斑马鱼微卫星分子标记检测鲤鱼种间多态性

用6072对斑马鱼（Danio rerio）微卫星引物对黑龙江鲤（Cyprinus carpio haematopterus Temminck et Schlegel）、荷包红鲤（Cyprinus carpio var．wuyuanensis）和柏氏鲤（Cyprinus pellegrini Tchang）进行种间遗传差异分析，共发现563个斑马鱼微卫星标记在鲤鱼种间表现出多态性。从中随机抽选25个标记，对斑马伍和3种鲤鱼的遗传多样性进行分析，使用Phylip3．63软件按照Nei氏标准遗传距离计算种间遗传距离，再用MEGA3．0软件绘制NJ系统发生树，并进行1000次bootstrap检验系统树。结果显示，黑龙江鲤与荷包红鲤首先聚类，然后是柏氏鲤、斑马鱼。这些具有多态性的斑马鱼微卫星标记可用于鲤鱼种间种质鉴定，同时，借用模式生物斑马鱼的丰富遗传标记资源，增加同科的鲤鱼遗传连锁图谱上遗传标记的密度，必将对鲤鱼遗传育种进入分子育种时代起到促进作用。     

黄河鲤全同胞家系的微卫星标记亲子鉴定

本研究利用11个高度多态的微卫星标记对26个黄河鲤(Cyprinus carpio haematoperus)全同胞家系中的417尾体重较大的正常体色个体(黄河鲤较大个体)和52尾红色个体(红鲤表型个体)进行了亲子鉴定。11个微卫星座位的平均等位基因数、平均观测杂合度(H_o)、平均期望杂合度(H_e)和平均多态信息含量(PIC)分别为8.2、0.792、0.792和0.76。当双亲未知且置信度为95%时,11个座位的累积排除概率达99.79%。除29尾黄河鲤较大个体以外,388尾黄河鲤较大个体和52尾红鲤表型个体准确地找到了其父母本,实际鉴定率为93.82%。通过对子代数目超过20尾的7个黄河鲤家系生长性状的比较分析,本研究成功鉴定出子代生长性状优良的黄河鲤亲本组合及其家系,同时也鉴定出包含隐性红色基因的黄河鲤杂合亲本,为进一步选育表型和体色纯正且生长快速的黄河鲤新品种提供了理论依据和技术手段。     

长江刀鲚选育群体转录组EST-SSR的分布特征分析

本研究利用MISA软件挖掘长江刀鲚(Coilia ectenes)肌肉和肝脏转录组中的微卫星标记,为刀鲚选育群体的种质资源评估和分子标记辅助育种奠定基础。结果显示,从71869条Unigenes中共获得33896条重复单元长度为1~6碱基的微卫星序列;刀鲚转录组中不同类型微卫星的重复基序具有不同的分布特征,其中,单核苷酸重复、二核苷酸重复和三核苷酸重复为主要的微卫星重复类型,分别占总微卫星数目的34.94%、49.47%和13.34%;不同微卫星重复类型的优势重复基序亦有所不同,其中,A/T为单核苷酸重复基序的优势重复基序占86.25%,AC/GT为二核苷酸重复基序的为优势重复基序占75.25%,AGG/CCT为三核苷酸重复基序的优势重复基序占28.57%;不同微卫星重复基序核苷酸的数量和重复次数亦有所不同,重复次数伴随着重复单元中核苷酸数量的增加而呈现降低的趋势;从100对四核苷酸重复的SSR引物中筛选获得了16对多态性微卫星标记,并以此为基础,对长江刀鲚选育群体(F3)的遗传学特征进行了初步评估,结果显示,长江刀鲚选育群体F3的平均有效等位基因数(Ne)、平均观测杂合度(Ho)、平均期望杂合度(He)和Shannon多样性指数I分别为1.7580、0.3414、0.3977和0.6278。以上结果表明,基于刀鲚转录组数据批量开发微卫星是切实可行的,所开发的多态性微卫星标记能够应用于长江刀鲚选育群体的遗传背景评估和进一步的遗传育种研究。     

PCR-based segregation of one hybrid variety of Labeo rohita and Catla catla from their wild-types

