High-throughput Pyrosequencing-based Transcriptome Analyses And Application Of Liaoning Cashmere Goat

Liaoning cashmere goat, one of the best cashmere goat breeds in the world, is famous forits fine cashmere quality, high cashmere yield, well adaptability and stable genetic character.However，because of absence of reference transcriptome data, little is known regarding themolecular mechanism and regulation of cashmere growth in this breed. Thus, in this research,we performed de novo transcriptome sequencing to generate the first expressed sequence tagdataset for the Liaoning cashmere goat, using454GS-FLX. Then, we performed the geneprediction, functional classification and pathway analysis, SSRs detection and comparativegenome analysis for the Liaoning cashmere goat transcriptome sequence. Finally, we screenedthe skin differentially expressed genes between2type of Liaoning Cashmere goat and2typeof Shaanbei White Cashmere goat, by using Liaoning cashmere goat transcriptome sequenceas reference gene. The main results are as follows:(1) Transcriptome sequencing of Liaoning cashmere goat on a Roche454platformyielded804,601high-quality reads, with an average size of403bp. De novo assembly usingNewbler Software v.2.5produced a non-redundant set of117,854unigenes, comprising13,194isotigs and104,660singletons.(2) All unigenes were analyzed by EMBOSS software for generation of putative proteinsequences. Then all putative protein sequences were subjected to a similarity search againstthe non-redundant protein database of NCBI,24,614unigenes had specific biological function.All putative protein sequences were further evaluated and functionally annotated bycomparing them with existing protein databases, such as Swiss-Prot and TrEMBL database,the COG database, and the KEGG protein database.17,356unigenes were assigned to6,700GO categories, and the terms were summarized as three main GO categories and59sub-categories.46,778unigenes were classified into25COG categories and3,548unigeneswere assigned to300KEGG pathways.(3)2,781potential simple sequence repeats (SSRs) were initially identified from allunigenes using a microsatellite identification tool (MISA). We also designed the primer pairsfor958SSRs using primer3. After comparative analysis,1,326SSRs were mapped to goatgenome,93SSRs were matched to goat protein-coding genes.(4) All Liaoning cashmere goat unigenes were compared with the sequences in the NCBInon-redundant nucleotide databases，42,254unigenes were aligned to17,532different specific genes. The coverage of every gene was also calculated。(5) All Liaoning cashmere goat unigenes were searched against the sequences of goatgenomes,97,236(82.51%) unigenes were mapped to30chromosomes of goat. All unigeneswere compared with the reported goat protein-coding genes,35,551(30.17%) unigenes werematched to11,438goat protein-coding genes. The remaining non-matched unigenes werefurther compared with cattle and human reference genes,67putative new goat genes werediscovered.(6) We obtained1,748skin differentially expressed genes between Perennial typeLiaoning Cashmere goat and Seasonal type Liaoning Cashmere goat, using Illumina/Solexaplatform. Among the skin differentially expressed genes,31keratin and keratin-associatedprotein genes may be important for the formation of cashmere.(7) Skin gene expression profiles sequencing of ShaanBei White Many-Cashmere goatand Shaanbei White Cashmere goat on Illumina/Solexa platform yielded5,614,110and7,167,309clean reads, respectively. Then, the clean reads were mapped to the Liaoningcashmere goat transcriptome sequence. By comparing the gene expression abundance, weobtained108skin differentially expressed genes between Perennial type ShaanBei WhiteCashmere goat and Seasonal type Shaanbei White Cashmere goat. Furthermore, weperformed the Gene Ontology (GO) and Pathway enrichment analysis for the differentiallyexpressed genes. Most of the significantly enriched GO terms were related with extracellularstructure and its composition. Pathway enrichment analysis of differentially expressed genesshowed that4Pathway were significantly enriched pathway, including Amino acidmetabolism, Immune diseases, Signaling molecules and interaction, and Carbohydratemetabolism.The transcriptome of Liaoning cashmere goat was deep sequenced, de novo assembled,and annotated, providing abundant data to better understand the Liaoning cashmere goattranscriptome. This result will facilitate the research on molecular mechanism and regulationof cashmere growth in Liaoning cashmere goat. The potential simple sequence repeatsprovide a material basis for future genetic map construction, quantitative trait loci analysisand genetic diversity evaluation. In addition, the screening and analyzing of skin differentiallyexpressed genes between2type of Liaoning Cashmere goat and2type of Shaanbei WhiteCashmere goat will promote the study on the molecular mechanism of Cashmere goatcashmere growth.