Construction Of Genetic Linkage Map Of RAPD, SSR And QTL Analysis In Larch (Larix Kaempferi×Larix Gmelini)

Larch (Larix spp) is a main species of afforestation and timber, it plays an important role in buliding and paper pulp timber. Larix kaempefri, Larix gmelinii and their 145 full-sib F1 individuals in Qingshan forest farm Linkou county Mudanjiang city Heilongjiang province were used as the mapping population, and RAPD (Random Amplified Polymorphic DNA) and SSR(Simple sequence repeat) were used to construct the genetic linkage map of L. kaempefri and L.gmelinii. It was the first time to QTL mapping analysis combination with growth characters (height,diameter,average crown and volume),wood characters(Wood Specific Gravity,Tracheid Length,Tracheid Width and TL/TW ratio) and it provided theoretical basis for molecular marker-assisted breeding of larch.1. SSR primers that suitable for larch were obtained. We got 118 pairs SSR primers by searching in literatures; and searching 40605 ESTs by logging in dbEST database of NCBI, searching EST-SSR loci by using SSRIT software on internet,then it designed 796 pairs EST-SSR primers using Primer3.2. Through the orthogonal design,the Larix RAPD-PCR and SSR-PCR reaction systems was optimized. RAPD-PCR reaction system:20μl total volume for RAPD reaction contained 1.0U Taq DNA polymerase,3.0 mmol·L-1 Mg2+,0.25 mmol·L-1 dNTP,0.5μmol·L-1 primer,50 ng DNA template; SSR-PCR reaction system:20μl total volume for SSR reaction contained 1.0U Taq DNA polymerase,3.0 mmol·L-1 Mg2+,0.25 mmol·L-1 dNTP,0.45μmol·L-1 each upstream and downstream primer,50 ng DNA template.3.210 RAPD primers and 17 pairs SSR primers were obtained through the optimized reaction system of larch RAPD-PCR,SSR-PCR respectively, and total 603 polymorphism loci (581 loci of RAPD,22 loci of SSR) were identified. The average number of markers produced by per RAPD primer was 2.8, and one pairs SSR primers amplified 1.3 markers in average. The size of amplified DNA bands ranged from 150 to 2000 bp.χ2test (χ20.05(1) =3.84) showed that 444 loci belonged to 1:1 pseudo-testcross separation (220 belong to Larix Kaempferi while 224 belong to Larix gmelini),46 belonged to 3:1(29 belongs to Larix Kaempferi while 17 belong to Larix gmelini), while the other 113 loci belonged to segregation distortion among all. There were 51.8%(308 loci) from L. kaempferi while the other 48.92% (295) originated from L.gmelinii.4.The pseudo-testcross separated loci were carried out by a two-point linkage analysis using JoinMap 3.0 with a LOD threshold of 3.0 and a recombination threshold 0.45, and then the genetic linkage map of Larix kaempefri and L.gmelinii have been constructed. The maternal Japanese larch map consisted of 48 linkage markers placed in 5 linkage groups(four or more sites per group),4 triplets,6 linkage pairs, and 168 non-linkage markers, the average map distance was 11.9 cM. The paternal Chinese larch map consisted of 91 linkage markers in 9 linkage groups (four or more sites per group),2 triplets,5 linkage pairs, and 128 non-linkage 'markers, the average map distance was 6.1 cM. The genetic map framework length and linkage group total length of Larix Kaempferi were 290.1cM and 546.5 cM respectively, while the ones of Larix gmelini were 147.7 cM and 262.0 cM respectively.5. Measured the growth traits of composition population, which included tree height, diameter, crown width and volume, and also measured Wood Specific Gravity,Tracheid Length,Tracheid Length and Tracheid Length to Tracheid Width ratio. Variances result shown: the difference of growth traits and timber characteristics mainly existed between individuals in the F1 population of Japanese Larch X Chinese Larch, and the growth traits were related to timber characteristics.6.With the association analysis between markers and characters, QTL loci that ralated to growth character and wood character were obtained. WinQTLCart2.5 were taken to composite interval mapping of population quantitative characters, it setted LOD≥1.5 to mapping analysis of QTL. With the association analysis between markers and characters,4 QTL loci that ralated to growth character were obtained;20 QTL loci that ralated to wood character were obtained,of which,each 1 QTL related to Tracheid Length and TL/TW ratio of L. Kaempferi; 9 QTL related to Wood Specific Gravity,4 QTL related to Tracheid Length,5 QTL related to Tracheid Width of L. gmelini were detected. The LOD values in the 1.5-11.9, the contribution rate of 0.0105%-45.9986% of each QTLs.