| Betula alnoides Buch.-Ham.ex D.Don is one of the indigenous broad-leaved tree species with the largest plantation area in tropical and subtropical regions of China.Its wood has good properties such as beautiful texture,moderate density,low crack and deformation rate,and easy processing,and is suitable for floor board,high-grade furniture and overlaid veneer making,and high-class interior decoration.In addition,as one of the main species of ecological public welfare forest in South China,B.alnoides also has important ecological value.It normally takes a long time to obtain excellent germplasm resources of tree species through traditional breeding,especially for valuable timber species.Therefore,it is necessary to combine conventional breeding and molecular marker techniques so as to shorten breeding cycle and improve breeding efficiency.In the present study,growth(height,diameter at breast height,volume,crown diameter and crown base height),form quality(crown type,stem form and branchiness)and wood property(basic density)traits of 364 individuals from 153 families of 26 provenances were investigated in 10-year-old B.alnoides provenance-family test plantation,and their variation was analyzed;A large number of polymorphic SSR markers were developed by high throughput sequencing of the genome for B.alnoides;These SSR markers were then used to estimate genetic diversity and genetic structure of these germplasm resources;Association analysis was further conducted to detect the SSR markers which significantly correlated with the target traits,and thus to explore genetic basis of these traits;and Functional genes that significantly correlated with the traits were identified and molecular marker-assisted selection was carried out according to the results of association analysis.The main results are as follows:(1)The variations of growth,form quality and wood traits were revealed,and the joint selection of excellent germplasm was carried out for B.alnoides.Variation coefficients of each trait ranged from 14.84% to 72.61%,the variation coefficients of volume,crown type,stem form and branchiness were more than 50%,indicating that there exsited abundant variation of these traits in tested germplasm resources of B.alnoides,and these resources had great potential for selection.The correlation analysis showed that significant positive correlation existed between most traits at the levels of 0.05 and 0.01.Branchiness and basic density had little correlation with other traits,infering that independent selection was needed for both traits.There was a significant positive correlation between height and DBH,so one of them could be selected alternatively for breeding.Crown type was closely related to other indexes,and could not be considered in the selection of superior trees.Two optimum selection methods,comprehensive score and principal component analysis were used to determinesuperior trees,36 superior trees was selected with 10% selection rate by each method,and 20 high-quality and fast-growing trees were finally screened out through comparing the results of both methods.The actual gain of each trait was ranged from 26.68% to 145.24%,and these individuals can be popularized and applied through tissue culture.(2)Genome microsatellite molecular markers were developed for B.alnoides using high-throughput sequencing technology.164,357 candidate SSR loci were obtained through De Novo sequencing.Among the derived SSR repeats,mono-nucleotide was the main type(77.05%),followed by di-(10.18%),tetra-(6.12%),tri-(3.56%),penta-(2.14%)and hexa-nucleotide(0.95%).A total of 2549 primer sets were designed from the identified putative SSR regions of which 900 were randomly selected for evaluation of amplification successfulness and detection of polymorphism if amplified successfully.Three hundred and ten polymorphic markers were obtained through testing with 24 individuals.The average number of alleles(NA),observed(HO)and expected(HE)heterozygosities,Shannon-Wiener diversity index(I)was 5.14,0.64,0.57 and 1.12,respectively.Cross-species transferability was further examined for 96 pairs of SSR primers randomly selected,and showed a high level.The obtained SSR markers can be used to study population genetics and molecular marker assisted breeding of these species in the future.(3)Genetic diversity of birch germplasm resources were studied with these SSR primers.A total of 189 alleles were revealed by 32 pairs of SSR primers.The average values of NA,HO,HE and I at 32 loci were 5.91,0.67,0.58 and 1.15,respectively.The average values of NA,HO,HE and I at provenance level were 6.30,0.66,0.60 and 0.96,respectively.The genetic diversity of provenance Shuangjiang,Yunnan was the highest,and the genetic parameters of 20 superior trees were slightly lower than those of species level.Clustering analysis showed that the 19 provenances could be divided into two categories,but the results were not in accordance with geographical distribution.The phylogenetic dendrograms of 19 provenances and superior trees eventually clustered on a larger genetic distance,also indicating that there existed high levels of genetic variation in provenances and superior trees,and these germplasm materials could be used as an association analysis population.(4)The association analysis of growth(height,DBH,volume,crown diameter and crown base height),form quality(crown type,stem form and branchiness)and wood property(basic density)traits was carried out with 208 SSR markers for 364 B.alnoides individual trees from provenance-family trial plantation.These individuals were divided into three subgroups through population structure analysis.The association analysis was thus conducted using GLM and MLM models,it was showed that there were 15,9,7,11,17,5,9,6 and 18 markers significantly associated with height,DBH,volume,crown diameter,crown base height,crown type,stem form,branchiness and basic density at 0.05 level,and 4,4,2,2,3,0,2,0,and 6 markers associated with these traits at 0.01 level,respectively.Among these markers,17 were associated with two or more traits.These markers can explain 1.1%?9.1% of the phenotypic variation.From calculation of alleles with the positive and negtive effect and their average effects of the associated markers for each trait,it was found that there were differences in the phenotypic effects of each locus and allele.The mean values of allelic plus effect and minus effect could achieve 64.22% and 50.67% respectively.There were six significantly associated markers with corresponding functional annotations,which were homologous to carboxylesterase,peptidyl-prolylcis-trans isomerases,WRKY transcription factor,kinesin,ribonuclease and b HLH transcription factor genes,respectively.These proteins mostly of involve and regulate growth and development,secondary metabolism,morphogenesis and hormone signaling substance metabolism,and response to biological stress.The loci associated with growth,form quality and wood properties of B.alnoides were discussed,which lay a foundation for its molecular marker assisted breeding.(5)The results of association analysis were applied to screen out and unite the advantage and disadvantage loci,and molecular marker-assisted selection was thus carried out combining with traits of growth,form quality and wood properties..The advantage allele-block BG0005(193 and 195 bp),BG0577(135 bp),BG0285(235 bp),BG0029(246 and 266 bp),BG0227(271 bp)and BG0091(147 bp)were obtained as well as the disadvantaged allele-block BG0547(112 bp),BG0574(207 bp),BG0023(260 bp),BG0009(234 bp),BG0067(254 bp)and BG00205(153 bp).Eventually,20 superior trees were selected by these allele-blocks.Compared with the previous results of phenotypic selection,the accuracy of molecular marker-assisted selection was 65%.The actual gain of each trait ranged from 13.37% to 126.74%.This showed that results of molecular marker-assisted selection were reliable.The obtained advantage allele-block can provide evidence for early breeding and hybrid breeding for B.alnoides in the future. |
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