The Study On Genetic Diversity Of Three Wild Cherries And Identifying S-genotypes Of Prunus Humilis Bunge

The variations of fruit traits obtained from 207 wild cherry seedlings including Prunus humilis, P.glandulosa and P.japonica ect. were studied , to provide the scientific basis for resource protection and utilization. S-genotypes of six Prunus humilis accessions native to China were detected using S-allele-specific PCR with primer combinations EM-PC2consFD + EM-PC3consRD. The nine S-RNase alleles and other S-RNase alleles of prunus were used to form the phylogenetic tree in this work. In order to provide basic information for further research of SI in Prunus humilis, and to study the genetic relationship of P. salicina, P.armeniaca, P.mume, P.humilis, P. amygdalus and P.avium. The results were as follows:1. Fruit mature periods for P.humilis and P.glandulosa were mainly in mid July and late July, with few rareripe and late mature types mature in early July and mid and late September, respectively, while fruit mature periods for P.japonica is mainly in September, with few late mature types mature in late October;2. The coefficient of variations of vertical diameter, horizonta size and vertical / horizonta size of fruit(stone) for these three species were all less than 20%, being a relatively steady botany character;3. There were larger genetic variations between different types in a species in per fruit weight, pedicle size, content of Ca and SSC in fruits, and the coefficient of variations were all more than 20%, manifesting the abundant genetic diversity. Therefore, wild Prunus humilis, P.glandulosa and P.japonica have large potential in selection of big fruit, high content of Ca, short pedicle, rareripe and late mature types;4. Using S-allele-specific PCR with primer combinations EM-PC2consFD + EM-PC3consRD, nine S-RNase alleles were identified and were defined as S1~S9 , S1was 266bp, S2 was 371bp, S3 was 309bp, S4 was 684bp, S5 was 441bp, S6 was 806bp, S7 was 889bp, S8 was 270bp, S9 was 667bp. Homology searches for the S1~S9 DNA sequences of the putative S-alleles were performed by BLAST software at NCBI and were confirmed new genes of P. humilis and deposited in GenBank under accession numbers EF569602, EF569603, EF577404, EF577405, EF595836, EF595837, EF601047, EF653137, EF653138 respectively.5. Combined with amplification and sequence of twelve S-alleles, S-genotype of six Prunus humilis accessions were identified, they were showed as follows: superior strain No.1 (S8; S9), superior strain No.2 (S1; S2), superior strain No.3 (S5; S7) , superior strain No.4 (S8; S9), superior strain No.5 (S1; S4), superior strain No.6 (S3; S6).6. For each S-allele, the position of the intron insertion was determined by comparing the genomic DNA sequences obtained with the cDNA sequences from the prunus S-alleles. And there was a intron between C2 and C3 of each DNA sequences. Each intron contains GT at the 5'terminal and AG at the 3'terminal. The intron regions have high AT contents 68.2%~76.7%, which coincidence the rule of eukaryote encoding proteins. There have significant differences in the size of intron and the sequence of bases among the nine cloned S genes.7. The nine S-RNase alleles of Prunus humilis and 69 other S-RNase alleles of prunus were used to form the phylogenetic tree according to the amino acid sequences. Culstering analysis displayed that P. salicina, P.armeniaca, P.mume, P.humilis, P. amygdalus and P.avium were crossed together, indicating that S gene of stone fruit origined from the same ancestor and the stone fruit belongs to Prunus. The classification of Prunus humilis was preliminary discussed.