Microsatellite loci for the reef-building coral, Acropora millepora, from the Great Barrier Reef

Nine novel, polymorphic microsatellite loci were developed for the scleractinian coral Acropora millepora (Acroporidae) from the Great Barrier Reef. The specificity and polymorphism of 5 microsatellite loci previously developed for a Caribbean congener, and one locus developed for an Indo-Pacific congener were also assessed - only one of those 6 loci produced reliable results: yielding a total of 10 polymorphic microsatellite loci for Acropora millepora. To construct a microsatellite library, genomic DNA was isolated from sperm (which lacks algal symbionts) of an Acropora millepora colony collected in December 1998 at Magnetic Island, an inshore island in the central Great Barrier Reef. The partial genomic library was constructed using hybridization capture for enrichment of microsatellites using the following probes: dimers (AC) 13 and (AG) 12, trimers (AAC) 6 and (ATC) 8, and tetramers (AATG) 6 and (ACAG) 6. Of 397 clones sequenced, 110 contained microsatellite repeats (excluding a small percentage of repeated clones) after only one round of enrichment. Polymerase chain reaction (PCR) primers were designed from 31 of those clone sequences.53 Acropora millepora individuals (colonies) were tested, 10 from each of 3 locations (Magnetic Island, Davies Reef and the Keppel Islands), and 23 from Great Palm Island. 31 loci were tested: 20 were abandoned due to the lack or non-specificity of the amplification, one was abandoned because only 2 alleles were observed among the 53 individuals; 3 of 5 Mendelian loci developed for the Caribbean Acropora palmata were initially found to amplify reasonably well and highly polymorphic in Acropora millepora, but nonspecific products were visible when analysed on a capillary sequencer for 2 of the 3 loci making an objective interpretation of allelic patterns impossible. The microsatellite marker developed for Acropora formosa yielded specific and polymorphic products in Acropora millepora, but was discarded as a relatively large fraction of non-amplifying individuals suggested a high frequency of null alleles for this locus.Nine novel and polymorphic microsatellite loci for Acropora millepora were presented. An additional microsatellite locus previously developed for the Caribbean congener Acropora palmata produced specific and polymorphic products. There was no evidence of linkage disequilibrium among these 10 loci as tested in the online software package genepop; of the 45 pairwise comparisons, one pair of loci showed significant linkage, but this statistical significance was lost after sequential Bonferroni correction.GenBank Accession numbers for the 9 novel loci: EF014480, EF014481, EF014482, EF014483, EF014484, EF014485, EF014486, EF014487, EF014488.
To construct a microsatellite library from sperm (lacking in algal symbionts) of an Acropora millepora colony.To test variability on different populations resulting in 5-20 alleles per locus.

本研究为采自大堡礁的石珊瑚目（scleractinian coral）鹿角珊瑚科（Acroporidae）米氏鹿角珊瑚（Acropora millepora）开发了9个全新的多态性微卫星（microsatellite）位点。同时，研究人员还评估了此前为加勒比海同属物种开发的5个微卫星位点，以及为印度洋-太平洋同属物种开发的1个微卫星位点的特异性与多态性；最终仅6个位点中的1个获得了可靠结果，累计得到适用于米氏鹿角珊瑚的10个多态性微卫星位点。
为构建微卫星文库，研究人员于1998年12月从大堡礁中部近岸岛屿磁岛（Magnetic Island）采集的一株米氏鹿角珊瑚的精子（不含藻类共生体）中提取了基因组DNA。
研究人员采用杂交捕获法富集微卫星序列以构建部分基因组文库，所用探针包括二核苷酸重复探针(AC)₁₃、(AG)₁₂，三核苷酸重复探针(AAC)₆、(ATC)₈，以及四核苷酸重复探针(AATG)₆、(ACAG)₆。
仅经过一轮富集后，在测序的397个克隆中，共110个含有微卫星重复序列（已剔除少量重复克隆）。
研究人员从其中31个克隆序列中设计了聚合酶链式反应（PCR）引物。
研究人员对53株米氏鹿角珊瑚个体（即珊瑚群落）进行了检测：其中3个采样点各取10株，分别为磁岛、戴维斯礁（Davies Reef）与凯珀尔群岛（Keppel Islands），另有23株采自大棕榈岛（Great Palm Island）。
共计对31个位点进行了检测：其中20个因扩增失败或扩增非特异性被舍弃，1个因在53株个体中仅观测到2个等位基因而被舍弃；此前为加勒比海掌状鹿角珊瑚（Acropora palmata）开发的5个孟德尔位点中，有3个最初在米氏鹿角珊瑚中表现出良好的扩增效果与高度多态性，但其中2个位点在毛细管测序仪上分析时出现了非特异性扩增产物，导致无法对等位基因图谱进行客观解读。
此前为美丽鹿角珊瑚（Acropora formosa）开发的微卫星标记在米氏鹿角珊瑚中扩增出了特异性且具有多态性的产物，但由于大量个体未出现扩增条带，提示该位点存在高频率的无效等位基因，因此该标记也被舍弃。
本研究共报道了9个适用于米氏鹿角珊瑚的全新多态性微卫星位点，另有1个此前为加勒比海同属物种掌状鹿角珊瑚开发的微卫星位点，其扩增产物同样具备特异性与多态性。
借助在线软件包Genepop对这10个位点进行连锁不平衡检测，结果未发现显著的连锁不平衡现象；在45组两两比较中，仅有1组位点表现出显著连锁，但经过序贯邦弗朗尼校正后，该统计学显著性消失。
9个全新位点的GenBank登录号依次为：EF014480、EF014481、EF014482、EF014483、EF014484、EF014485、EF014486、EF014487、EF014488。
从米氏鹿角珊瑚群落的精子（不含藻类共生体）中构建微卫星文库。
在不同种群中检测位点的变异度，最终每个位点可检测到5~20个等位基因。