High consumer preference together with its polyculture potential has undoubtedly driven Rohu (Labeo rohita) and Catla (Catla catla) to top the list of the most preferred fishes among the Indian major carps. Commonly found in these fishes are hybrids that can be natural or man-made. Increasing emphasis on biodiversity issues has necessitated proper stock management of these through molecular genetics techniques. Also with few morphological differences that can be used to differentiate wild types and hybrids properly, the problem demands a straightforward molecular approach. Here, we report a simple PCR-based technique that can differentiate the hybrid variety from wild types easily using three different microsatellite markers. Three sets of primers were used to amplify three different microsatellite markers from the genomic DNA isolated from pectoral fins. When the PCR products using all three primer sets were analyzed, ‘hybrid–Rohu’ could be distinguished from wild types. Whereas the hybrid–Rohu DNA yielded specific PCR products with all three primer pairs, only two PCR products were obtained either from wild-type Catla DNA (by primer sets 1 and 2) or from wild-type Rohu DNA (by primer sets 1 and 3). This study clearly demonstrates that a simple PCR-based technique will help the fish breeders and hatcheries to identify and differentiate suspected hybrid–Rohu carp from the wild types within a few hours.     

草鱼生长相关的微卫星标记在选育群体中的验证

生长性状是水产动物遗传育种中的重要经济性状,利用与性状相关的分子标记与育种相结合的手段,可以大大加速育种进程。在对草鱼生长性状的前期研究中,采用数量性状位点(QTL)定位的方法,在1号连锁群中发现了2个与生长相关的QTL。在此基础上,实验利用这2个QTL侧翼的2对微卫星标记(CID391_2、CID1512、CID973_1和CID254_1),对长江草鱼选育群体的480个个体进行分析,以期基于草鱼QTL定位结果,对草鱼生长相关的微卫星标记在选育群体中进行验证。结果显示:(1)4个微卫星标记在该群体中均具有高度多态性,其中各位点观测等位基因数(N_a)为12～23个,有效等位基因数(N_e)为4～12个,观测杂合度(H_o)为0.607～0.904,期望杂合度(H_e)为0.751～0.902;(2)利用方差分析及多重比较对4个多态性的微卫星标记与选育草鱼群体的生长性状(体质量和体长)进行关联分析,发现CID391_2在雌性个体中,各基因型与体质量和体长之间均无显著差异;而在雄性个体中,各基因型与体质量和体长之间差异显著。CID1512、CID973_1和CID254_1在雌性或雄性个体中,各基因型与体质量和体长之间均具有显著差异。研究表明,对草鱼生长相关的微卫星标记在选育群体中的验证结果,为进一步开展草鱼生长性状QTL定位研究和基于QTL结果的分子标记辅助育种(MAS)实践奠定理论基础。     

斑马鱼SSLP标记检测鲤鱼种间的遗传多态性

用500对斑马鱼SSLP标记引物对黑龙江野鲤（Cyprinus carpio haematopterus Temminck et Schlegel)和柏氏鲤（Cyprinus Pellegrini pellegrini Tchang)进行种间遗传差异分析，共发现110个斑马鱼标记在鲤鱼种间表现出差异，故可作为黑龙江野鲤和柏氏鲤间的分子标记，也可作为建立遗传连锁图谱的遗传标记。     

云斑尖塘鳢微卫星分子标记的筛选与检测

用磁珠富集法分离云斑尖塘鳢的微卫星序列,由所获得的1032个克隆中筛选出146个阳性克隆,经测序81个含有微卫星序列,52个为完美型,27个为非完美型,2个为复合型,其中43个微卫星序列重复次数在10以上。所获得的云斑尖塘鳢微卫星序列中除探针中使用的CA重复单元和GA重复单元外,还有TAC等其他类型的重复单元。设计合成38对微卫星引物,其中29对引物可稳定扩增出条带,使用这些引物对云斑尖塘鳢48个个体进行检测显示:观测杂合度平均值为0.63,期望杂合度平均值为0.43。29对引物中1对引物表现为单态,7对表现为高度多态,14对表现为中度多态,7对表现为低度多态,多态性较为丰富,说明本研究开发的绝大部分微卫星分子标记较为理想。     

Characterization of 95 novel microsatellite markers for Zhikong scallop Chlamys farreri using FIASCO-colony hybridization and EST database mining

