| Wood production and improvement are critical to tree genetic breeding. wood formation is a complex process controlled by multiple genes. Dissecting the regulator mechanisms and core regulators controlling wood formation would facilitate the tree breeding by taking advantage of marker assisted breeding. Japanese larch(Larix kaempferi)is one of the most important conifer tree species in North, Northeast and Southwest alpine regions of China, and it is an major pulp wood. To found more functional markers associated with wood growth and quality properties,and to establish a foundation for Japanese larch molecular breeding, we constructed an association papulation contains 603 individuals representing a 17-years-old natural provenance/family trial, evaluated the phenotype diversity, genetic diversity, population structure, single nucleotide polymorphism(SNP) and linkage disequilibrium(LD), analyzed the associations between 21 phenotypes(related to tree growth, wood form quality and wood fiber and wood chemical properties) and genotypes(genes related to growth traits and wood properties) with 100 SNP and 20 SSR markers. The main results are as follows:1. Phenotype diversity analysis revealed that there were abundant phenotype variations in association population. The phenotype variation coefficents(CV) were in the rage of 3.08% to54.84%. growth trait>fiber trait>timber form trait>wood chemical trait. 134 pair positive and negative correlations were detected in the correlation ananlys between phenotype traits and geographical environment factors. Principal component analysis validated the correlation among the characters effectivly dividing 21 phenotypic traits into ten main components.Phenotypic variance analysis showed that there were not significant difference among different provenances for the crown and sooner or later wood microfibril angle but there were significant or extremely significant differences for the other traits. The result of phenotypic variation coefficient and phenotypic differentiation coefficient analysis indicated that there were abundant phenotypic variation in the Japanese larch natural population. The clustering analysis of phenotypic traits divided six natural population into two groups. Comparison betweenmen-made population and natural population show the higher performance in men-made population in most traits with lower permance in patial wood fiber traits(for example: late wood fiber length, late wood fiber length/width).2. The SNP analysis in 20 growth and wood properties related genes showed moderate level of nucleotide diversity. A total of 1698 SNPs were found within 27301 bp of DNA sequence. The frequency of SNP were 1/16 and the level of nucleotide diversity θw and πT were in the range of 0.0035 to 0.2167 and 0.00224 to 0.03045 respectively. Neutral test results imply purifying selection is the major driving force during the gene evolution. The further linkage disequilibrium analysis suggested that candidate-gene-based association analysis may be feasible and particul early useful for searching functional SNP locus of L. kaempferi. The LD in many genes are declined rapidly(R2<0.1) when nucleotide sequences changed between250 and 1000 bp, except Lac2 and AE2 gene is slow down when nucleotide sequences increased to 1500 bp and R2 <0.1. The tissue expression pattern of 20 growth and wood properties related genes were detected by utilizing real-time PCR technology in root, mature xylem, immature xylem, cambium, phloem and leaf. The results indicated that 20 candidate genes are all expressed in six tissues and organs, however the expression level varied significantly, and the lowest abundance are detected in the roots. The research also showed the growth related genes are expressed predominantly in leaf tissues, and wood properties related genes are expressed mainly in mature and immature xylem. These results suggested 20 candidate genes are mainly participated in the tree growth and wood properties develop in L.kaempferi.3. The genetic diversity were determined by SSR and SNP markers. Japanese larch has high level of genetic diversity in conifer(He=0.626) based on the biological characteristics of L. kaempferi(phenological differences, pollen and seeds flows), complex terrain changes of distribution area and the effects of habitat fragmentation. The high genetic diversity of men-made population(He=0.613)indicates that the progeny maintain the high genetic diversity after high intensity of growth traits artificial selection. The results of molecular variance analysis and genetic differentiation coefficient showed that genetic variation mainly variedwithin population(genetic distance: 0.05-0.093). Natural population genetic structure of analysis based on SSR, SNP, SNP+SSR divided six natural populations into two groups.Based on the results of natural population structure division, principal component analysis and genetic structure of association population, the association population were divided into 2groups. Groups I is mainly composed of KS and AM, and groups Ⅱ is mainly composed of FJ,NK and part of men-made populations.4. Association analysis between phenotype and genotype were analyzed using MLM model taking the genetic structure(Q matrix) and kinship(K matrix) as covariables. The results showed that only BC241 marker was significantly associated with 3-years-old DBH and3-years-old tree volume among 20 SSR. 25 SNP markers in 15 genes were significantly associated(p<0.01) with 19 phenotypes(among which contained 13 wood properties and 6 tree growth and form traits) within all 100 SNP of 20 candidate genes. The phenotype variance explained by each SNP ranged from 1.09% to 4.75%. A total of 10 SNP in 6 tree growth related genes are significantly associated with 13 traits, within which the AE2 and AE3 gene belonging to AE gene family were significantly associated with form traits and wood physical properties;among 10 wood properties related genes, 12 SNP belonging to 8 genes were detected significantly associated with 12 phenotype traits. The phenotype variance explained by each SNP ranged from 1.09% to 4.75%, the average of variance explaination rate was 2.23%. In the transcription and regulation factors, only one SNP in miR397 a were significantly associated with one early wood traits(early wood microfibril angle) and four late wood traits(late wood wall cavity ratio, late wood fiber length, fiber width of late wood and late wood rate); and two SNP locus(MYB1-337 and MYB1-392)were significantly associated with wood basic density.Totally, AE2, AE3 and POD gene not only significantly associated with early/late wood traits and chemical properties, but also associated with tree growth and form traits, while miR397 a were significantly associated with late wood properties. CESA2 and CSL gene were mainly associated with late wood development, which could be used as an important function marker for late wood breeding improvement; MYB gene were mainly associated with wood density,which could be applied for density breeding improvement; and the POD gene weresignificantly associated with form traits, which could be taken into account in form traits improvement in L. kaempferi. |
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