Research On The Transcriptome Of Pyrus Pyrifolia Nakai 'Huanghua' During Color Formation And Analysis Of Color Formation Genes

Pear(Pyrus spp.)is one of the most important and widespread fruit trees in the world.Fruit color is a very important agronomic trait and appearance quality of pear which can affect its market price,and also consumers' first sense before their purchasing decisions.Yet the color formation mechanism of pear is still not clear.Study on russet pears during fruit color development can help us deeply understand the color formation mechanism of the pear.It is also very significant for molecular breeding of pear fruit color.But the insufficiency of transcriptome data in public databases during the pear fruit color formation is an obstacle for our study.The high-throughput RNA sequencing(RNA-seq)is a fast,cost-effective,and reliable approach to generate large expression datasets for functional genomic analysis.To facilitate research on color formation of pear,we performed a comprehensive analysis of the transcriptome of Pyrus pyrifolia Nakai 'Huanghua' peel using the Solexa/Illumina HiSeqTM 2000 RNA-seq platform,which will provide rich resources for genome annotation and analysis of the color formation genes.In addition,we also mined a lot of molecular markers of pear from the transcriptome data.The major results are as follow:1.Using high-throughout Solexa/Illumina HiSeqTM 2000 RNA-seq platform,53 856740 clean reads were obtained,trimmed and assembled into 75 764 unigenes,with an average length of 735 bp and N50 size of 1 310 bp.Sequence similarity analyses against six public databases(Nr,Nt,Swiss-prot,KEGG,COG and GO)with a cut-off E-value above 10-5 found 53 268 unigenes that could be annotated with gene descriptions,conserved protein domains,metabolic pathways,clusters of orthologous groups categories,or gene ontology terms.Out of 75 764 unigenes,27 492 unigenes(36.3%)had significant matched in the KEGG database and were assigned to 128 pathways.After the pathway analysis we found that several secondary metabolic pathways that maybe critical to the color formation of pear,such as 'phenylpropanoid biosynthesis'(ko00940)and 'flavonoid biosynthesis'(ko00941).We also predicted the coding region sequences(CDS)of all 75 764 unigenes.By this way,a large,high-quality set of 72 673 protein sequences were obtained and 3 091 protein sequences predicted by program ESTScan.2.From the Biosynthesis of secondary metabolites,Phenylpropanoid biosynthesis and Flavonoid biosynthes,we selected 20 genes to performed qRT-PCR analysis during'Huanghua' pear peel color formation.The results showed that 17 genes presented similar expression leve variation in this period.The overall trend showed its first rise after falling trend.To some extent,this also shows that 6-9 weeks have basically covered the entire process of peel color formation.It also further validates our judgment is accurate of the critical period of color formation.During 7th week and 8th week,biosynthesis of secondary metabolites,phenylpropanoid biosynthesis and flavonoid biosynthes activities were more active.3.A total of 10 622 novel simple sequence repeats(SSR)markers were identified in 9154 unigenes,accounting for 14.02%of all the unigenes.The average length and distribution distance of these SSRs are about 16 bp and 5.24 kb,respectively.Dinucleotide repeat motifs are the main type,accounting for 55.87%of all the SSRs.There are 159 kinds of repeat motifs existing in the pear transcriptome.AG/CT is the most frequent motif,accounting for 49.64%of all the SSRs.All the 9 154 SSR-containing unigenes were functionally annotated using the GenBank Nr database,Nt database and and Swiss-prot database.These unigenes also further classified using the gene ontology(GO)terms and clusters of orthologous groups(COG)categories.Kept the SSRs which the length of both ends on the unigene more than 150 bp.We used these sequences to design primers.Finally,4 300 primer pairs were obtained.We selected 40 primer pairs at random for PCR amplification and PAGE analysis.Among the 40 primer pairs,31 primer pairs were successful in PAGE.Of the 31 successfully amplified primer pairs,28 amplified PCR products at the expected size,3 primer pairs generated smaller products than what expected.Using the 75 764 unigenes from deep transcriptome sequencing,a total of 162 048 novel SNP were identified,including transiton 102 342 and transversion 59 706.The frequency of pear transcriptome SNP is 1/344 bp.A/G and C/T are the main types,accounting for 31.66%and 31.50%of all the SNPs,respectively.All the 25 589 SNP-containing unigenes were functionally annotated using the GenBank Nr database,Nt database and and Swiss-prot database.These unigenes also further classified using the gene ontology(GO)terms,clusters of orthologous groups(COG)categories and KEGG pathways.There are 1178 SNPs,which may link with the key genes of color formation,were identified.