Genetic Relationship By Genome-wide Rad Sequence Data And Establishment Of Genetic Maps Based On Molecular Markers Of Eriobotrya Plants

Loquat(Eriobotrya japonica),a subtropical evergreen fruit tree,originated in southwest China and has been cultivated for over 2,000 years.It has been introduced to more than 30 countries while leading loquat production countries still is China.Althout there are more than 26 species,varieties or forma in Eriobotrya,only E.japonica is cultivated for its fruit.Several technologies were used to evaluate the genetic relationships of Eriobotrya genus;but little is known about this.Based on abundant loquat germplasm collected and genome sequenced by SCAU,we used RAD-seq to illuminate the genetic relationships of Eriobotrya genus and used both SLAF and SSR markers to establish genetic maps of Eriobotrya plants.The mapping population consisted of E.japonica cv.Jiefangzhong × E.prinoides Rehd.et Wils.and their 150 progenies from the F1 population.Main results are as follows:1.RAD-seq was used to illuminate the genetic relationships of Eriobotrya genus.After the raw data were filtered,and 220.97 million clean reads were used for further analysis.Neighbor-Joining phylogenetic tree of 23 Eriobotrya species and two relative genera was generated with 42,526 SNPs.The results showed that the Chinese and Vietnam accessions were distributed throughout the dendrogram.A correlation between genotype and the geographical source was not evident.However,it is worthy to mention that the clustering results were highly correlated with the size of leaves.The Eriobotrya species could be divided into three groups: one group has small leaves which the leaf length is less than 10 cm,the second group has mid-size leaves which the leaf length is ranged from 10 cm to 20 cm,and the third group has biggest leaves which the leaf length is bigger than 20 cm.2.In the establishment of genetic maps based on molecular markers of Eriobotrya plants by SLAF markers,the enzyme projections for reference genome sequences was performed and fragments ranging from 264 to 464 bp were identified as SLAF markers and was digested with RsaI and HinCII enzymes,which 266539 SLAF markers were predicted.Repetitive SLAF percentage was 6.89%.Markers evenly distributed in chromosomes of a genome.We developed 489,334,306 reads(97.76Gb)data,the average Q30 sequencing was 85.81%,the average GC content was 39.28%,so the GC data distribution was acceptable,which the quality and quantity both match the research requirement.And,paired-end mapped reads of Control sequencing was 82.82% and the enzyme efficiency of control data is 88.75%.SLAF library construction was acceptable.And then,415,739 SLAF markers were developed,which the polymorphic SLAF markers were 142,905 and the effective polymorphism for the mapping construction is 2.71%.Finally,the high-density genetic map composed 4,756 SLAFs in 17 linkage group and spanned 2782.15 cM in total.The average integrity of the markers on the map was 96.82%.The average sequencing depth of the parent on the map was 79.97 X,and the average sequencing depth of the population was 10.99 X.3.About 150 small seedlings were gotten from E.japonica cv.Jiefangzhong ×E.prinoides Rehd.et Wils.and transplanted to pot at the greenhouse.Quantity and quality of genomic DNAs were checked by several SSR primers.Suitable 92 SSR primers were found.Electrophoresis PCR products of all genotypes to 6% denaturing polyacrylamide gel to investigate polymorphism among genotypes.Data generated for segregating markers will be analyzed using JOINMAP 4.0.The map contained 5 linkage groups with an average genetic distance of 14.52 cM between neighboring markers and covered 566.623 cM.Each linkage group contained from 2 to 15 markers,with a length ranging from 22 to 183 centimorgans(cM).the linkage groups ranged in size from 22.838 cM(LG 4)to 183.900 cM(LG 1-2),and the number of markers on each group varied from 15 for LG 1 to 2 for LG3,LG 4,LG 5.LG 1 possessed the highest marker density with an average locus distance of 11.992 cM,while the other LGs had relatively lower marker density(22.98 cM).The LOD scores at which these linkage groups appeared ranged 4.The final genetic map was 566.623 cM in length.The largest linkage group covered 183.900 cM(an average marker interval of 12.260 cM)and the shortest 22.838 cM(average marker interval of 11.419 cM).The largest linkage groups(LG 1-2)contained 15 loci and the smallest groups(LG 4)contained only two.The average number of loci in each linkage group was 14.528 cM.The minimum distance between two loci was 11.419 cM.This result is most likely due to the large genome size and the insufficient number of markers for complete coverage.This deficiency leads to gaps too large for statistical linkage between markers that may,in fact,be linked.