Construction of High Density Linkage Map in Alfalfa Using GBS.

Fall dormancy (FD) and winter injury are yield-limiting factors in the cultivated alfalfa (Medicago sativa L.). Therefore, understating the genetic basis of these two traits through quantitative trait loci (QTL) mapping is crucial for alfalfa improvement. The objective of this study was to construct high-density genetic map and find QTLs associated with those two traits. A bi-parental F1 mapping population of 184 plants was developed by crossing two cultivars contrasting in FD: 3010 (FD= 2) and CW1010 (FD=10). The mapping population along with the two parents, and standard checks were planted using RCBD design with three replications at two locations: Athens and Blairsville in GA. The FD was measured as canopy height after 28 days of an autumn clipping on 21^st September and in mid-winter to confirm the dormancy level of the segregated genotypes. Chlorophyll content was also notified for FD phenotyping. An indoor screening of the mapping population in a cold chamber was also optimized using standard checks to score freeze sensitivity of the genotypes in a greenhouse. Regrowth height and biomass of indoor cold temperature treated and control samples were compared. the FD levels of parents and mapping progenies were estimated using regression equation derived via check cultivars, which displayed segregation of mapping population into various FD levels. Similarly, frost damage visual scoring data also showed a distinct segregation of the mapping population from cold sensitive (score 1) to cold susceptible (score 5). The GBS library was prepared using a single digestion of DNA by ApeKI. We received 2032 million raw reads from Illumina NextSeq PE75 High Output Flow Cell out of which 1008 million paired reads were usable. The sequencing data were processed in the Tassel Uneak pipeline using only the R1 reads of the pair-end sequencing data. The loci that were present in at least 80% of the total population were considered for the genotyping. About 2400 single dose allele (SDA)-SNPs for paternal parent, CW1010, and ~5000 SNPs for the female parent, 3010, were filtered from the raw maker file obtained from UNEAK. Of the total filtered markers, 645 SNPs of CW1010 and 1028 of 3010 parents were mapped into 8 linkage groups of respective parents. The average density of markers on CW1010 haplotype map was one marker per 1.9 cM and the average density of marker on the female map was one marker per 1.34 cM. The markers will be subsequently used for QTL mapping for fall dormancy and winter hardiness in the population.