis. In this study, we tried to identify and exclude non-parental pear hybrids from a crossing between‘Mantianhong＇and‘Hongxiangsu＇without emasculation for the the female flowers and analyze the population structure before genetic analysis of the progeny.【Methods】A combined method of CTAB and adsorption column were used to extract genome DNA from‘Mantianhong＇,‘Hongxiangsu＇and their 348 offspings. DNA quality and concentration were detected by agarose gel electrophoresis and Nano Drop absorbance detection. Then DNA samples were preserved at-20 ℃ in refrigerator. Nine polymorphism genic-SSR primer pairs developed from unigenes of pear pericarp andflesh transcriptome data and one S-RNAse gene primer pair were employed to perform hybrid identification,segregation distortion analysis and cluster analysis of the progeny. Primers were synthesized by Ge-newiz Biotechnology Co., Ltd. PCR amplification was performed in a 20 μL volume, consisting of 20 ng ge-nomic DNA, 1×PCR buffer（Mg2＋plus）, 0.2 mmol·L^（-1） d NTPs, 0.5 U Ex Taq DNA polymerase（Ta Ka Ra,Dalian）, 0.3 μmol·L^（-1） forward primer and 0.3 μmol·L^（-1） reverse primer. SSR PCR amplification was per-formed using a PCR program of 98 ℃ for 20 s; followed by 30 cycles of 98 ℃ for 10 s, annealing tempera-ture for 30 s, 72 ℃ for 45 s, followed by 10 min at 72 ℃. S-RNAse gene PCR amplification was per-formed using a PCR program of 98 ℃ for 20 s; followed by 10 cycles of 98 ℃ for 10 s, 50 ℃ for 50 s, 70 ℃for 50 s, followed by 25 cycles of 98 ℃ for 10 s, 48 ℃ for 50 s, 70 ℃ for 50 s, followed by 10 min at 70 ℃.PCR products were detected by 10 percent non-denatured polyacrylamide gel electrophoresis and stainedusing silver staining protocol. The genotypes of hybrid individuals and the two parents in the ten markersites were recorded according to the＂CP＂population segregation type of Join Map 4.0 software for markersegregation distortion. And a＂0/1＂matrix files（in which the number＂1＂means there is one band in a lo-cation, and＂0＂means there is no band） were established according to NTSYSpc 2.10 e software for clusteranalysis. 【Results】Results of hybrid identification showed that 339 individuals among 348 offspringplants had the characteristic bands of both parents. These 339 individuals were true type hybrids of‘Man-tianhong＇and‘Hongxiangsu＇. The other nine individuals were not the true type hybrids of‘Mantian-hong＇and‘Hongxiangsu＇due to lack of the characteristic bands of the male parent‘Hongxiangsu＇. Twoof the nine plants might be selfing seedlings of‘Mantianhong＇, and the other seven individuals might beoffsprings of‘Mantianhong＇crossed with other cultivars. The result of chi-square test showed that sixmarker sites could inherit in accordance with Mendel＇s law of inheritance and separation in 339 true typehybrids, and four marker sites showed segregation distortion. According to the cluster analysis, the twoparents and 339 true type hybrids formed three groups. Among them, 77 plants were clustered togetherwith the female parent‘Mantianhong＇, 183 plants were clustered together with the male parent‘Hongx-iangsu＇, and the rest 79 plants were clustered together with themselves. The number of the markers of thesegregation type＂lm × ll＂were over that of＂nn × np＂type, resulting in that most of the hybrids were clus-tered together with the male parent‘Hongxiangsu＇.【Conclusion】Non-parental pear hybrids from a cross-ing between‘Mantianhong＇and‘Hongxiangsu＇were sucessfully identified by using genic-SSR. Segrega-tion distortion of loci of S-RNAse and SSR were tsetified by cluster analysis and more than half of the hy-brid individuals were clustered with thw male parent‘Hongxiangsu＇.