| The tea plant,[Camellia sinensis(L)O.Kuntze] originated from China,is a perennial evergreen woody plant.Tea plant is one of the most economically valuable tree species in China.Anthracnose is one of the main diseases of the tea plant.This disease caused death and loss on leaf,reducing the quality and limiting yield of tea.Tea plant anthracnose was caused by multiple Colletotrichum fungi.Colletotrichum fructicola and C.camelliae are important pathogens of Colletotrichum fungi.At present,the researches about anthracnose of tea plant are mainly focused on the identification of pathogens and the screening of effective agents.Little is know about genetic diversity of Colletotrichum spp.causing anthracnose on Camellia sinensis.Information on pathogen genetic diversity and population structure on temporal and spatial scales are important to understand the potential of pathogen populations to spread,develop fungicide resistance and overcome host resistance.This study aims to study genetic diversity of C.fructicola and C.camelliae isolates from tea plantations of Anhui,Fujian and Yunnan using SSR molecular markers in order to provide theoretical basis for epidemiology and management strategy of anthracnose.These results are as follows:1.The C.fructicola genomic sequence were used to develop SSR markers in this study.Two or three nucleotide repeat primitives were selected for searching SSR loci using SSRHunter software.Primers were developed using Primer v 6.0.Oligo Analyzer v 3.1 and Blast were used to analyze and check primers,respectively.Ten SSR primers were obtained finally.Meanwhile,18 pairs SSR primers of closely related species of C.fructicola and C.camelliae in published article were picked.The polymorphisms of these primers were detected by PCR amplification and 2.5%agarose gel electrophoresis.The result showed that fourteen and thirteen polymorphic primers were screened for C.fructicola and C.camelliae,respectively.2.The 14 polymorphic SSR markers detected a total of 51 alleles.The number of alleles per locus ranged from two to eight.The polymorphism information content(PIC)varied from 0.305 to 0.769,with an average of 0.534 per marker.Fourteen SSR loci were above reasonably informative;The 13 polymorphic SSR makers detected a total of 41 alleles.The number of alleles per locus ranged from two to four.The PIC values varied from 0.245 to 0.617,with an average of 0.490 per marker.Twelve SSRloci were above reasonably informative.3.Fourteen polymorphic SSR markers were used to investigate genetic diversity of C.fructicola isolates from three geographic populations.The number of different alleles(Na),Shannon's Index(I),expected heterozygosity(He)averaged across all loci ranged from 1.92 to 2.92,0.517 to 0.955,0.341 to 0.583;Thirteen polymorphic SSR makers were used to investigate genetic diversity of C.camelliae isolates from two geographic populations.The number of different alleles(Na),Shannon's Index(I),expected heterozygosity(He)averaged across all loci ranged from 2.92 to 3.0,0.853 to 0.912,0.496 to 0.554.It indicated that genetic diversity of each population of C.fructicola and C.camelliae was high.4.UPGMA cluster analysis showed that the similarity coefficients of C.fructicola ranged from 0.62 to 1.00.The strains were divided into five groups with a similarity coefficients of 0.70.All isolates were divided into two sub-populations based on PCo A and STRUCTURE analysis.Analysis of AMOVA revealed that genetic variance within populations accounted for 87% of the total genetic variance.The pairwise PHi PT value showed that genetic differentiation between Anhui and Fujian population was smalle while genetic differentiation between Yunnan and Fujian,along with Anhui population was large;UPGMA cluster analysis showed that the similarity coefficients of C.camelliae ranged from 0.60 to 1.00.The strains were divided into six groups with a similarity coefficients of 0.68.Analysis of AMOVA revealed that genetic variance within populations accounted for 95% of the total genetic variance.The pairwise Fst value showed that genetic differentiation between Anhui and Fujian population was small. |
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