Optimal set of microsatellite markers required to detect illegitimate progenies in selected oil palm (Elaeis guineensis Jacq.) breeding crosses

Oil palm is continually being improved via controlled crossing of selected palms to ensure sustainable yields and productivity. As such, correct parental assignment is important as the presence of illegitimates will compromise the progress of improvement. In the present study, we determined the optimal number of microsatellite (SSR) markers for detection of illegitimates in selected oil palm crosses with high confidence. Determining the optimal number of markers to assign parentage will ensure that the DNA fingerprinting will be cost effective for routine use as a quality control tool in oil palm improvement programs. Here, we evaluated a wide range of crosses that included a cross derived from wild germplasm palm. The results revealed that markers with high PIC are informative and detect most of the alleles present in a cross, including those exhibited by the illegitimates. A larger number of optimum sets of markers are needed to detect all illegitimates for crosses with higher levels of genetic diversity. The optimal number of polymorphic SSR markers determined in the present study can ensure that appropriate quality control is implemented for oil palm improvement programs.     

缢蛏微卫星多态性分析及其在亲子鉴定中的应用

利用磁珠富集法,构建了缢蛏(Sinonovacula constricta)微卫星富集文库。开发并筛选出18对微卫星引物,其中8对微卫星引物亲缘排除率较高,选取这8对引物对家系进行了亲子鉴定。结果表明,18对微卫星引物在12个个体中等位基因为2~6,平均等位基因为3.00;观测杂合度在0~1.000之间,平均值为0.581 3;期望杂合度0.290~0.779,平均值为0.528 4;多态信息含量为0.239~0.703,平均值为0.432 2。当两个亲本基因型未知时,8对微卫星引物在整个家系中单个亲本排除率(E-1P)为0.583~0.916,平均值为0.790 0;当一个亲本基因型已知时,另一个亲本的排除率(E-2P)为0.406~0.830,平均值为0.653 9。同时可知在子代个体数量不断增加的情况下,排除率仍在较高范围,因此可以作为亲子鉴定的分子标记。这对于缢蛏规范化养殖、家系选育的开展具有重要意义。     

畜禽群体中基于SNP标记的亲子鉴定及亲本推断方法

利用双亲高密度SNP标记信息,在畜禽群体中进行亲子鉴定及亲本推断新方法的计算机程序开发,并使用模拟的600 000个SNP标记对该程序进行测试。结果表明:对系谱正确性进行鉴定时,SNP标记数大于100即可达到100%的亲子鉴定准确率;对错误的系谱关系进行潜在亲子关系推断时,SNP标记数大于300可保证100%的推断准确率。标记平均MAF较低会降低亲子推断准确率。在程序运行效率方面,当使用50 000的SNP标记对1 000条错误率为10%的系谱进行亲子鉴定和推断时,共耗时332.87s,且计算耗时与标记数及个体数呈线性变化。本研究基于孟德尔遗传原理,设计并开发了基于双亲及后代基因型的亲子鉴定及亲本推断程序,该方法运行速度快,操作简单,准确性高,值得在畜禽群体基因组相关研究方面进行推广应用。     

大黄鱼微卫星多重PCR体系的建立及其应用

利用本实验室开发的微卫星标记,通过优化退火温度、引物浓度、循环次数等条件,建立了3组大黄鱼微卫星多重PCR体系,每组包含3个微卫星位点,用3组微卫星多重PCR分析了一个大黄鱼选育群体JD-01的遗传多样性。结果显示,该群体的平均等位基因为12.111,平均有效等位基因为7.408,平均多态信息含量为0.846,平均观测杂合度和期望杂合度分别为0.825、0.868,香农多样性指数为2.165。运用Cervus 3.0软件,对已知系谱关系的9个大黄鱼家系及对应亲本进行了亲子鉴定分析,以验证3组微卫星多重PCR在亲子鉴定中的准确性。结果显示,使用该3组微卫星多重PCR体系进行亲子鉴定准确率为100%。大黄鱼微卫星多重PCR体系的建立为分析群体的遗传多样性、亲子鉴定和辅助家系管理提供了一种高效的技术手段。     

Microsatellites markers to depict the reproductive and genetic patterns of farmed gilthead seabream (Sparus aurata): illustration by a case study on mass spawning

In the absence of breeding strategy, natural spawning constitutes the breeding ground for fish farmers to empirically manage their commercial broodstock. In this context, we used six microsatellite markers to characterize the genetic pattern of six commercial seabream broodfish tanks having a common history spanning four generations. The progeny of one tank single‐day mass‐spawning event, reared in two different environments, was used to estimate the genetic parameters for body weight. Limited genetic differentiation was observed among broodfish groups. A panel of nine loci allowed us to unambiguously assign 95.4% of the offspring (1692) and identify 37 parents (65% of the total broodfish). The limited effective population size (N_e = 15.3) was due to the elevated variance of parental contributions and to broodfish failing to contribute to the progeny. The fluctuation of the allele frequency highlighted the risks of genetic drift and reduction in the heterozygosity in the next generations. Heritability for body weight was moderate at commercial size (0.40 ± 0.10) and the high genetic correlation at later stages laid the groundwork for precocious selection criteria for growth. The discussion opens on the opportunity to use mass spawning for selective breeding.     

