Genome and comparative transcriptomics of the African wild rice Oryza longistaminata and development of a fast and efficient plant CRISPR-Cas9 vector cloning system
长雄野生稻（Oryza longistaminata）是分布于非洲大陆及马达加斯加岛的具有AA基因组的野生稻，拥有众多优异的性状，如地下茎性状、自交不亲和性状以及对众多非生物与生物胁迫的抗性等。解析长雄野生稻的基因组将是理解这些优异性状并加以利用的关键一步，然而基因组的高杂合度是解析这些性状的很大障碍。在本次工作中，我们使用了大量的二代测序数据(~396× Illumina 短读长 以及 ~5.9× Roche GS FLX+ 长读长) 组装了长雄野生稻的基因组。利用目前已经发布的其他3种稻属物种基因组数据，我们比较了4种稻属物种中抗性基因的数目，发现长雄野生稻拥有最多的抗性基因。为了揭示地下茎及自交不亲和性状的机制，我们测定了4组共8个组织的转录组数据（地下茎vs. 地上茎，地下茎尖vs. 地上茎尖，长雄野生稻的雄蕊vs. 自交亲和后代的雄蕊，长雄野生稻的雌蕊vs. 自交亲和后代的雌蕊；此处的自交亲和后代是指长雄野生稻与水稻籼稻RD23杂交后代中可以自交亲和的单株）并进行了比较转录组学的研究，鉴定了可能影响地下茎和自交不亲和性状的候选基因。同时，我们使用长雄野生稻作为外群，分析了亚洲栽培稻驯化基因的进化历程，发现：PROG1可能是从普通野生稻中进化出来的新基因，使其进化出了匍匐株型来适应沼泽的生长环境；而后功能丧失型的PROG1又在亚洲栽培稻的驯化过程中受到强烈的人工选择以满足人工密植的直立株型的需求，第一次在植物中展现了一个新基因能够控制植物关键的适应性形态性状。本文研究得到的长雄野生稻基因组及相关组织的转录组数据为我们研究并挖掘长雄野生稻优异的基因资源及稻属的进化提供了重要的数据。 CRISPR-Cas基因组编辑系统已经在模式植物及主要作物中广泛应用。 对于农杆菌转化体系，通常将cas9元件与gRNA元件整合入双元载体需要多步克隆，实验周期长且实验成本高。在本次研究中，我们开发了一套基于一步克隆法的植物CRISPR-Cas克隆体系PLANTCRISPR-CLONING，并且在水稻和烟草中验证了其高效的基因打靶效率。同时，我们对模式植物水稻（粳稻和籼稻）、拟南芥和烟草基因组中所有注释基因的潜在PLANTCRISPR-CLONING靶点进行了特异性分析，发现基因组中绝大部分基因都拥有特异性的靶点。植物PLANTCRISPR-CLONING克隆体系的应用将对植物学家研究基因功能及作物改良提供新的选择。; Oryza longistaminata is a close wild relative of the African cultivated rice and has several hallmark biological traits, such as rhizomatousness, self-incompatibility and high resistances to biotic and abiotic stresses. Deciphering genome of O. longistaminata has been hindered by the high heterozygosity of this species, which is a result of its inherent self-incompatibility. Here, we present a draft O. longistaminata genome assembly using a large quantity of combined sequencing data (~396× Illumina short reads and ~5.9× Roche GS FLX+ long reads). We compared the number of resistance (R) genes among other 3 published genomes of Oryzas species and revealed more R genes in O. longistaminata. By comparing transcriptomes of 4 pairs of tissues (rhizome vs. stem, rhizome-tips vs. stem-tips, pistils of O. longistaminata vs. pistils of a self-compatible hybrid line and stamens of O. longistaminata vs. stamens of the self-compatible hybrid line; here hybrid line refers to the offsprings between the cross of O. longistaminata and O. sativa ssp. indica variety RD23) related to the presence/absence of rhizomes or self-incompatibility, we identified candidate genes related to rhizomatousness and self-incompatibility. Using this genome as an outgroup we analyzed evolution of domestication genes identified in the Asian cultivated rice, and revealed an striking evolutionary pattern of origin and loss of prostrate plant architecture in Asian wild rice (O. rufipogon) and cultivated rice (O. sativa) through the birth and death of the domestication gene, PROG1. Our findings into PROG1 provide the first piece of plant evidence that shows how a newly evolved gene can control a striking adaptive morphological trait. In aggregate, the draft genome and large quantity of transcriptome data of O. longistaminata provide a basic evidentiary foundation for targeted studies into genes underlying valuable phenotypic traits, gene mining or breeding efforts, and evolutionary studies of African rice and the genus Oryza. CRISPR-Cas system has been widely used in plants to target mutagenesis or modify the DNA sequence. For the Agrobacterium-mediated transformation, it is necessary to combine the cas9 cassette and gRNA (guide RNA) cassette into one binary vector, in which usually takes steps to construct the CRISPR-Cas vectors. Here, we report a new CRISPR-Cas cloning system PLANTCRISPR-CLONING for plants, which takes only one step for cloning. High efficiency for targeting mutations was validated in rice and tobacco and multiple mutated alleles were found in the T0 transgenic mutated plants. Meanwhile, all the potential specific target sites across all genes in rice, Arabidopsis and tobacco were identified for PLANTCRISPR-CLONING system. The PLANTCRISPR-CLONING system we report here will promote plant functional studies as well as crop improvement.
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