| Forest,as the most indispensable part of terrestrial ecosystem,plays an important role in wide range of economic and important ecological value for human.In recent year,with the rapid population growth and the development of the social economy,the demand for lumber has been increasing year by year.At present,our country imports 250m cubic meters of timber annually and relies on imported timber for up to 50%.Therefore,it is important to improve the biomass and quality of wood.Previous reports revealed that tree growth and wood formation are complex and dynamic processes which controlled by multiple genes and underlying regulation networks.So,identifying the wood formation related genes,addressing the underlying genetic mechanism of wood growth and property traits,and uncovering the genetic variations in candidate genes will improve the quality of wood and provide an important basis of genic marker-assisted selection breeding system for sustainable forest resources management in China.Thus,in this thesis,author used the most important commercial tree species(Populus tomentosa Carr.)as model tree of woody plants,and created a large-scale method that combined RNA-seq,population resequencing profile and bioinformatics methods to systematically identify differentially expressed(DE)genes underlying wood formation.Furthermore,the InDel variants within these genes and diversity test and linkage disequilibrium(LD)were analyzed using InDel variants.On the basis of these.multi-InDel association mapping was performed,joining additive,dominant,and epistatic effects to decipher the genetic architecture of growth and wood properties traits in a P.tomentosa population which consisted of 435 unrelated natural individuals.The main results and conclusions in this study as follows:1.High throughput RNA-seq and bioinformatics analysis of three tissues woody including mature xylem,developing xylem,and cambium tissues in P.tomentosa revealed 697 differentially expressed genes(FC?0.5 or?2 at P<1.0e-03 with FDR q<0.10)as the candidate genes of wood formation.Subsequently,11 genes were randomly selected and successfully validated in tissue-specific differential expression by Realtime-qPCR among seven tissues including apex tissue,developing leaf,mature leaf,cambium tissue,developing xylem tissue,mature xylem tissue and root tissues,suggesting their potential functional role during wood formation2.Combining RNA-seq with P.tomentosa population resequencing data of 435 unrelated natural individuals,a total of 2,565,583bp sequences for 679 differentially expressed genes were obtained,covered all 19 chromosomes of P.tomentosa genome.Gene ontology(GO)analysis revealed 77 significant GO terms in which differentially expressed genes for glucan metabolism polysaccharide biosynthesis,UDP-glucosyltransferase activity,and molecular binding were over-represented.Further functional annotation showed that these genes were related to the wood formation genes such as cellulose synthase(CesA)family proteins,sucrose synthase(SuSy),caffeoyl-CoA O-methyltransferase(CCoAOMT),phenylalanine ammonia-lyase(PAL),aux/IAA protein(Aux/IAA),and auxin response factors(ARF).Regulatory motif enrichment analysis in the promoters of differentially expressed genes found several motifs which were specifically bind to WRKY,MYB,bHLH and bZIP transcription factors.All of these suggested 679 differentially expressed genes were participated in cellulose and lignin biosynthesis pathway,and were regulated by some related transcription factors3.Using bioinformatics analysis,5,482 high-quality InDels,including 2,495 insertions and 2,987 deletions,were identified with an average density of 2.14 InDels per kb.InDels were mostly occurred within noncoding regions,including promoter(21.9%),intron(36.8%).5'UTR(7.7%).and 3'UTR(29.4%).Besides,about 4.3%InDels located in exon,71%of which were three or three-fold nucleotides insertions or deletions,indicating a strict purifying selection on coding regions.Further analysis of sequences of 5,482 high-quality InDels identified two major InDels classes:5,260 diallelic InDels and 222 complex InDels4.InDels diversity and linkage disequilibrium test were evaluated in the whole P.tomentosa population and three climatic regions using a total of 5.259 diallelic InDels.Results showed that these InDel loci had high diversity,where ?T=0.05434 and ?w=0.09078.Among these,InDel diversity of exon regions was significant lower while promoter regions hold the highest InDel diversity.Genetic differentiations showed that levels of InDel variation within and among the three climatic regions(Northeastern,Northwestern and Southern)of the P.tomentosa population varied,but exhibited similar patterns of ?T and ?w.Tajima's D test was negative signifying an excess of low-frequency InDel polymorphisms and purifying selection in the population.Linkage disequilibrium level was valuated.Results showed that the extent of linkage disequilibrium declined rapidly within scales comparable to single genes,suggesting a feasible way to perform an InDel-based association mapping study within candidate genes for forest trees.5.Single InDel-based association mapping was performed with ten tree growth and wood property traits using the mixed linear model in TASSEL software.Totally,119 significant associations(FDR q<0.10)were identified,representing 89 InDels in 81 DE genes.The individual InDel explained 10.4%to 24.7%of the phenotypic variation,suggesting that these InDels might play important roles in tree growth and wood properties.The detailed additive and/or dominant effects for these 119 significant associations were further calculated,and 74 unique InDels from 68 genes(FDR q<0.10)demonstrated notable additive or dominant effects across ten traits.Potential epistatic interactions of InDels for growth and wood property traits were examined by EPISNP package,and revealed 1.004 significant InDel epistatic pairs(P<1e-04)which representing 517 InDels from 290 differentially expressed genes.Only 58(11.3%)displayed significant additive/dominant effect,implying a possibility that epistatic interactions commonly occurred between mutations.Dissecprofile and bioinformatics methods to systematically identify differentially expressed(DE)genes ting of the gene-gene epistatic interaction networks revealed that the two epistatic interactions of PtoCCoAOMT6 with Pto4CL7 and PtoCCoAOMT4 with Pto4CL9 showed significant epistatic effects for lignin content.This study was the first InDel-based association mapping within candidate genes on a long-lived perennial species,and provides a theory and techniques assistance for genic marker-assisted improvement of wood properties and the breeding of industry commercial forest. |
PDF Full Text Request