Larch Genes Mining And Mechanism On Differetial Gene Expression And Heterosis Formation

Genomics resources play an important role in forest genetic improvment.Yet, Larch genusis poorly characterized at molecular level with little sequence information available in publicdatabases, therefore hindered the application of genomics-assisted breeding approaches.Nowadays, the much-needed improvement for larch genomics-assisted breeding is acquiring anabundance of Unigenes and molecular markers which will provide critical information to helpidentify target genes for functional analysis, association studies and integration breeding. As acommon and important biological phenomena, heterosis has extensive application in the fieldof larch breeding, yet little consensus has been reached about the genetic basis of heterosis. Inthis study, we first used454GS FLX Titanium pyrosequencer to produce larch’s Unigenes, andconducted molecular marker and miRNA mining. Finally, differential expression gene ingenome-wide scale was screened between Larix.princips-rupprechtii, L.kaempferi and theirhybrids using digital gene expression technology. The main reasults were as follow:(1) Normalized cDNA collections from multiple tissues and genotypes were used tosample large numbers of expressed genes for L.principis-rupprechtii and L.kaempferi. Weobtained over1,300,000sequencing reads (mean length:345base pairs) for L.principis-rupprechtii, and1,240,000sequencing reads (mean length:330bp) for L kaempferi. De novoassembly yielded75,597(mean length:466bp) and80,246Unigenes (mean length:436bp),more than4,000Unigenes were longler than1kb and contain CDS. Based on sequencesimilarity with known proteins, these sequences represent approximately26,000unique genesand cover a broad range of Gene Ontology categories, and1,123Unigenes were assigned tospecific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Similarresults were achived for L.kaempferi. Our data provide the most comprehensive sequenceresource and an extensive collection of potential genetic markers currently available forL.principis-rupprechtii and L.kaempferi, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, theUnigene set has permitted significant enhancement of the number of publicly-availablemolecular genetic markers as tools for improvement of these species.(2) We located and characterized1,473simple sequence repeats (SSRs) and16,436singlenucleotide polymorphisms (SNPs) as potential molecular markers in our assembled andannotated sequences for L.principis-rupprechtii, and1,498SSRs and16,437SNPs forL.kaempferi. Most of larch genes belong to diversifying evolutionary patten.(3) In order to identify putative novel microRNAs belonging to evolutionary conservedfamilies, the larch transcriptome was compared with known microRNA hairpin sequences inmiRBase. A total of22significant local alignments between Unigenes and hairpin sequenceswere identified. After secondary hairpin strctures analysis,11conserved miRNAs comprising6miRNA families from L.principis-rupprechtii and L.kaempferi were in silico identified, andthirty-seven and thirty target mRNAs from L.principis-rupprechtii and L.kaempferi,respectively, were predicted by psRNATarget program. These results lay a solid foundation forfurther studying the regulative roles of miRNAs in the development, growth and responses toenvironmental stresses in larch.(4) Differential expression gene in genome-wide scale was screened in L.princips-rupprechtii, L. kaempferi and their hybrids using digital gene expression technology, and geneexpression mode was categorized. We found that non-additive gene expression, especiallyover-dominance mode played an important role in heterosis manifestation. Differetialexpressed gene are over-represented in extracellular region, terpene synthase activity, lyaseactivity, ion binding, hydrolase activity, transferase activity, oxidoreductase activity,beta-glucosidase activity, carbohydrate metabolic process, phenylpropanoid metabolic process,plant-type cell wall biogenesis, biosynthesis of secondary metabolites, protein processing,starch and sucrose metabolism. Function and pathway analysis of heterosis--associated geneswill lay a foundation for shed the relationship of gene differetial expression and heresosis.