Identification Of Self-incompatibility Genotypes Of Sweet Cherries And The Genetic Relationship By SSR Analysis

The plantation of sweet cherry is one of the most economic benefit fruitindustries, and known as "golden industry". Sweet cherry is worth to planting because its fruit is not only fresh and beautiful, but also nutritional. While the correct evaluation and rational utilization on sweet cherry cultivars has been limited because of the miscellaneous name and the self-incompatibility. In order to supply rational collocation for orchards pollination cultivars and theoretical information for evaluation, utilization resources and genetic and breeding research, using 5 pairs of prunus primer combinations to amplify S-allele-specific PCR for 24 kinds of sweet cherry and S-genotypes of sweet cherry cultivars were identified,meanwhile the genetic diversity and relationship were studied by SSR marker technique. The conclusion as follows:1. By TA,10×PCR Buffer concentration, dNTP concentration, enzyme concentration, and the concentration of the test DNA, suitable for the laboratory to obtain a SSR-PCR reaction system.Amplification procedure is as follows: PCR reaction system:20ul of each amplification reaction mixture comprising 10× PCR Buffer 2.5 μL, dNTPs (2.5 mmol.L-1) 1.6 μL, primer 1 (10 umol.L-1), primer 2 (10 umol. L-l) are 0.8 μL, DNA (50 ng-100 ng) was 0.5 μL, TaqDNA polymerase (5 UL-1) was 0.2 μL.PCR response procedures:Degeneration 3 min 94 ℃,94 ℃,30 s, 32 cycle (30 s,56℃ 72 ℃ for 2 min),72℃ for 5 min,12 ℃.2. The effect of PCR amplification with 3 primer combinations is different.the primer combination PruC2+PruC4R is the best one for Sweet cherry, All can amplify the two clear S genetic stripe, the primer combination EM-PC2consFD+EM-PC3consRD next one is the combination, Only part of sweet cherry cultivars can swell out.the last one is the combination BFP93+ BFP94, sweet cherry cultivars basic can’t swell out.. S-allele S1、S5-specific amplification with primer BFP208+BFP209 and BFP212+BFP213 in sweet cherry cultivars;’Brooks’,’Lapins’,’Early Ruby’, ’Ruby’ as same as ’Hongshouqiu’ can augment s1,’Victor’ can augment s5,3. Using primer combination PruC2 +PruC4R to havePCR amplified and the S-gene amplified fragments were cloned and searched in GenBank. As a result, the same S genes display the same sizefragments in electrophoretogram. The sizes of S-gene amplificationfragments are:S1 was 800 bp,S3 was 762 bp, S4 was 962 bp, S5 was 300 bp,S6 was 456 bp,S9 was 650 bp;4. Combined with three amplification, S-genotype of 24 cultivars wereidentified, they were showed as follows:’Hongshouqiu’,’Early Ruby’ were S1 S3;’Lapins’ was S1S4;’Ruby’ was S1S6;’Brooks’ was S1S9;’Napoleon’ was S3S4;’Qinlin’ ’Tartarian’,’Van’,’Zaodaguo’,’Lizhu’,’Meizao’,’5-106’ and ’Satonishiki’ as same as ’Santina’ were S3S6;’Heizhenzhu’,’Hongdeng’,’Samituo’ and ’Qinying’ were S3S9;’Victor’ wasS5S9;’Mingzhu’,’Hongmi’ and ’Rainier’ as same as ’Bing’ were S6S9;5. In the 20 pairs of SSR primers, UDP97-403 primers is the best one for Sweet cherry.Clustering results showed that 24 species divided into three large groups, genetic diversity was abundant.