牙鲆渤海自然群体的遗传多样性分析

为分析牙鲆自然群体的遗传多样性并筛选出能有效鉴定群体遗传特征的特异性微卫星标记,实验收集了渤海流域74尾野生个体组成实验群体。选择牙鲆24个连锁群上不同区域的72个微卫星标记进行遗传分析,其中,近着丝粒区域包含17个标记,连锁群中部包含19个标记,远着丝粒区域包含36个标记。结果显示:在连锁群不同区域上等位基因数(A)介于6.400～7.389,有效等位基因数(N_e)介于4.469～5.129,Shannon多样性指数(I)介于1.565～1.683;观测杂合度(H_o)、无偏倚杂合度期望值(H_e)和多态信息含量(PIC)的范围分别为0.568～0.593、0.738～0.753和0.707～0.746;Hardy-Weinberg遗传偏离指数(d)在-0.200以下;群体内个体之间的遗传距离在0.620以上。各项遗传参数表明该实验群体具有较为丰富的遗传多样性,个体之间具有相对较远的遗传距离,适宜作为基础群体进行选育。位于连锁群不同区域的微卫星标记获得的遗传参数并无明显差异,验证了标记所在位置与标记多样性高低不存在必然联系,但这些多态性标记可以作为分析牙鲆群体遗传结构的候选标记。     

偏分离条件下牙鲆生长性状QTL的主成分定位

本研究利用有丝分裂雌核发育建立牙鲆(Paralichthys olivaceus)双单倍体(Doubled Haploids,DH)群体,对牙鲆体重、全长、背鳍长、腹鳍长、体高、尾柄高、头高和躯干长共8组表型性状标准化处理后,进行主成分分析,得到可解释全部性状90.4%的表型主成分性状。然后,基于JoinMap 4、MapDisto、JoinMap 4-DistortedMap、MapDisto-DistortedMap构建4个连锁图谱,用偏分离标记矫正数量性状位点(Quantitative Trait Locus,QTL)基因型条件概率,采用Bayesian模型选择方法定位牙鲆表型主成分性状的加性QTL和上位性QTL。结果表明,用不同方法构建的遗传图谱和表型主成分性状QTL定位结果均有所不同。在图谱构建中,与基于JoinMap 4构建的图谱相比,基于MapDisto构建的图谱中偏分离标记的相对位置和遗传距离都发生了变化,甚至有5个偏分离标记没有被定位到相应的连锁群上;相对于基于JoinMap 4和MapDisto构建的图谱,经DistortedMap校正后的图谱中偏分离标记的相对位置没有发生变化,但是偏分离标记间遗传距离发生了变化。另外,在4个图谱中都检测到3个加性QTL,分别位于6号连锁群上,可解释表型变异的12.95%、14.85%、11.56%和11.76%,9号、22号连锁群上,具有负向加性效应;9号连锁群上,可解释表型变异的13.86%、13.27%、11.17%和11.25%,具有负向加性效应;22号连锁群上,可解释表型变异的5.68%、4.36%、4.97%和3.58%,具有正向加性效应。同时,分别检测到28对、19对、29对和20对上位性QTL,主要分布在6号、7号、9号、17号、20号和22号连锁群上,可解释表型主成分性状变异的2.19%~17.62%、2.40%~22.26%、2.08%~26.0%、3.16%~22.05%。研究结果表明,基于MapDisto软件构建、经DistortedMap软件包矫正后的图谱,定位结果更加准确,但仍需进一步验证。     

微卫星用于凡纳滨对虾育种过程中亲权分析及遗传多样性的变化监测

利用7个微卫星标记分析了6个凡纳滨对虾家系的亲本（G₀）及其后代（G₁）的基因型分离情况,同时对G₀和G₁的所有座位期望杂合度进行配对比较分析,并且通过对G₁群体每个位点的F-statistics分析检验群体的遗传分化,以期对凡纳滨对虾育种进行遗传监测。结果表明：共检测到44个等位基因。G₀和G₁的平均每个座位的等位基因数目分别为6.14和6.27,平均期望杂合度为0.786和0.733,平均多态性信息含量为0.709和0.695。7个微卫星座位的累计排除概率为0.99,并且在有亲本存在的情况下,能够将6个家系分开。G₀与G₁的所有座位期望杂合度的配对比较分析结果表明G₀平均期望杂合度要显著高于G₁(P<0.01)。G₁各座位的遗传分化指数F_ST在0.270 3～0.465 4,平均分化系数为0.358 4。说明亚群间属于高度遗传分化。最后,根据6个家系的遗传距离,利用UPGMA法对G₁个体聚类分析,结果表明亲缘关系较近的家系A和B以及C和D的相似系数很高分别各聚为一类,E和F分别聚为一类。     

