Molecular Studies of Citron (Citrus medica L.) from China using Simple Sequence Repeat Markers and Single Nucleotide Polymorphism Analysis

Citron (Citrus medica L.) is believed to have originated in China. The wild citrons in China exhibit considerable phenotypic diversity. Our dataset for this study had 50 accessions including exotic fingered citrons, over-sized citrons and other local varieties collected mainly from the Yunnan province of China in addition to representative citrons from various parts of the world, available at the Citrus Variety Collection, Riverside, CA. We conducted molecular analysis using simple sequence repeat (SSR) markers and single nucleotide polymorphisms (SNP), two types of co-dominant markers. SSRs distributed throughout the genome were studied by PCR amplification and gel electrophoresis to detect polymorphic alleles. We tested 37 SSR markers and identified 23 that could discriminate among the different taxa. Seventy five alleles were identified and the band sizes obtained for the various accessions in the study were recorded. Citrons are generally homozygous but we found considerable heterozygosity in this dataset. As part of the SNP analysis, we sequenced a 1650 bp region of a single copy nuclear gene, malate dehydrogenase from all the accessions used for SSR analysis. About 80 parsimony-informative single nucleotide polymorphisms and eight indels were detected. We have also sequenced a chloroplast gene, rps16 and identified SNPs in the data set. We have conducted analysis using SSR data, SNP data from nuclear gene and chloroplast gene and clustered the various citrons in the study. STRUCTURE analysis was used to understand the population structure and phylogenetic trees were constructed using SNP data.