Identification of new polymorphic SSRs, fingerprinting and cluster analysis in sweet cherry

Abstract: 【Objective】In order to meet the rapidly increasing demands of identifying new sweet cherry(Prunus avinum L.) varieties, new polymorphism molecular markers and fingerprint data obtained. In this paper, a strategy of gaining new polymorphism SSRs by digging sweet cherry genome sequencing result was tested by S-RNase genotype and was used in sample identification and further fingerprint construction. Then, fingerprint data of 48 sweet cherry varieties were constructed from S-RNase genotypes and SSRs.【Methods】SSRs, which had 2-3 bp repeat units with more than 15 repeats, were selected from sweet cherry genome sequencing data of 8 chromosomes with 12 SSRs on each chromosome.Fragment AnalysisTMbased long gel capillary electrophoresis was used to separate the amplification productions of these SSRs in 4 sweet cherry varieties. Only those SSRs that showed polymorphism in 4 varieties and had target product size were selected for further ABI3730 XL based capillary electrophoresis to check more details such as peak pattern. In addition, 305 reported Prunus SSRs were used to get polymorphism SSRs in 4 sweet cherry varieties by the same method. DNAs were isolate from leaves of 122 individual plants of 52 varieties by CTAB method. The S-RNase special gene primer set(universal primer PaConsI-F/PaConsIR2 and special primers for S4′, S2, S7, S12) was used to identify the true single plant by comparing with reported S genotype. Newly Selected polymorphism SSRs were amplified in true single plants and amplication products were separated by ABI 3730. The peak values were read and analyzed by SSR analyser software. SSR alleles were directly recorded by production size. And single peak was identified as homozygous. Power maker v3.25 software was used to analyze allele frequency, PIC and genotype frequency. Then, distance was calculated by Nei1983 method and phylogenetic tree was drawn by UPGMA method. The least molecular markers needed for separating 48 sweet cherry varieties was gained by adding markers one by one in a sequence according to their PIC.【Results】From sweet cherry genome sequencing data, 96 SSRs were selected to test polymorphism. In which, 9 SSRs were identified polymorphic and suitable for ABI3730 platform detection in 4 sweet cherry varieties. Among them Pav62, Pav64 and Pav612 were on the 6 thchromosome and Pav82, Pav89 and Pav182 on the 8 th chromosome, Pav12 on the 1 stchromosome, Pav10 on the 5 thchromosome and Pav78 on the 7 thchromosome. In addition, another 9 polymorphic SSRs were obtained from 305 reported Prunus SSRs. The SRNase genotypes of'Miniroyal','Royal Lee'and'Black York'had not been reported before and were identified as S1/S3,S4/S6 and S1/S3 respectively here. The S-RNase genotypes of'Mashad Black''Caixia''Mingzhu'and'Hanxiang'were corrected as S3/S12 S2/S9 S6/S9 and S3/S9 respectively. Four pairs of the 52 varieties couldn't be separated by S-RNase and 18 SSRs. Then each one was excluded from the 4 pair varieties for further fingerprint construction. Finally, molecular fingerprint of 48 varieties were gained by using S-RNase and 11 high polymorphic SSRs, 8 of which came from sweet genome sequencing and 3 from the 305 Prunus SSRs. The range of PIC value of the 12 molecular markers was0.81-0.33. And S-RNase had the highest PIC. Forty-eight sweet cherry varieties could distinguish by 5 markers, which were PaConsI, Pav82, Pav62, Pav78 and Ma073. The sweet cherry varieties were divided into 4 groups by cluster analysis, namely, Low-chilling group, China group, Northern American group and Eastern European group. The low-chilling group included'Miniroyal'and'Royal Lee'which came from the US and had the low-chilling reqirement. China group contained 16 varieties, in which 7 came from China ('Wanhongzhu''Zaodan''Hongyan''Caihong''Caixia''Hongdeng''Mingzhu'), 4 from the US('Benton''Chelan''Brooks' and'Tieton'), 3 from Eastern Europe('Hanxiang''Крупноплодная'and'Valerij Cskalov') and 2 from France('Bigarreau Moreau'and'Earlise').'Caihong'and'Caixia'are sister lines and theses two varieties and'Zaodan'were released by IFP in Beijing.'Hongdeng''Hongyan''Mingzhu'and'Wanhongzhu'were all bred by Dalian fruits Institute. One of the parents of'Brooks' and'Tieton'is'Early Burlat', a mutation of French variety'Burlat'. The Northern American group contained 19 varieties, of which 11 came from Canada('Sweet Heart''Sandra Rose''Sunburst''Sonnet''Skeena''Cristlina''Van''Canada Giant''Sam''Lapins' and'Santina') and 4 from the US('Selah''Bing''GilPeck'and'Rainier'). The main parents of these verities are'Van''Stella'and'Bing'. This group also included a Germany variety('Royal Ann') and 2 Chinese varieties('Jiahong'and'Fuxing'). The Eastern Europe group included 11 varieties, in which 7 came from Eastern Europe such as Ukraine ('PyσИНОВаЯРаННЯЯ''Космическая'), Hungary('Margit''Katalin''Linda'), Romania ('Rubin') and Iran('Mashad Black'). In addition, 2 Germany varieties'Munchebergi Korai'and'Hedelfinger'and one Chinese variety'Longguan'were also included in this group. These results of cluster analysis could be verified by the genetic relationship and regional origin among varieties. So, the new SSRs and sweet cherry fingerprint should be reliable.【Conclusion】In this paper, S-RNase genotypes of 3 new sweet cherry varieties were reported. A way of efficiently gaining polymorphic SSRs was tested and 9 new polymorphic SSRs were identified. A fingerprint of 48 sweet cherry varieties was constructed by using 12 molecular markers.