牙鲆遗传连锁图谱的构建

利用基因组测序得到的大量微卫星序列,以681383B为父本、6812E36为母本杂交获得的F1为作图群体,构建了牙鲆(Paralichthys olivaceus)微卫星标记(SSR)遗传连锁图谱。雌雄图谱共定位SSR标记529个,其中雄性连锁图谱包括418个标记,分布在24个连锁群上,总长度1 418.1 cM,标记平均间隔3.62 cM,图谱覆盖率为88.7%;雌性连锁图谱包括437个标记,分布在24个连锁群上,总长度1 298.1 cM,标记平均间隔为3.16 cM,图谱覆盖率为89.1%。牙鲆中密度遗传图谱的构建为QTL分析以及分子标记辅助育种进一步奠定基础,并可以有效推动牙鲆的遗传改良工作,推动牙鲆养殖业的可持续发展。     

黄颡鱼遗传图谱构建及生长相关性状的QTL定位

以野生(♂)和人工养殖(♀)黄颡鱼杂交的100个F1个体为作图群体,用SSR、SRAP和TRAP3种DNA分子标记技术构建黄颡鱼的遗传连锁图谱。图谱整合了13个SSR标记,89个SRAP标记,26个TRAP标记。其中雌性框架图谱包括16个连锁群,图谱的长度为585.5cM;雄性框架图谱包括15个连锁群,图谱的长度为752.3cM;共享框架图谱包括5个连锁群,图谱的长度为231.3cM。用该连锁图谱对黄颡鱼的5个生长相关性状进行QTL扫描,在雌性图谱上检测到1个头宽的QTL,定位于第七连锁群上,LOD值为3.2,可解释的表型变异为13%。在雄性图谱上分别检测到1个体高和体长的QTL,均定位于第一连锁群上。体高QTL的LOD值为2.4,可解释的表型变异为12%。全长QTL的LOD值为2.1,可解释的表型变异为11%。3个QTL均可用于黄颡鱼的生长性状的标记辅助育种。     

长牡蛎壳橙性状遗传参数评估及与生长性状的关联性

长牡蛎壳橙快速生长品系是结合群体选育与种内群体间杂交方法,以壳色性状和生长性状为选育指标,经过连续3代选育获得的新品系。本研究以第1代长牡蛎壳橙快速生长品系(G₁)为亲本,采用巢式设计建立了48个混养家系,共获得863个个体。利用微卫星标记对混养家系进行了系谱分析,并基于REML法评估了长牡蛎壳橙快速生长品系壳色性状的遗传参数及与生长性状的相关性。结果显示,863个个体中,有851个个体被准确鉴定其所属家系;长牡蛎壳橙快速生长品系壳色性状颜色参数指标a^*(红绿轴色品指数)、b^*(黄蓝轴色品指数)和ΔE (色差)等均具中高等遗传力,大小依次为0.47±0.23、0.42±0.21和0.56±0.29。4个颜色参数指标间的遗传相关和表型相关范围分别为-0.79~0.86和-0.45~0.48。壳色性状与各生长性状的遗传相关和表型相关较低,分别为-0.33~0.17和-0.04~0.11。研究表明,在长牡蛎壳橙快速生长品系育种过程中,对壳色性状进行选育可以得到预期的改良效果。此外,壳橙性状与生长性状应分别作为目标性状进行协同选择,以实现同时改良两个性状的目的。本研究可为长牡蛎壳橙快速生长品系选育提供基础资料。     

中华绒螯蟹37个多态性微卫星标记在亲子遗传中的分离方式分析

为了评估基因组扫描方法获得的中华绒螯蟹微卫星标记的遗传方式,随机选择60个候选三核苷酸微卫星标记,首先利用中华绒螯蟹F1家系双亲及其6个F1子代共8个样品进行PCR扩增验证和多态性检测,随后对多态性标记位点在80子代个体中的亲子遗传分离类型及连锁关系进行了分析。结果显示,42个（70.00%）位点得到清晰扩增产物,其中有5个单态微卫星位点和37个多态微卫星位点。多态位点中23个位点子代基因基因型为1：1：1：1分离类型,11个位点属1：1分离类型,余下3个位点为1：2：1分离类型。37个多态位点中35个位点（94.59%）的分离符合孟德尔分离比（P>0.05）,scaffold430598_213690和scaffold21303_16865位点显著偏离1：1：1：1分离比。35个分离比符合孟德尔分离比的位点中,scaffold240262_150253、scaffold216209_138892、scaffold293154_172768三个标记发生连锁关系,scaffold285640_169721和scaffold427534_212914发生连锁关系,scaffold507500_231891和scaffold92860_68250标记发生连锁关系。以上结果表明开发的候选微卫星标记适用于中华绒螯蟹亲子鉴定和遗传图谱构建。     

