Development Of Genetic Linkage Map Based On EST-SSR Markers In Rubber Tree(Hevea Brasiliensis)and QTL Mapping For Identification Of Yield-Related Traits

As a result of crossbreeding between relative species,a technique used most frequently for rubber tree（Hevea Brasiliensis）breeding over the past few centuries,the consequent narrowing of genetic pool has caused prolonged breeding cycle and slowed down breeding of new variety.The genetic maps constructed by different study groups on different populations or markers featured relatively lower density and saturation,and thus resulting in a lack of generality.In order to shorten breeding times and improve breeding efficiency,it is now necessary to construct a genetic linkage map with higher density and saturation with the aids of molecular markers developed by EST database,as well as the subsequent integration with the genetic maps reported previously.In the present study,by designing a large number of EST-SSR primers using the NCBI database,the genetic diversity in Hevea Brasiliensis was studied;by using the markers with separation difference in offspring,a genetic linkage map on the population Reyan 5-11×PR107 was constructed.In addition,by integrating the obtained map with the genetic maps reported in published studies,QTL mapping was conducted to identify the traits related to rubber yields.1.A total of 38815 EST sequences were downloaded before June 1^st,2012 and processed by Seqclean and CD-HIT software;the subsequently obtained 27865 sequences were then spliced by using the CAP3 software,and 3519 splicing sequences and 7532singlet sequences were obtained.MISA software was then used to identify SSR loci,1134and 1413 SSR loci were detected,respectively,corresponding to an occurrence of 23.9%and 15.1%,respectively,a distribution frequency of 32.2%and 18.8%,respectively.In average,a new EST-SSR was detected for every 2.59kb and 2.51kb.2.There were 1-6 repeat types of SSRs in Hevea Brasiliensis.The most frequently repeated motif was mononucleotide（mainly（A/T）n）,which accounted for 38.89%and45.65%among the splicing and the singlet sequences,respectively;while dinucleotide repeats（mainly（AG/CT）n）accounted for 36.95%and 34.39%,respectively,trinucleotide repeats（mainly（AAG/CTT）n）accounted for 18.17%and 15.71%,respectively.There were 55 and 44 types of repeat motif among the splicing and the singlet sequences,with a total SSR length of 24859 bp and 33996 bp,respectively.The average lengths of the SSRs were 21.92 bp and 24.06 bp,respectively.3.By using primer 3 software,739 pairs and 687 pairs of primers were designed,respectively.After preliminary selection,180 pairs and 89 pairs of primers were found to possess polymorphism.By adopting 38 pairs of SSR primers,the genetic diversity of 14cultivated varieties and 16 wild species was analyzed.A total of 233 alleles were obtained after amplification;223 of the 233 alleles showed polymorphism,showing a polymorphic rate of 95.7%.In the cultivated varieties,171 alleles were polymorphic,showing a polymorphic rate of 73.4%;while in the wild species,213 were polymorphic,showing a polymorphic rate of 91.4%.The wild species showed a higher degree of polymorphism than the cultivated varieties.The results of cluster analysis showed that the 30 materials were at a level with a similarity coefficient of 0.70 and were divided into 6 groups.5 pairs of primers were randomly selected to undergo electrophoresis;35 of the 39 recovered fragment possessed SSR loci,showing a SSR detection rate of 89.7%.4.The genetic linkage map of rubber tree population Reyan5-11×PR107 based on SSR and AFLP markers was constructed,consisting of 132 markers contained in 19 linkage groups.Among the markers,88 were SSR markers and 44 were AFLP markers.The genetic map had coverage of 954.9c M Hevea Brasiliensis genomes.The average length of linkage groups was 50.26c M,while each linkage group contained 2-40 markers with an average inter-marker distance of 7.23c M.5.A total of 12 RGA markers were developed,two of them,which obtained four loci,were located at two linkage groups.One is the CF18-2 were located at the linkage groupⅡ,with a distance of 4.5c M with H36-3,jz54-2-1,jz54-2-1and jz54-2-1 were located at two linkage groupsⅢ.6.The obtained linkage map was integrated with the genetic maps reported in the previously published studies.The integrated map contained 19 linkage groups,consisting of 143 molecular markers（including 99 SSR markers and 44 AFLP markers）.The integrated genetic map had coverage of 1298.5c M Hevea Brasiliensis genomes.The average length of linkage groups was 68.34c M,while each linkage group contained 2-40markers with an average inter-marker distance of 9.08c M.The lengths of various linkage groups ranged between 23.2 and 398.4c M.7.QTL analysis was conducted by using the Map QTL6 software with both the Multiple QTL Mapping（MQM）and Interval Mapping（IM）methods.The QTLs related to the girth,the dry rubber content,the dry latex yield per tree,dry latex yield of the length of cutting line,conducting phloem laticifer and yellow phloem laticifer was identified.Under the confidence LOD 2.5,two QTLs were related to the girth,located in the 5^th and 14^thgroups,the phenotypic variation explained（PVE）of the QTL was 5.4%-5.8%and6.6%-6.7%,respectively,near C2037-1 and C483-2.Three QTLs were related to the dry rubber content,located in the 2^nd,5^th and 14^th groups,the PVE was 5.0%,4.5%and 6.1%,respectively,near h43,C2037-1 and C483-2.Three QTLs were related to the dry latex yield per tree,located in the 1^st,5^th and 14^th groups,the PVE was 4.5%,5.1%-6.2%and4.3%-6.8%,respectively,near E7/M14-1200,C2037-1 and C483-2.Four QTLs were related to the dry latex yield of the length of cutting line,located in the 1^st,5^th,7^th and 14^thgroups,the PVE was 4.3%,5.0%-8.6%,5.4%and 6.0%-9.4%,respectively,near E7/M14-1200,C2037-1,C1047-3-2 and C483-2.Five QTLs were related to conducting phloem laticifer,located in the 1^st,5^th,10^th,13^thand 14^th groups,the PVE was 4.3%,5.1%,6.7%,5.8%and 5.3%,respectively,near E7/M14-230,HBE1253,HBE207-1,CF077-2and C367-2.Four QTLs were related to the yellow phloem laticifer,located in the 1^st,5^th,14^th and 16^th groups,the PVE was 3.9%,5.1%,6.8%and 6.1%,respectively,near E8/M8-400,HBE1253,C367-2 and C1704-1.