| High-density linkage map is a valuable tool for genome assembly,studying genomic variation and the genetic basis of complex traits in model or non-model species.Comprehensive genetic map for conifer tree species has been hindered by the large genome sizes,high contents of repetitive sequences and difficulties of obtaining large number of segregating markers across the genome.High-density genetic mapping for conifer still remain to be advanced.The next-generation sequencing technologies provide effective means to generate larger number of markers.RNA sequencing(RNA-seq)technology has been widely used to generate a large amount of variants from expressed regions of the genome,in addition to gene expression profiling.In this study,we utilized megagametophytes(haploid)populations from three maternal trees from three different geographical regions(Qinghai Province,Inner Mongolia,Shanxi Province)of Chinese pine(Pinus tabuliformis)to establish linkage maps.RNA-seq technology was applied to obtain large numbers of single nucleotide polymorphism(SNP)markers to construct three single-tree maps and a consensus linkage map.The comparative analyses among the three maps allowed for the eharaeterization of synteny and structural variations in P.tabuliformis.The main results are:1.We detected large amount of genetic variation from coding regions of P.tabuliformis genome.Sequence reads of the three families mapped to 174,727 scaffolds of the Pinus taeda reference genome,and harbored 12,090,260 variations.These markers were located in 28,500 different genes.After filtering the low-quality loci,1,405,060 SNP makers from 15,936different genes were remained for following genetic mapping.2.We constructed high density genetic maps.The segregation of 41,462 markers in Hz(from Qinghai Province)population was used to construct a high-density genetic map.This map contains 3,616 frame markers mapped to the expected 12 linkage groups(LGs)and with an average distance of 1.29 cM between adjacent markers.The map for the NC(from the east of Inner Mongolia)population consisted of 30,720 segregating markers.This map contains 5,842 frame markers mapped to 12 LGs with an average distance of 1.21 cM between adjacent markers.For the LKS(from Shanxi Province)population.41,736 segregating markers in 61 haploid samples were used to construct a high-density genetic map which contains 4,178 frame markers mapped to 12 LGs and with an average distance of 0.84 cM between adjacent markers.The consensus linkage map contains 12 LGs,corresponding to the haploid chromosome number of Pinus(n=12),with total length of 3,741.978 cM and 81,941 markers distributed across 15,062 unique loci,and an average distance of 0.25 cM betiween adjacent markers.The longest linkage group was 351.6 cM and contained 1,402 frame markers,and shortest linkage group was 208.9 cM which contained l,067 frame markers.This is the first high-density SNP linkage map for Chinese pine based on the functional genome sequences.3.The comparative analysis among the three linkage maps identified synteny and structural variation.164 markers were found in common,and 107 were in synteny on homeologous chromosomes from Hz and NC populations,and 57 markers may have positional variation among them.172 markers were found in common,and 118 were in synteny between Hz linkage map and LKS linkage map,and 54 markers may have positional variation.181 were found in common between NC and LKS,and 117 were in synteny,64 markers may have positional variation.This study provides a basis for the study of geographical provenance variation of Pinus tabuliformis from the molecular levelThis study may provide a useful resource for adaptive genetics/genomics research and breeding for this important pine species from northern China. |
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