长牡蛎出肉率与壳形性状的 QTL 定位分析

本研究以长牡蛎(Crassostrea gigas)F1全同胞家系为作图群体,在已构建的基于120个微卫星和66个SNP标记的长牡蛎性别平均连锁图谱上,利用PROC QTL 2.0软件对出肉率和壳形(壳宽和壳深)性状进行QTL定位分析。结果表明,共检测到13个相关的QTL,分布在3个连锁群上;其中,与出肉率相关的4个QTL定位在1号和3号连锁群上(LG1和LG3),表型解释率为0.25%~47.53%;与壳宽相关的3个QTL定位在10号连锁群上(LG10),表型解释率为0.71%~45.39%;与壳深相关的6个QTL也定位在LG10,表型解释率为3.37%~24.78%。根据QTL连锁群分析和性状相关性分析结果可以推测,出肉率与糖原含量性状以及壳宽与壳深性状分别具有相近的遗传特征,利用与相关性状共同关联的分子标记可以同时对出肉率与糖原含量性状、壳宽与壳深性状进行遗传改良。本研究结果为今后长牡蛎相关性状候选基因克隆和分子标记辅助育种提供了参考。     

牙鲆抗淋巴囊肿病相关SSR标记的筛选与鉴定

为了筛选出中国牙鲆群体的抗淋巴囊肿病相关SSR标记,实验采用分群分析法对102个牙鲆个体(感病56个,抗病46个)进行了抗淋巴囊肿病相关SSR标记的筛选。首先构建了抗、感基因池(抗病个体和感病个体各15个),并利用178对微卫星引物对其进行了扫描;对于在抗、感池间扩增出差异条带的引物,用构建基因池的30个个体对其进行了第一次单个体验证;对于在第一次单个体验证中差异显著的引物用102个个体进行了第二次验证。结果显示,有4对引物(scaffold440_22585、scaffold826_5003、scaffold703_4284和scaffold185_597)在抗、感基因池间扩增出了差异条带;经第一次单个体验证,表明scaffold826_5003(P=0.023)和scaffold185_597(P178bp=0.028,P173bp=0.009)的差异条带在抗、感个体中呈显著性差异;经第二次单个体验证,发现scaffold185_597的差异条带在抗、感个体中出现的频率分别为60.9%和14.3%(P=0.001),差异显著;对scaffold185_597在10个抗病个体中扩增出的差异条带进行了克隆并测序,经BLAST比对,证实其为黄海水产研究所水产基因组与细胞工程研究室公布的牙鲆微卫星标记scaffold185_597中的一段,同源性达到96%。研究表明,微卫星标记scaffold185_597可能与牙鲆抗淋巴囊肿病相关。     

半滑舌鳎抗哈维氏弧菌病相关微卫星标记筛选及QTL定位

为了筛选出与抗哈维氏弧菌病相关的微卫星标记并进行QTL定位,以2014年感染实验中存活率为52.22%的半滑舌鳎(Cynoglossus semilaevis)家系F1412(Family+年份+家系号)为材料,采用混合分离子分析法(bulked segregant analysis,BSA)对169个微卫星标记(SSR)进行筛选,得到1个可能与哈维氏弧菌病相关的微卫星标记scaffold479_23523。用scaffold479_23523所在的连锁群LG18上的37个SSR对94尾抗病、感病个体进行基因分型,得到3个图谱:整合图谱(LG18)、雌性图谱(LG18F)和雄性图谱(LG18M),并用两种不同的模式进行单标记分析和QTL定位。模式一得到3个显著性相关标记(P0.05)和1个极显著性相关标记(P0.01),图谱LG18F定位出一个区间qE-F1;模式二得到4个显著相关标记(P0.05)和1个极显著相关标记(P0.01),图谱LG18M定位出两个区间qE-M1、qE-M2。在全基因组序列的分布范围上,区间qE-F1包含了qE-M1及qE-M2,因此qE-F1更可能与抗病性状相关。本研究开发出了抗哈维氏弧菌病性状相关微卫星标记及QTL区间,为抗病新品种的培育奠定了基础。     

Quantitative trait locus mapping of growth‐related traits in inter‐specific F1 hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) in a tropical climate

Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F₁ hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species.     

Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino

The locations and effects of quantitative trait loci (QTL) were estimated for nine characters for growth‐related traits in the Pacific abalone (Haliotis discus hannai Ino) using a randomly amplified polymorphic DNA (RAPD), amplification fragment length polymorphism (AFLP) and SSR genetic linkage map. Twenty‐eight putatively significant QTLs (LOD>2.4) were detected for nine traits (shell length, shell width, total weight, shell weight, weight of soft part, muscle weight, gonad and digestive gland weight, mantle weight and gill weight). The percentage of phenotypic variation explained by a single QTL ranged from 8.0% to 35.9%. The significant correlations (P<0.001) were found among all the growth‐related traits, and Pearson's correlation coefficients were more than 0.81. For the female map, the QTL for growth were concentrated on groups 1 and 4 linkage maps. On the male map, the QTL that influenced growth‐related traits gathered on the groups 1 and 9 linkage maps. Genetic linkage map construction and QTL analysis for growth‐related traits are the basis for the marker‐assisted selection and will eventually improve production and quality of the Pacific abalone.     

Genotyping‐by‐sequencing for construction of a new genetic linkage map and QTL analysis of growth‐related traits in Pacific bluefin tuna

Pacific bluefin tuna (Thunnus orientalis) has high market value, but its wild populations have decreased in recent years. The broodstock of Pacific bluefin tuna that were hatched artificially and reared under aquaculture conditions is beginning to be used for production. The creation of broodstock with commercially valuable traits, such as rapid growth, is therefore of great interest. Genetic linkage map‐based identification of markers associated with quantitative trait loci (QTLs) facilitates marker‐assisted selection (MAS) breeding and allows efficient genetic improvement of broodstock. Single nucleotide polymorphism (SNP)‐based genetic linkage map construction using the genotyping‐by‐sequencing method can expand the number of mapped markers and help identify growth‐related QTLs. In this study, we constructed sex‐specific maps for 24 linkage groups consisting of 677 SNP and 651 microsatellite markers. The total lengths of 93 progenies in the mapping population followed normal distribution, with an average length of 9.4 mm. We performed composite interval mapping in the mapping population. QTL analysis revealed one significant QTL in LG10 on the female linkage map. The genetic linkage map—the second such map generated for Pacific bluefin tuna—and the growth‐related QTLs detected in this study will be useful for tuna aquaculture MAS programs.     

鲤头长、体厚、体高性状的QTL定位及遗传效应分析

用174个SSR、41个EST、345个SNP标记对以镜鲤良种后代为祖父母本所培育的杂交F2群体的68个个体进行基因型检测,运用JoinMap4.0软件包构建遗传连锁图。利用MapQTL5.0区间作图法(interval mapping,IM)和多QTL区间定位法(MQM mapping,MQM)进行QTL检测,通过置换实验(1000次重复)确定连锁群显著性水平阈值。在对体高、头长、体厚的区间定位中,共检测到6个与体高性状相关的QTLs区间,分布在LG1(SNP1339-SNP1490)、LG10(HLJE469-SNP1491)、LG12(SNP0922-HLJ1316)、LG13(SNP0937-HLJ328)、LG25(SNP1041-HLJ594)、LG35(SNP1425-SNP0389)等6个连锁群上,解释表型变异范围为20.0%～43.3%。其中,SNP1339-SNP1490区间LOD值最大为3.64,解释表型变异35.4%。6个与头长相关的QTLs,分布在LG1(SNP1339-SNP1490)、LG12(HLJ071-HLJ336)、LG13(SNP0937-HLJ328)、LG24(SN...     

Identification of quantitative trait loci for growth‐related traits in the swimming crab Portunus trituberculatus

The swimming crab (Portunus trituberculatus) is an economically important species in Asian aquaculture. Implementing growth‐related traits of P. trituberculatus into genetic breeding programmes is an ongoing effort. We used a previously published genetic linkage map of P. trituberculatus, containing 55 simple sequence repeat (SSR) markers and 172 amplified fragment length polymorphism (AFLP) techniques to map quantitative trait loci (QTL) for growth‐related traits in a single full sibling F₂ family. Ten growth‐related traits were measured for QTL mapping. Composite interval mapping identified 9 QTL on the female map and 16 on the male map. Individual QTL with additive effects explained 11–38% of the phenotypic variance for various traits using the female parent's map, and from 1% to 21% using the male parent's map. Two QTL explaining a large percentage of variation in body weight were detected on chromosome 17 on the female map, and on chromosome 16 on the male map, and contributed 38% and 18% of the phenotypic variance respectively. This is the first study to report the detection and positioning of major QTL affecting growth in a true crab species (Brachyura). The mapping of growth‐related QTL in this study raises the possibility of improving the growth of P. trituberculatus through marker‐assisted selection.