ABSTRACT:   In order to construct a simple sequence repeat (SSR)-based genetic linkage map and to promote molecular marker-assisted selection (MAS) in scallop breeding, the methods of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO)-colony hybridization and expressed sequence tag (EST) database mining were modified and used to develop 95 novel microsatellite markers for Zhikong scallop. The SSR-enriched library constructed by the FIASCO method consisted of 830 clones, and 295 (35.5%) positive clones were identified after colony hybridization. One hundred and fifty clones were randomly sequenced and the results showed all clones contained at least one microsatellite. Of 91 primer pairs designed, 72 were amplified scorable polymerase chain reaction (PCR) products and 70 were polymorphic with the allele number range of 3–16 alleles/locus (average 7.0 alleles/locus). When EST database mining was performed, 66 microsatellites containing ESTs were identified from 3467 sequences deposited in GenBank. Based on cluster analysis of length and GC content of the flanking regions, 47 primer pairs were designed and 23 scorable EST SSRs were obtained. Compared with genomic SSRs developed in this study, EST SSRs showed lower genetic variability with an average of 4.2 alleles/locus. The results in the present study demonstrate that modified FIASCO-colony hybridization is an efficient and low-cost method for the isolation of large numbers of microsatellite markers for scallop species.     

中国明对虾单个家系中与生长性状相关微卫星标记的初步筛选

以1个人工授精获得的中国明对虾(Fenneropenaeus chinensis)F2家系为材料,对其中80尾中国明对虾进行正态分布检验,选择19个稳定扩增的微卫星位点对每一个体进行扩增和基因分型,分析家系遗传信息.利用最小二乘法对微卫星位点与中国明对虾体长、全长、体质量性状进行关联性分析.采用SPSS 11.5软件作微卫星分子标记与经济性状的方差分析,考察各微卫星位点对体长、全长、体质量3个经济性状的影响程度.结果表明,中国明对虾体长、全长、体质量3个性状均呈正态分布(P＞0.05);相关分析得到,19个微卫星位点中RS0683和FC019两个微卫星位点与体长、全长、体质量呈显著相关(P＜0.05);同时对差异显著的位点进行不同基因型间经济性状的多重比较,RS0683标记座位AA基因型在体长、全长、体质量的表型效应上极显著高于AB、BB基因型(P＜0.01),表明该标记位点基因型AA与3种经济性状正相关;FC019标记座位的AA基因型体长、全长、体质量显著低于AB、BC基因型(P＜0.05),表明该标记位点基因型AA与3种经济性状呈负相关.     

镜鲤与建鲤生长性状共享 QTL 标记及优势基因型

本研究以1个镜鲤(Cyprinus carpio L.)全同胞家系(190个个体)构建的微卫星遗传图谱(992个标记)为基础,从体重、体长、体高和体厚的QTL区间内发掘了54个标记,其与性状具有显著相关性,进而通过对不同基因型性状间的比较,筛选出83个优势基因型。在此基础上,用54个镜鲤QTL标记分析了建鲤(Cyprinus carpio var.jian)作图群体,其中40个标记在建鲤中表现出多态性,比例为74.07%;相关性分析结果显示,其中22个标记与建鲤家系的体重、体长、体高或体厚性状具有显著相关性(P0.05),占多态标记的55.00%;镜鲤与建鲤共享的22个QTL标记中,18个标记与至少1个相同的性状具有显著相关性(P0.05),从中筛选出建鲤性状具有优势的基因型30个,可用于指导建鲤的选育。品种间共享QTL的发掘能够扩展QTL标记的使用空间,减少新品种重新构建图谱进行QTL标记定位的工作量和成本。     

Genetic linkage map of the pearl oyster,Pinctada martensii (Dunker)

Genetic linkage maps were constructed with amplified fragment length polymorphism (AFLP) and microsatellite markers for the pearl oyster, Pinctada martensii (Dunker), the main bivalve used for marine pearl production in Asia. Twenty‐four AFLP and 84 microsatellite primer pairs were used for linkage analysis in a full‐sib family with two parents and 78 offspring. Of the 2357 AFLP fragments generated, 394 (16.7%) were polymorphic and segregating. Most (340 or 86.2%) of the markers segregated according to expected Mendelian ratios. Female and male linkage maps were constructed using 230 and 189 markers, including 15 and 10 microsatellites respectively. The female map consisted of 110 markers in 15 linkage groups, covering 1415.9 cM, with an average interval of 14.9 cM. The male map consisted of 98 markers in 16 linkage groups, with a total length of 1323.2 cM and an average interval of 16.1 cM. When unlinked doublets were considered, genome coverages were 78.5% for the female and 73.5% for the male map. Although preliminary, the genetic maps constructed here should be useful for future linkage and quantitative trait loci mapping efforts.     

