宽口光唇鱼胚胎发育的研究

在涪江下游宽口光唇鱼的产卵时间为4—5月,自然产卵水温为17℃—23℃。产卵场位于底质为卵石和砾石的浅水滩,分散产卵,卵弱粘性,金黄色,沉性,卵径1.55—2.11mm,卵膜外径2.27-2.55mm。在水温19.5—21.2℃条件下,胚胎从受精到孵化出膜共历时56h30min,初孵仔鱼全长6.0-6.2mm,肛后长/全长=24.14％,卵黄囊前部为膨大的球形,后部为均匀的棒状。     

氯化钠浓度对宽口光唇鱼精子活力的影响

本文观察了不同氯化钠浓度对宽口光唇鱼Ａｃｒｏｓｏｃｈｉｌｕｓｍｏｎｔｉｃｏｌａ精子活力的影响,并进一步建立数学模型进行分析论证,得出结论：在氯化钠浓度０—０５１％的范围内,精子快速运动时间和寿命相对延长,但在浓度超过０５１％,精子活动开始受到抑制,活动强度减弱,寿命缩短。     

中华金沙鳅多态性微卫星位点的筛选与特征分析

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基于高通量测序的厚唇裸重唇鱼微卫星分子标记筛选

本研究对厚唇裸重唇鱼(Gymnodiptychus pachycheilus)基因组进行Illumina HiSeq高通量测序,共获得85 905条序列,包含567 200个微卫星位点,从中筛选出15个微卫星位点,采用雅砻江新龙种群和黄河渭河种群对其多态性进行了验证。新龙种群中,15个位点的平均等位基因数为6.9(3 ~ 13),观测杂合度(Ho)为0.712 4,多态信息含量(PIC)为0.630 3,有8个位点显著偏离哈迪-温伯格平衡,1对位点表现出连锁。渭河种群中,15个位点的平均等位基因数为8.4(4 ~ 16),观测杂合度(Ho)为0.719 5,多态信息含量(PIC)为0.680 7,有7个位点显著偏离了哈迪-温伯格平衡,4对位点表现出连锁。筛选获得的15个微卫星位点多态性较高,适合用于厚唇裸重唇鱼种群遗传学研究。     

克氏光唇鱼线粒体基因组测定及光唇鱼属的系统发育分析

根据侧条光唇鱼(Acrossocheilus parallens)线粒体基因(mt DNA)序列设计引物,采用引物步移和PCR扩增产物测序,获得了克氏光唇鱼(A.kreyenbergii)的mt DNA全序列。结构分析表明,克氏光唇鱼mt DNA为首尾闭合的环状基因,全长16 596 bp,编码37个基因,包括13个蛋白编码基因、22个t RNA基因、2个r RNA基因和两段非编码区(D-loop和轻链复制起点OL),碱基组成具有明显的A+T偏好和反G偏倚现象。13个蛋白编码基因中,除COⅠ的起始密码子是GTG,其余基因的起始密码子均为ATG;终止密码子包括完全的终止密码子TAA(38.46%)和TAG(7.69%),不完全的终止密码子TA(15.38%)和T(38.46%)。在D-loop区的811~837 bp区间发现了一段"TA"短串联重复序列。从蛋白编码基因所包含的信息量、变异位点和变异率看,ND5、ND4、COⅠ和ND2最适合作为光唇鱼属种间系统发育分析的分子标记。采用贝叶斯法利用13个蛋白编码基因所构建的系统发育树显示,光唇鱼属和白甲鱼属(Onychostoma)的24种鱼类各自没有聚为单系群,相互间不能明确区分。     