大额牛与婆罗门牛种间杂交的亲子鉴定*

 利用9个微卫星座位对一例大额牛与婆罗门牛杂交后代进行亲子鉴定,所有座位都呈孟德尔共显性遗传,计算的父权相对机会为99.994 9 %,证实此例亲子鉴定的确是一个三联体家庭成员,也证实大额牛（Bos frontalis）与婆罗门牛（Bos indicus）种间杂交的可行性。     

Parentage determination of the mud crab Scylla paramamosain using microsatellite markers

This study evaluated the properties of nine Scylla paramamosain microsatellite loci screened by us previously for inclusion in a parentage assignment marker suite. These nine highly polymorphic markers (mean He=0.847 and PIC=0.830) were determined as being suitable for parentage assignment. Simulations based on allele frequency data from 15 known maternal families (165 individuals) demonstrated that at least four loci were required to assign >95% of offspring to maternal parents with 95% confidence. In actual parentage assignments, all progenies were assigned to the maternal parents with six or more loci, which was similar to the simulation predictions. Our results suggest that this set of microsatellites provide a powerful and efficient tool for identifying pedigree information for selective breeding programmes of S. paramamosain.     

利用微卫星DNA标记进行黄牛的亲子鉴定*

 亲子鉴定在畜牧业生产中具有重要实际应用价值,常见其用于建立准确的系谱档案和家畜归属的诉讼案件。其中标准的三联体（即在母子关系是已知的情况下,鉴定假设父亲与子畜是否有亲生关系）和单亲［即父子（女）或母子（女）亲生关系的确定］两种情况下的亲子鉴定在畜牧生产中具有普遍意义。以云南文山黄牛为试验材料,采用10对常染色体微卫星DNA标记对这两种情况下的亲子关系进行了检测分析。结果表明,常染色体微卫星DNA标记在标准的三联体和单亲两种情况下都能用来进行亲子鉴定,结果较准确,但标准三联体情况下的亲子鉴定更准确。确定的不同模式下亲子鉴定的方法,可为家养动物亲子鉴定标准方法和流程的建立提供依据。     

苹果属观赏海棠实生单株亲本的AFLP鉴定

应用AFLP技术, 对以苹果属观赏海棠品种王族(Malus ‘Royalty’) 为母本, 以5个材料为父本的38个实生单株进行多态性分析。结果表明: 筛选出的9对分辨率高的引物共扩增出349条带, 其中多态性带303条, 多态位点百分率达86.8%。根据聚类分析和电泳图谱扩增得到亲本材料的特征带, 鉴定出19株实生单株的父本可能为‘宝石’ (Malus‘Jewelberry’) ; 7株实生单株的父本可能为海棠花(Malus spectabilis Borkh. ) ; 3株实生单株的父本可能为草莓果冻(Malus‘Strawberry Parfait’) ; 不能确定其父本的实生单株有9株。     

Evaluating spawning performance among captive Florida pompano Trachinotus carolinus broodstock using microsatellite‐based parentage assignment

Florida pompano has been identified as a promising candidate for commercial‐scale aquaculture production, but to date, little information is available regarding captive broodstock spawning characteristics. Genetic markers were tested for their power in monitoring mating outcomes and potential in analysing heritability of rapid growth trait in Trachinotus carolinus. A total of 20 unrelated adults (10 females and 10 males) were chosen for a hormone‐induced mass spawning event. The 515 fastest growing and 485 slowest growing fish of the total 4852 offspring were considered a selected progeny stock, and fish were collected at 45 days post hatch based on their growth traits. Parentage analyses based on the 20 breeders and 1,000 selected progeny were performed using a total of nine microsatellite markers, a 100% assignment rate was achieved, and a four‐marker set was the minimum number for the parentage assignment. The effective breeding number for the selected progeny was 11 (six females and five males), among which three females and two males were predominant contributors with the total contribution of 95.8% and 94.7% respectively. The proportion of fast‐growing offspring from broodfish and each mating cross (sire/dam) was used for detecting whether variation in growth of the offspring was related to parental stocks. Results showed that three adults and their mated combination exhibited the greatest fast‐growing offspring proportion (69.73% and 55.95%). This research provided new information regarding spawning performance and parental contribution during mass spawning events; both important first steps towards developing improved management strategies for captive Florida pompano broodstock.