DNA markers for estimation of inbreeding depression and heterosis in the guppy Poecilia reticulata

Molecular markers have significant potential for use in precise breeding programmes in aquaculture. This paper reviews the use of DNA markers to estimate inbreeding depression and heterosis in the guppy Poecilia reticulata. Full‐sib matings revealed that inbreeding causes declines in survival and salinity tolerance, but not in undwarfism, growth and high water temperature tolerance, indicating the effects of inbreeding differ among fitness‐related traits. Salinity tolerance was used to quantify the level of inbreeding depression and heterosis because the trait is strongly sensitive to inbreeding and shows a linear decrease with an increase in inbreeding coefficient. A positive correlation was observed between heterozygosity at microsatellite loci and salinity tolerance among 17 guppy populations. This indicates that heterozygosity estimated from microsatellites is a useful indicator for the estimation of inbreeding depression, suggesting that overall heterozygosity is important for fitness‐related traits that show inbreeding depression. Use of DNA markers to estimate the amount of heterosis in various strain combinations was examined using diallele and reciprocal crosses among four domestic strains. The amount of heterosis differed among the strain combinations and correlated with Nei's genetic distance measured by microsatellites and also by dissimilarity using random amplified polymorphic DNA (RAPD) analysis. This indicates that microsatellite and RAPD markers are useful for estimating the amount of heterosis in various strain combinations, further suggesting that the amount of heterosis depends on the genetic differences between the strains. The present study showed that DNA markers are useful tools for estimating inbreeding depression and heterosis in guppy breeding.     

吉富罗非鱼不同选育群体的遗传多样性

用微卫星和扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分子标记对海南、广州和青岛吉富品系尼罗罗非鱼的遗传多样性和遗传分化进行了研究。9对微卫星引物和4对AFLP引物的分析结果一致。微卫星分析表明,青岛吉富鱼的平均等位基因数(4.8)、平均观测杂合度(0.528)、平均多态信息含量(0.605)最高,海南吉富鱼的平均等位基因数(4.4)、平均观测杂合度(0.479)、平均多态信息含量(0.549)最低。AFLP分析显示,青岛吉富鱼的多态位点比例(48.4%)和基因多样性(0.245)最高,海南吉富鱼的多态位点比例(36.3%)和基因多样性(0.147)最低。这些表明,青岛吉富鱼的遗传多样性最高,海南吉富鱼最低。遗传分化分析表明,两两群体间遗传分化显著(微卫星FST为0.07~0.11,P<0.01;AFLPFST为0.24~0.29,P<0.01)。AMOVA分析显示,大部分遗传变异(微卫星的分析结果为91.26%;AFLP的为67.6%)来源于群体内个体间,表明吉富鱼选育品系尚具有进一步选育的潜力。     

翘嘴鳜养殖与野生群体及其家系的遗传多样性分析

通过5个翘嘴鳜(Siniperca chuatsi)的微卫星标记,对收集的湖北、广东翘嘴鳜养殖与长江野生3个群体及其家系的遗传结构进行了分析和比较。结果表明:筛选的5个微卫星位点具有高度可提供信息性,平均多态信息含量(PIC)0.5,三个群体遗传多样性关系为:湖北养殖群体(BF)长江野生群体(WBF)广东养殖群体(DF),不同家系的期望杂合度在0.4～0.7之间,家系间的遗传分化显著。分子方差分析结果显示,家系群体内的变异(83.33%)是总变异的主要来源。可见,湖北养殖群体具有较高的遗传多样性,具备进一步繁育筛选优良群体的潜质,微卫星分子标记技术可用于翘嘴鳜家系育种过程中的亲子鉴定。     

黄鳍鲷基因组微卫星的分离

采用磁珠富集法构建黄鳍鲷(Acanthopagrus latus Houttuyn)基因组微卫星富集文库。共挑选60个克隆进行测序,分析发现58个克隆分别含(GA)n或(CA)n两碱基重复单元。进一步通过序列比对,最终获得41个具有特异微卫星序列的阳性克隆。其中,23个克隆含有(GA)n或(CT)n两碱基重复序列,17个克隆含有(GT)n或(CA)n重复序列,另1个含有以上两种重复类型。获得的微卫星序列中,单一型及间断型序列各有20条,另有1条属于复合型序列。序列长度为117~512 bp,平均259 bp。微卫星核心序列两碱基重复5到38次,绝大多数序列重复次数大于10。基于微卫星两端的侧翼序列设计并获得了3对能够在黄鳍鲷基因组有效扩增的微卫星引物。本研究旨为进一步开展黄鳍鲷分子育种及资源评价分析提供基础资料。     

