| Chaenomeles speciosa(Sweet)Nakai,as a traditional Chinese medicine,is widely distributed in China.Due to the lack of unified classification standards among varieties,it is very difficult for resource protection and product in-depth development,which seriously restricts the variety selection and industrialization development of C.speciosa.Therefore,In order to investigate the genetic diversity in molecular level of C.speciosa germplasm resources,the genetic diversity was analyzed by sorting out leaf phenotypes,flower color and fruit characteristics.At the same time,RAD-seq(restriction-site associated DNA sequencing)was used to conduct simplified genome sequencing for Hong Xiangyu samples.The SSR sequences were identified by MISA and the characterizations of SSR sequences and their polymorphisms were analyzed by bioinformatics methods.168 C.speciosa varieties were collected and used TP-M13-SSR markers combined with capillary electrophoresis to analyze the genetic diversity and the degree of genetic differentiation.The results are as follows:(1)Among the 5 morphological markers,flower color and fruit shape were two traits with high discrimination.Flower color has the highest proportion of light red and white(30.95%),and fruit shape has the highest proportion of spherical shape,which is 36.90%.According to morphological markers,168 unfolded C.speciosa germplasms were divided into 4 categories.(2)A total of 10,427 SSRs were detected in the 141,788 C.speciosa gene sequences assembled,among which the mononucleotide repeats had the highest frequency(51.90%).The repeated motifs of SSRs were dominated by(A/T)n,showing biases in the SSR sequences.Except for mononucleotide and dinucleotide repeat types,there were generally between 5 and 6 SSR motifs repeats,and with an increase in the number of repeats,the frequency of repeat types of SSRs showed a downward trend.As the number of repetitions increased,the occurrence frequency of each SSR sequence gradually decreased.(3)In total,8,475(82.28%)pairs of primers were successfully designed according to different types of SSRs.The polymorphism analysis of 26 SSR markers showed that the average number of alleles was 4.846,and the PIC(polymorphism information content)values ranged from 0.275 to 0.842.Among them,16 pairs showed high polymorphism and 10 pairs showed moderate polymorphism.(4)The results showed that 26 pairs of primers amplified 304 alleles in 168 C.speciosa varieties,with an average of 11.577 per locus;the average expected heterozygosity(He),I and PIC were 0.748,1.731 and 0.607.The genetic similarity coefficient ranged from 0.029(“Yate149” and “Yate201”)to0.971(“Su02” and “Yate028”),with an average of 0.540,indicating that there was little genetic difference and close genetic relationship among the tested C.speciosa varieties.(5)11 pairs of core primers(Nak2,Nak3,Nak5,Nak7,Nak8,Nak10,Nak11,Nak12,Nak14,Nak17 and Nak25)were selected using genetic diversity index(H),number of alleles(Na)and polymorphism information content(PIC)as indexes for DNA fingerprint database construction of C.speciosa varieties.(6)According to the results of cluster analysis,the population can be divided into 2 groups,and further into 6 groups.Based on population structure analysis,the tested materials were divided into two subgroups.Three methods(characteristic band method,primer combination method and core primer combination method)were used to construct the fingerprint of 168 C.speciosa germplasm resources,which could provide reference basis for the varieties identification,genetic resource management and the construction of germplasm resources database of C.speciosa. |
PDF Full Text Request