Development of a genetic method to assess bleaching tolerance in corals (MTSRF Project 2.5i.2c)

Next generation DNA sequencing was used to scan a large proportion of the transcribed coding DNA in Acropora millepora for polymorphisms. Transcribed coding DNA (messenger RNA - mRNA) was extracted from 8 colonies sampled at three thermally distinct habitats along the GBR (Wilke Island Reef in Princess Charlotte Bay, Nelly Bay at Magnetic Island, and Miall Island in the Keppel Islands). The mRNA extractions from each population were pooled and translated back to the complementary DNA (cDNA), which was sequenced using 454 pyrosequencing by the Australian Genome Research Facility. This transcriptome consists of over 500 000 DNA sequences that are assembled into 55 000 contigs, of which over 11 000 aligned to known genes (8 000 unique genes). A database was generated consisting of almost 90 000 SNPs (Single Nucleotide Polymorphisms/gene variants).26 SNPs from 16 genes were investigated, from which one SNP from each of 15 genes were deemed suitable for large scale genotyping and a genotyping assay was developed.20 individuals from 17 reefs throughout the Great Barrier Reef were genotyped. Four from the 15 show a geographic pattern.The relative frequency of each of the nucleotides was measured in each SNP (A, T, C, G). To investigate the variation in gene coding regions and their frequency distributions along a themal gradient on the Great Barrier Reef with the aim of using such genes as markers to map thermal tolerance across the Great Barrier Reef.To describe DNA sequences for genes that are likely to be involved in thermal stress responses.To describe gene variants and their potential function in the coral host.To map allele frequencies of these genes at select locations on the GBR. Reef locations: Boulton Reef, Calder Island, Darley Reef, Emily, Goble Reef, High Peak Island, Holbourne Island, Night Island, North Keppel, Reef 20-344, Reef 21-121, Ross Reef, Sudbury Reef (I and II), SW Pelorus, Wilkie Island, Wallace Island.15 selected genes: Argenine Kinase, Beta Gamma Crystalin, Coatomer, Complete Component C3, Feritin, Galaxin, Gene of unknown function, Hsp60, Ligand of number X2, Mn Superoxide Dismutase, Thioredoxin, Transcription factor 1_1, Transcription factor 1_5, Ubiquitin like protein.The image is a portayal of one of the gene distributions.

本研究采用下一代DNA测序技术，对千孔珊瑚（Acropora millepora）的转录编码DNA的大片段区域开展多态性扫描。
转录编码DNA即信使RNA（messenger RNA, mRNA），样本采集自大堡礁（Great Barrier Reef, GBR）三个热环境差异显著的生境：夏洛特公主湾的威尔基岛礁、磁岛的内利湾以及凯珀尔群岛的迈尔岛，共获取8个珊瑚群落样本。将每个种群的mRNA提取物混合后反转录为互补DNA（complementary DNA, cDNA），并由澳大利亚基因组研究中心（Australian Genome Research Facility）采用454焦磷酸测序技术完成测序。
该转录组包含超过50万条DNA序列，经组装得到55000个重叠群（contigs），其中超过11000个重叠群可比对至已知基因，对应8000个独特基因。研究构建了包含近9万个单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs，即基因变异）的数据库。
本研究对16个基因的26个SNP位点进行分析，最终筛选出15个基因各1个SNP位点，适用于大规模基因分型，并开发了配套的基因分型检测方法。研究人员对大堡礁全境17个礁区的20个个体进行基因分型，其中15个位点中有4个呈现出明确的地理分布模式。
研究测定了每个SNP位点的四种核苷酸（A、T、C、G）的相对频率。本研究的核心目标为：探究基因编码区的变异特征及其沿大堡礁温度梯度的频率分布规律，以期将此类基因作为标记物，绘制大堡礁区域的热耐受能力图谱。具体研究方向包括：1. 描述可能参与热应激响应的基因的DNA序列；2. 阐释珊瑚宿主中的基因变异及其潜在功能；3. 绘制大堡礁特定区域内上述基因的等位基因频率分布。
本次研究涉及的礁区包括：博尔顿礁、考尔德岛、达利礁、艾米莉礁、戈布尔礁、高岛、霍尔本岛、奈特岛、北凯珀尔礁、20-344号礁、21-121号礁、罗斯礁、萨德伯里礁（I和II区）、西南佩卢罗斯礁、威尔基岛、华莱士岛。
本次筛选的15个基因包括：精氨酸激酶、βγ晶状体蛋白、包被蛋白复合体、补体成分C3全长、铁蛋白、半乳凝素、功能未知基因、热休克蛋白60（Hsp60）、X2号配体、锰超氧化物歧化酶、硫氧还蛋白、转录因子1_1、转录因子1_5、泛素样蛋白。
附图展示了其中一个基因的分布情况。