Application of microsatellite DNA parentage markers in the swimming crab Portunus trituberculatus

Microsatellites have been a powerful genetic tool in determining pedigree relationships among individuals. A microsatellite marker suite can be used to reliably assign parentage in the Portunus trituberculatus selective breeding programs. Thirty published microsatellites were screened for their suitability to be included in a DNA parentage marker suite. Nineteen microsatellite markers that exhibited significant stutter, poor allele resolution, null allele, and significant deviation from Mendelian expectations were eliminated from the marker suite, and the left eleven markers were ranked by PIC. Six most polymorphic markers with mean He?=?0.928 and PIC?=?0.901 from the original eleven were further assessed for the efficiency of pedigree identification. Total predicted power of the markers to exclude false parents was higher than 99%. Both simulations and real data analysis confirmed the high statistical power of six microsatellites suite in parentage exclusion, which permit us to trace back all offspring to single pairs of parents.     

草鱼全基因组微卫星特征分析与亲子鉴定

为分析草鱼全基因组微卫星特征并开发高多态性微卫星标记亲子鉴定平台,实验利用已发布的草鱼全基因组序列,开发高度多态、准确度高、重复单元在4～6碱基范围的微卫星标记.结果显示,在草鱼900.51 Mb基因组序列中共筛选到微卫星序列677363个,总长度12 835 407 bp,占全基因组长度的1.4254％,平均跨度为1...     

Sequence characterized amplified regions (SCAR) reveal candidate markers linked to growth traits in the Chinese shrimp Fenneropenaeus chinensis

Growth traits in 150 Chinese shrimps Fenneropenaeus chinensis were analyzed by the sequence characterized amplified regions (SCAR) technique in two populations of sixth-generation cultured shrimps with different ranges of body lengths (CP-a and CP-b) and in the wild-type population (WP). First, 240 random primers were used to screen polymorphic fragments by random amplified polymorphic DNA (RAPD). Population analysis revealed nine RAPD markers correlated with growth traits, including seven positively and two negatively correlated ones. The sequences of the RAPD markers were then used to design longer primers for SCAR. Six primer pairs were obtained, and two of these produced polymorphic fragments among the three groups. One amplified 39, 26 and 27 polymorphic fragments, with band frequencies of 78, 52 and 54% in the CP-a, CP-b and WP groups, respectively. The distributions of polymorphic fragments in the three groups were significantly different (P < 0.05) according to a χ ² test, indicating that they may be candidate markers linked to growth traits. Another primer pair amplified three alleles, resulting in six combinations of genotypes among three groups. Since allele A₁ was only found in the population of shorter shrimps, it may be a negative growth marker.     

条斑紫菜6个品系的SRAP分析

为鉴别条斑紫菜不同品系的种质,使用相关序列扩增多态性(sequence-related amplified polymorphism,SRAP)标记对条斑紫菜的5个选育品系和1个野生品系进行遗传分析,结果从35对引物组合中筛选出可扩增出稳定清晰条带的组合11对,共获得131个扩增位点,其中多态性位点125个,多态性比例高达95.42%。6个品系 间的遗传距离为0.364 3～0.867 9,平均为0.593 0。用UPGMA法进行聚类分析,结果将6个品系分为2个群,所反映的亲缘关系与各品系的来源基本一致,说明SRAP 标记技术可以成为条斑紫菜品系间遗传分析的有效工具。在131个多态性位点中,选择扩增出的4个位点构建了6个品系的指纹图谱。另外,通过ME1/EM6引物组合 扩增得到耐高温品系TM-18的特异性条带,经回收测序和重新设计引物,该条带在其丝状体和叶状体DNA中均能稳定地被扩增出来,可用于该品系的种质鉴别 。     

Genetic characterization of wild Dutch common carp (Cyprinus carpio L.)

Six male carp, caught in the water system surrounding the Anna Paulowna (AP) Polder in The Netherlands, were characterized using allozyme and microsatellite markers. At the sMDH‐A1,2* loci an allele was found, which has previously only been found in wild River Rhine and wild Vietnamese common carp. Microsatellite allele frequencies showed that these AP carp were significantly different from a group of carp originating from several different domesticated strains. Based on both allozyme and microsatellite data, the AP carp probably originated from a wild or feral self‐sustaining population.