Construction of a genetic linkage map based on RAPD, AFLP, and SSR markers for tea plant (Camellia sinensis)

Abstract

Based on double pseudo-testcross theory, a population of 79 F1 clones, which was derived from a crossing of two tea cultivars using a Japanese tea cultivar “Fushun” (Camellia sinensis) as female parent and a Korean tea cultivar “Kemsull” (C. sinensis) as male parent, was applied to construct a genetic linkage map with different molecular markers. Previously, 143 random amplified polymorphism DNA (RAPD) markers and 11 simple sequence repeat (SSR) loci developed from 41 decamer random primers and 60 published SSR primer pairs, respectively, were used into our mapping analysis. In the study of amplified fragment length polymorphism (AFLP), 2439 bands were generated from 27 primer combinations with an average number of 90.3 bands for each, of which 495 (20.3%) were polymorphic. The majority of those polymorphic markers segregated in accordance with Mendelian segregation ratios at p < 0.01 for 400 (80.8%) or at p < 0.05 for 382 (77.2%), of which 136 (34.0%) or 131 (34.3%) were at 3:1 segregation ratios and 264 (66.0%) or 251 (65.7%) were at 1:1 segregation ratios at p < 0.01 and p < 0.05, respectively. For developing more SSR markers, the transcriptome sequences of flowers and leaves of two parents were obtained using high throughput RNA sequencing and compared. Afterwards, 1800 potential polymorphic SSR markers were successfully developed and 296 of them were selected and experimentally validated with a subset of tea plants (including two parents and six F1 offspring), from which 75 (25.3%) were repeatably amplified and polymorphic between two parents. From that, 29 (38.7%) newly mined SSR markers were heterozygous in “Fushun” and/or “Kemsull” and showed segregant genotypes in F1 seedlings and were adoptable by JoinMap 4.0. Totally, 678 markers including 143 RAPDs, 11 public SSRs, 495 AFLPs, and 29 newly mined SSRs were conjointly used to construct a combined linkage map for tea plant. The new genetic map located 79 RAPDs, 5 public SSRs, 214 AFLPs, and 11 new SSRs developed from RNA seq technique and covered 1441.6 cM with an average distance of 4.7 cM between two adjacent markers. This map will lay a foundation for qualitative or quantitative trait loci (QTLs) analysis of important agronomic traits for tea plant in the future study.