细角螺微卫星DNA 富集文库构建及特征分析

采用生物素磁珠富集法,用生物素标记的(GT)15和(CT)15两种探针与细角螺基因组 MseI 酶切的300~1200bp 片段杂交,杂交复合物与链霉亲和素磁珠结合,捕获含有重复序列的微卫星片段。最后将磁珠捕获到的重复序列与PMD19-T载体连接后克隆到DH5α中构建微卫星基因组文库。通过PCR检测出354个阳性克隆,从中随机选取248个片段大于500bp的阳性克隆进行测序,结果显示,在220个成功测序的阳性克隆中共获得278个微卫星序列,其中完美型171个,占61.5%;非完美型81个,占29.1%;复合型26个,占9.4%。除探针使用的GT和CT的重复序列外,还筛选到三碱基GTT、TGG、GAA、CAA;四碱基TCTA、ACAG、TAGA、GTGA、GTCT、GATA及五碱基TTTTG的重复序列。在278条序列中共有82条可以设计引物。     

Genetic differentiation between the New Zealand and Falkland Islands populations of southern blue whiting Micromesistius australis

The southern blue whiting Micromesistius australis (Norman, 1937) is found in two geographically distinct areas, the South Atlantic and south‐west Pacific Oceans. To date there has been no appraisal of the genetic relationships between the populations in these two areas. Here, we present analyses of New Zealand and Falkland southern blue whiting using mini‐ and microsatellite loci. Two out of six loci show highly significant allele frequency heterogeneities between the two areas, strongly suggesting genetically distinct populations.     

Assessing multiple mating and reproductive skew in three species of clinid fishes (Blenniiformes: Clinidae: Clinini), in reference to multiple mating in viviparous teleosts

This study investigated multiple mating in three species of intertidal klipfishes (family Clinidae): super klipfish Clinus superciliosus, bluntnose klipfish Clinus cottoides, and nosestripe klipfish Muraenoclinus dorsalis. These species display the rare reproductive mode of female vivipary with superfetation, a phenomenon where offspring at various stages of development are found within the female. Three or four microsatellite loci were used to genotype 25–30 adults of each species, as well as embryos of 131 C. superciliosus from six broods, 103 C. cottoides from six broods, and 63 M. dorsalis from five broods. The presence of multiple mating by females was assessed by maximum-likelihood sibship reconstruction implemented in the program COLONY, and the non-random distribution of reproductive success among individuals (reproductive skew) within broods was tested using the binomial skew index. Multiple mating by females was pervasive and observed in 83%, 100% and 80% of the broods of C. superciliosus, C. cottoides and M. dorsalis, respectively. The mean number of sires per brood was 4.3, 3.0 and 3.2, and, correspondingly, 5/5, 5/6 and 1/4 of multiply-sired broods showed significant reproductive skew in C. superciliosus, C. cottoides and M. dorsalis, respectively. The presence of pronounced reproductive skew may indicate post-copulatory processes taking place that bias paternity. We compared our results on multiple mating within the klipfish family, and with four other fish families that have vivipary with superfetation, and found the klipfish patterns were among those of live-bearing vertebrate groups with the highest average numbers of sires per brood.     

一种基于图像统计量的相对辐射纠正算法

线阵推扫式传感器，由于在信号传输和转换以及量化过程中产生的奇偶效应，和不同CCD探元的响应差异，导致原始图像出现明显的纵向条纹，相对辐射纠正的目的就是降低或者消除这些影响，以恢复图像原始信息。针对宽幅线阵推扫式传感器的特点，提出了一种基于图像自身统计量的相对辐射纠正算法——相邻列均衡法，消除了原始图像中明显的纵向条带效应。和常规的基于图像统计量的相对辐射纠正算法相比，这种算法的特点在于求解相对辐射定标系数时不依赖于均匀场景，并在处理北京一号小卫星图像中得到了验证。     

Establishment of microsatellite-based triplex PCR for parentage analysis of Chinese shrimp Fenneropenaeus chinensis

Through exploring the microsatellite primers from the random genome sequences of Chinese shrimp (Fenneropenaeus chinensis), some microsatellite primers were obtained with rich polymorphic genetic information, and a triplex PCR was established using three primers (RS1101, RS0683 and H081 primers). By adjusting the final concentration of Mg2 , dNTP and primers, and using a touch-town PCR program, the optimum amplification parameters of PCR system were obtained, which could successfully amplify the three primers in a PCR reaction. In the denatured PAGE gel, the amplified DNA fragments of three primers RS1101, RS0683 and H081 could be easily identified each other. For the triplex PCR system, the PPE (probabilities of paternity exclusion) is 0.967 9, and the DP (discrimination power) is 0.999 327. Using the triplex PCR to test ten individuals of a parentage and their parents, an individual was excluded from the parentage in all of the three microsatellite loci, which might be mixed into the parentage for some unknown reason such as factitious misplay. The triplex PCR will be of great practical value in identifying the parentages of F. chinensis.     

