RNA-Seq Analysis of Acacia crassicarpa

Acasia crassicarpa is a fast-growing leguminous tree widely cultivated in tropical areas including Indonasia, Malaysia, Australia and southern part of China. Acacia trees provide versatile utility in timber, furniture, and pulp production. Demands are growing for efficient breeding programs of Acacia trees based on the molecular genetic information, which enables the precise subspecies segregation, identification of useful genes, and altering the genetic traits by genome editing technology. In this study, complementary DNA was prepared from the callus-cultured cells of A. crassicarpa, massive parallel sequencing by paired-end sequencing, and the raw data of 124,410,892 reads containing 12,565,500,092 bases were filtered and assembled de novo into 93,319 contigs, with a total of 84,411,793 base. The unigenes were annotated based on sequence homology by BLAST searches on non-redundant database. Several K+ and Na+-transporter genes annotated in A. crasssicarpa revealed high sequence similarity to those of Prosopis alba, which has the characteristic high tolerance for the drought and barren salt land. Our RNA-seq analysis also revealed the microsatellites, composed of simple-sequence repeat (SSR) loci, representing di-, tri-, and tetra-nucleotide repeat units, in the open-reading frames. Remarkable polymorphism was produced when PCR amplification was applied to the microsatellite loci in auxin-response factors and other transcription factors, which successfully discerned 16 variant species of A. crassicarpa tested. The transcriptomic information of A. crassicarpa generated in this study presents a widely exploitable platform for efficient molecular breeding programs that potentially contributes to the forestry plantation and biomass exploitation.厚荚金合欢（Acasia crassicarpa）是一种速生豆科乔木，广泛栽培于印度尼西亚、马来西亚、澳大利亚以及中国南部等热带区域。金合欢属树木在木材、家具及制浆生产中具有多方面的应用价值。当前，基于分子遗传信息开展金合欢属树木高效育种项目的需求日益增长，这类项目可实现精准的亚种区分、功能基因鉴定，并可通过基因组编辑技术改造遗传性状。本研究从厚荚金合欢的愈伤组织培养细胞中制备了互补DNA（cDNA），采用双端测序技术开展大规模平行测序；对包含124,410,892条reads、总碱基数达12,565,500,092 bp的原始数据进行过滤后，通过从头组装得到93,319条重叠群，总长度为84,411,793 bp。本研究通过BLAST比对非冗余数据库，基于序列同源性对单基因序列进行功能注释。在厚荚金合欢中注释得到的多个钾离子（K+）与钠离子（Na+）转运蛋白基因，与具备极端耐旱及耐盐碱瘠地特性的白牧豆树（Prosopis alba）的同源基因序列相似度极高。本研究的RNA测序（RNA-seq）分析还在开放阅读框（ORF）中鉴定出了由二核苷酸、三核苷酸及四核苷酸重复单元构成的简单序列重复（SSR）位点，即微卫星序列。针对生长素响应因子及其他转录因子所在的微卫星位点进行聚合酶链式反应（PCR）扩增后，可产生显著的多态性，成功区分了本次测试的16个厚荚金合欢变种。本研究获取的厚荚金合欢转录组数据，可为高效分子育种项目提供通用性极强的可利用平台，有望为林业种植及生物质开发提供助力。