飘鱼微卫星位点的筛选及珠江流域5个地理群体的遗传多样性分析

为了解珠江流域飘鱼(Pseudolaubuca sinensis)群体间的遗传多样性及其分化程度,利用RAD-Seq技术开发出微卫星位点,并设计了100对微卫星引物,筛选出66个可稳定扩增出目的条带的位点,其中16个具有较高的多态性(PIC>0.5),以2碱基重复居多。利用其中10对多态性位点对珠江流域的郁江(YuJ)、左江(ZJ)、右江(YJ)、融江(RJ)和桂江(GJ)飘鱼群体进行遗传多样性研究,得到有效等位基因数、观测杂合度、期望杂合度范围分别为5.2028—6.3800、0.6773—0.7667和0.7975—0.8425,表明珠江流域5个飘鱼群体具有较高的遗传多样性,以YuJ群体遗传多样性最高, RJ群体遗传多样性为最低。且群体间各个遗传参数相差较小,说明他们的遗传多样性水平相近。AMOVA分析显示,遗传变异主要来自于群体内(98.45%),仅一小部分的变异来自于群体间(1.55%),总群体的遗传分化系数Fst为0.015,两两群体间的Fst在–0.0033—0.0295波动,属于低等程度的分化。群体间基因交流值(Nm)在8.2246—76.0075,表明群体间基因交...     

北太平洋柔鱼微卫星标记的筛选及遗传多样性

采用(AC)12、(AG)12两种生物素探针,通过磁珠富集法构建了柔鱼部分基因组微卫星富集文库。68个阳性克隆中有60个含有微卫星序列,重复次数在10次以上的占86.84%,最高重复次数为33次。其中,完美型微卫星占60.53%,非完美型微卫星占36.84%,混合型微卫星占2.63%。除探针使用的AC/TG、AG/TC重复外,还得到ACAG、AGAC重复序列。利用筛选出的8个微卫星位点对北太平洋柔鱼6个群体的遗传多样性及遗传结构进行分析。结果表明,8个微卫星位点均为高度多态性位点(PIC=0.787—0.987),位点Bo103与位点Bo105极显著偏离Hardy-Weinberg平衡(P0.01)。6个地理位置的柔鱼群体显示出较高的遗传多样性水平(Ho=0.672—0.761,He=0.808—0.851)。两两群体间的Fst值以及AMOVA分析结果均表明,群体间遗传分化不显著(Fst=0.00559,P0.05),遗传差异主要来自于个体间。基于Nei's遗传距离的UPGMA聚类树显示,北太平洋东北部2个柔鱼群体(NE1、NE3)聚为一类,西北部3个群体(NW1、NW2、NW3)与东北部1个群体(NE2)另聚为一类,且群体NW1与群体NE2亲缘关系最近,遗传距离与地理距离线性相关分析没有呈现出正相关性(R=0.175,P0.05)。遗传结构分析结果推断北太平洋柔鱼存在1个理论群。柔鱼个体具有较强的游泳能力,在海流的作用下,群体之间存在较强的基因交流。建议今后在柔鱼资源开发利用过程中将北太平洋柔鱼看作1个管理单元。     

基于转录组测序的缺须盆唇鱼微卫星标记特征分析

缺须盆唇鱼(Placocheilus cryptonemus)是云南省怒江流域特有的一种高原经济鱼类.通过在Illumi-naHiSeq 2500平台上对缺须盆唇鱼进行转录组测序(RNA-seq)分析,利用MicroSAtellite (MISA)微卫星识别工具对1～6个碱基重复、碱基数在10 bp以上的微卫星进行鉴定.结果 表明,在缺须盆唇鱼转录组中共发现分布在36764条Unigene上的30500个SSR位点,包含序列的SSR数量为18078,发生频率为49.17％.获得的单纯SSR中共有215种重复基元,其中单核苷酸重复基元类型数量最多,为88个,六碱基重复的SSR最少,其他的重复从二、三、四、五、六碱基开始,SSR的形成呈现递减规律.SSR位点的重复次数主要发生在6～10次和11～15次重复之间,发生频率分别为38.59％和36.02％.本研究对缺须盆唇鱼的转录组数据进行测序鉴定,得到了数量较多的SSR标记,同时对得到的缺须盆唇鱼SSR数据特点进行归纳总结.本研究不仅提供了一个有价值的转录组资源,而且建立了一套SSR标记,可以为缺须盆唇鱼的基础研究、应用研究提供有效的分子标记.     

