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

Abstract: 【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‘Hongxiangsu', 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 crossing 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’.