合浦珠母贝基因组微卫星富集文库的构建与分析

采用生物素标记的(AC)_(15)探针和磁珠富集法构建合浦珠母贝(Pinctada fucata)基因组DNA微卫星富集文库。随机挑选2097个克隆进行筛选,得到483个候选克隆(23.03%),对其中135个阳性克隆测序分析发现122个克隆含有微卫星重复单元(90.37%)。进一步通过序列比对,最终得到65个具有特异微卫星序列的阳性克隆(53.28%),其中包含85个微卫星DNA结构域,其中完美型(perfect)70个,占82.36%;非完美型(imperfect)7个,占8.24%;混合型(compound)8个,占9.41%,重复次数主要分布在5~20(95.74%),平均重复次数为7.83,(AC/GT)n重复所占比例最高(75.53%)。基于微卫星两端的侧翼序列,利用Primer Premier 5.0设计引物,获得11对具有多态性的微卫星引物。本研究为开展合浦珠母贝分子育种及资源评价分析提供了基础资料。     

拟穴青蟹(CA)n微卫星DNA 多态性引物筛选

利用FIASCO (Fast Isolation by AFLP Sequences Containing repeats) 技术建立拟穴青蟹Scylla paramamosain基因组文库, 并与生物素标记的(CA)15 寡核苷酸探针杂交, 联合磁珠富集法构建拟穴青蟹微卫星富集文库。测序194 个阳性菌落, 分析其中的150 条序列, 结果表明: 两碱基重复类型占90%以上, 其中重复拷贝数在30 以上的占27.45%; 含微卫星座位189 个, 其中完美型146 个、非完美型28个和复合型15 个。设计125 对引物扩增一个拟穴青蟹野生群体(含20 个个体), 其中的19 对引物能稳定扩增且片段大小基本符合理论长度。遗传变异分析表明, 17 个位点表现出高度多态性, 16 个位点显著偏离Hardy-Weinberg 平衡(P<0.05), 4 组两两位点间存在连锁不平衡现象(P<0.0026, 经Bonferroni 法校正), 7 个微卫星位点可能存在无效等位基因。若排除混合微卫星位点的引物对以及扩增位点PIC (polymorphism information content)值在0.5 以下的引物对, 则13 对引物能用于拟穴青蟹群体遗传学等研究。     

Abundance of fin whales (Balaenoptera physalus) in the north-western Mediterranean Sea,using photo-identification and microsatellite genotyping

The Mediterranean fin whale population, Balaenoptera physalus, is resident, with almost no exchanges with the Atlantic population. The entire population was estimated at 1300 or 13,300 individuals by a recent project depending on the platform used. This disparity shows the importance of long-term monitoring with a unique protocol of survey. Capture-recapture approaches using dorsal photographs and genetic identity collections over a 10 years period were used to estimate the abundance of the north-western Mediterranean fin whale. We identified 332 individuals using photographs and 470 using genotypes, with a total of 546 individuals identified between 2008 and 2019, when some whales were double-marked. The inter-annual percentage of recapture varied between 15% and 17% respectively for genotypes and photographs methods. Using Cormack-Jolly-Seber models, the abundance of fin whales in the north-western Mediterranean is estimated at 1295 individuals (95% CI: 1116–1474) with a survival probability of 0.945 (95% CI: 0.690–0.993) from genotypes. Abundance estimates from combined collections (photographs and genotypes) and corrected photograph estimates were similar to the genetic ones. Future studies might prioritize the genetic approach which is the least biased and with a narrower confidence interval. The genetic abundance estimates show relative stability over time, when compared to 1990 estimates, and should be included in future conservation actions.     

Parentage determination of black sea bream(Acanthopagrus schlegelii) for stock enhancement:effectiveness and loss of genetic variation

The stock enhancement programs for black sea bream Acanthopagrus schlegelii have been conducted in China for a fewyears.However,little information has been reported concerning the effectiveness and genetic effect of black sea bream stock enhancement.In order to detect the contribution of released individuals in Zhujiang River Estuary(ZRE) and Daya Bay(DB),six microsatellite markers were used to identify the hatchery-released individuals.In addition,this pedigree of hatchery populations(broodfish and hatchery-released offspring) was traced to detect the number of effective parents(N_e),the inbreeding coefficient and the decrease of genetic variability in the reproduction.The pedigree reconstruction showed that at least 69(out of 93) broodfish had offspring.The estimated N_e was 54.8,consequently the inbreeding coefficient was 0.91%.The genetic diversity of hatchery-released offspring was lower than that in that of broodfish(heterozygosity alleles,0.727-0.774),some alleles(number of alleles,61-69) and genetic variance were lost during reproduction.It was observed that wild samples had higher levels of genetic diversity compared with hatchery populations as well as recaptured samples in releasing area.A total of 128 hatchery-released black sea bream were identified among 487 recaptured samples in ZRE,while a total of 15 samples were identified among 96 samples in DB.In summary,there was a high survival of released fish.Nevertheless,the results provided evidence to consider a loss of genetic variation in hatcheryreleased stock and a negative genetic effect of the stock enhancement.     

中国鲎(Tachypleus tridentatus)微卫星标记筛选及种群遗传多样性和遗传结构分析

采用FIASCO方法构建了中国鲎(Tachypleus tridentatus)(CA)_n微卫星富集文库,对214个阳性克隆进行测序,筛选获得60条含有微卫星的序列,其中可设计引物的序列共38条。38对微卫星引物在37个中国鲎个体中PCR检测发现,只有9个位点具有多态性,其等位基因数为5—14个,每个位点平均具有8.1个等位基因。9个位点之间不存在连锁。利用该9个具有多态性微卫星标记分析了中国沿海9个中国鲎地理群体的种群遗传多样性和遗传结构。结果表明:中国沿海的中国鲎种群遗传多态性水平仍较高, 9个地方群体间无遗传差异,它们之间未见显著分化,且有着较高的基因流;推测人为的迁移是造成遗传无分化的主要原因。我们建议中国鲎已绝迹的海域可从其他海域引入中国鲎个体来恢复地方群体资源。