| Japanese larch,Larix kaempferi,is a endemic species to Japan,which was successfully transplanted in china more than 100 years.Compared with domestic larch,L.kaempferi has many attractive features,such as greater survival and wide adaption,faster growth in the early stage,high volume productivity,great wood properties.For these reasons,It is becoming a major tree for pulpwood in temperate zone and north sub-tropical area in China.With the increasing of the consumption of paper and paper products,breeding new varieties with fast growth,material quality,low lignin content has become the primary breeding objectives of L.kaempferi.The character of large genome and many traits such as wood properties and tree growth were quantitative characters lead to traditional breeding method time-consuming and inefficiency.Association analysis does not need specific population construction,multiple alleles can be analyzed synchronously,and mapping resolution is higher.So it is becoming the preferred approach to study the complex quantitative traits of allelic variation.In this study,the association mapping population which was consisted of 450 representative individuals in a15-year-old natural provenance/family trial was used for evaluating sequence diversity and linkage disequilibrium(LD)of nine wood quality-related candidate genes.Then,based on the population structure analysis,the association between SNP genotyping data in nine candidate gene and phenotypic data of growth and wood properties were used.Functional SNP markers were found by using association analyses,and this study provides an important genetic foundation for genic marker-assisted selection breeding in L.kaempferi.The main research results were as follows:1.The genetic diversity in association mapping population of L.kaempferi was studied.Result showed that the phenotypic variation in association mapping population was abundant,and most separation of 19 phenotypic traits were normally distributed.The variation coefficient of phenotypic trait was 3.57%-55.46%,with an average of 15.42%,the biggest variant coefficient was volume of wood and the least value was the content of holocellulose.The order of the mean variation coefficient of the phenotypic traits was as follows:4 growth traits(28.55%)>17 wood physical properties(13.71%)>3 wood chemical properties(4.78%).It indicated that the traits stability of wood chemical properties was higher than that of other traits.Correlation analysis indicated that the relationships among traits were different,i.e.,positive and negative correlations coexisted.The results of principal component analysis showed that the cumulative contribution ratio of the seven principal components to the total variation reached 73.1336%.The order of the contribution ratio of the principal component and the main phenotypic traits of each principal component were as follows:growth traits(20.408%)>fiber traits(15.783%)>microfibril angle and the ratio of wall to cavity(8.980%)>wood chemical properties(8.353%and 7.335%)>Latewood percentage and basic density(6.345%)>fiber width in early wood(5.932%).2.The diversity of association mapping population were determined using 20 SSR markers.The results were as follows:241 alleles were found among the materials with the average alleles per locus 12,which varied from 3 to 31.Shannon index(I)ranged from 0.445 2to 2.912 9,with an average of 1.481 9.The average observed heterozygosity(Ho)were 0.4869with a range of 0.119 2-0.896 7,and Expected heterozygosity(He)were 0.638 1 with a range of0.189 4-0.919 9.The polymorphism information content(PIC)value ranged from 0.183 to0.914,with an average of 0.599.Assessment of population structure showed that the tested materials can be classified into three groups.The materials of groupⅠmainly came from the provenances of Asama,Ina,Matsumoto where in the Nagano Prefecture of central Honshu,Japan and seed orchard in Japan;The materials of groupⅡmainly came from the provenance of Kusatsu where in the Gunma Prefecture of the Kanto region in Japan;The materials of groupⅢmainly came from the provenance of Fuji where in the Nagano Prefecture of central Honshu,Japan and seed orchard in Japan.3.In order to analysis the nucleotide diversity and linkage disequilibrium in L.kaempferi.A final set of 9 genes was selected to design PCR primers for sequencing.These included six genes(PAL,CAD,C3H,CCR,COMT and CCoAOMT)from the lignin biosynthesis pathway,two genes(SUS and Kor)involved in the synthesis of cellulose,and one gene(MYB)was transcription factors.Results show that,a total of 1,170 single nucleotide polymorphisms(SNPs)were found within 26,061 bp of DNA sequence from nine candidate genes,resulting in an average SNP frequency of 1/22 bp.Nucleotide diversity of candidate genes were moderately high with average valuesθ_w=0.011 29 andπ_T=0.007 23.The ratio of non-synonymous nucleotide substitutions with synonymous were lower than one.Tests of neutrality for each gene revealed that Tajima’s D was negative in eight genes except SUS gene,while statistics D*and F*was negative in all genes,indicating the action of purifying selection.LD was low and was showed to decline rapidly as distances between sites increased,with r~2 dropping<0.2by about 700 bp.The low linkage disequilibrium suggest that a candidate-gene-based LD approach may be feasible and particularly useful in association mapping for searching functional SNP markers of L.kaempferi.4.The results of association analysis between SNPs and 19 phenotypic traits indicated that 22 SNPs were significantly with 14 phenotypic traits.These SNPs are from five genes,namely CCR,COMT,Kor,PAL and MYB.Eleven SNPs sites in CCR gene were identified significantly to be associated with four traits(P<0.01).Of them,there were 11,9,3,and 6 sites were significantly associated with DBH,height,volume and the ratio of fiber length to width in early wood,respectively.The phenotypic variance explained by each SNP ranged from 2.16%to 3.61%.In COMT gene,the site of COMT338 and 869 were both associated with the ratio of wall to cavity in late wood,with 6.21%and 3.08%of explained phenotypic variation,respectively.COMT594 and COMT1204 were explained 2.65%and 2.49%of the phenotypic variance in height and late wood fiber length,respectively.Four SNPs sites in Kor gene were significantly associated with five traits,including the content of Lignin,late wood fiber width,the ratio of fiber length to width in late wood,late wood microfibril angle,DBH.For each trait,percentage of the phenotypic variance explained was ranged from 2.48%to 5.91%.A total of two sites in PAL gene were identified to be associated with eleven agronomic traits,with3.38%-12.09%of phenotypic variation explained.Among them,PAL1362 were significantly associated with fiber width and ratio of fiber length to width in late wood,while PAL1365 were significantly associated with fiber width,fiber length,ratio of fiber length to width in early wood,the ratio of fiber length to width and wall to cavity in late wood and the content of Holocellulose.One site of MYB gene which was a synonymous mutation was significantly associated with the ratio of wall to cavity in late wood,with 2.68%of explained phenotypic variation. |
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