光唇鱼消化道的形态结构特征

采用解剖及石蜡切片显微技术,观察研究了光唇鱼消化道的形态结构特征。消化道由口咽腔、食道、肠构成。口下位、马蹄形,无颌齿,具咽齿,齿式为4/4。舌较小,前端游离,舌粘膜表层为复层鳞状上皮,有较多的杯状细胞和味蕾。食道及肠均由粘膜层、粘膜下层、肌层及外膜构成。食道内皱襞发达,粘膜层有大量杯状细胞。肠道盘曲,由前、中、后肠组成,肠长/体长为1.84±0.24;前肠管腔较大,中、后肠管腔渐变小;前、中肠皱襞及纹状缘比后肠发达;前肠及后肠杯状细胞较少,中肠杯状细胞较多。光唇鱼消化道的形态结构特征与其食性相适应。     

黄颡鱼微卫星标记的筛选及三个野生群体的遗传结构分析

为了探明长江中上游流域黄颡鱼野生群体的遗传多样性状况和遗传结构,本研究采用磁珠富集法筛选出10个黄颡鱼微卫星标记,并利用其对长江中上游流域3个黄颡鱼(Pelteobagrus fulvidrac)野生群体(赤水群体、乐山群体、洞庭群体)的遗传结构进行分析。获得65个阳性克隆并测序,得到36个含有微卫星的序列,设计并合成了22对微卫星引物,经筛选得到10对多态性稳定的引物,其中高多态性位点6个,中多态位点3个。每对引物等位基因数2-9个,平均4.8。3个群体(赤水、乐山和洞庭)的平均多态信息含量(PIC)分别为0.5438、0.4568和0.3965,平均有效等位基因(Ne)分别为2.9928、2.5401和2.1713,平均期望杂合度(He)分别为0.6280、0.5277和0.4757,表明这3个群体遗传多样性水平较高,其中赤水群体遗传多样性最高,洞庭群体最低。种群分化指数(Fst)和遗传距离(Ds)分析表明,洞庭群体和乐山群体之间的亲缘关系最近,而与赤水群体的亲缘关系最远。聚类分析显示,乐山群体和洞庭群体聚为一支,赤水群体单独聚为一支。     

Isolation and Characterization of the Bovine Microsatellite Loci

Microsatellite loci were isolated using five repetitive probes for Korean native cattle. Eleven microsatellite loci were developed based on a biotin hybrid capture method, and enrichment of the genomic libraries (AAAT, TG, AG, T, and TGC repeats) was performed using Sau3AI adapters. The isolated markers were tested in two half-sib Korean cattle families and four imported breeds (Angus, Limousine, Holstein, and Shorthorn). Nine informative microsatellite loci were observed, and two microsatellite loci were revealed as monomorphic in Korean cattle. In the imported breeds, however, all of the markers were informative. In total, 213 alleles were obtained at the 11 loci across five breeds, and the average number of alleles found per locus, considering all populations, was 4.26. Heterozygosity was 0.71 (expected) and 0.57 (observed). The range of the polymorphic information content for the markers in all cattle populations was 0.43-0.69. Eleven percent of genetic variation was attributed to differentiation between populations as determined by the mean F (ST) values. The remaining 89% corresponded to differences among individuals. The isolated markers may be used to identify and classify the local breeds on a molecular basis.     

Isolation and Characterization of Twelve Microsatellite Loci for Rockfish (Sebastes)

We describe the first microsatellites for rockfishes in the diverse genus Sebastes. Clones containing microsatellites were isolated from the genomic library of a quillback rockfish, Sebastes maliger. Twelve microsatellites are characterized; six of these are polymorphic in quillback rockfish, and eight are polymorphic in at least one rockfish species on which they were tested. The number of alleles per variable locus ranged from 4 to 15 and averaged 6.8. The expected heterozygosities ranged from 0.38 to 0.79 and averaged 0.60 in these loci. These loci should prove valuable in studies examining species identification, population genetics, hybridization, paternity, kinship, and microsatellite evolution. Received September 8, 1998; accepted November 23, 1998.     

Isolation and Characterization of Microsatellite Loci for Striped Bass (Morone saxatilis)

Genetic variation has been difficult to detect in striped bass (Morone saxatilis). Therefore, we identified and characterized 13 microsatellite loci to provide additional genetic markers for striped bass. Microsatellites were identified by screening a striped bass genomic library or by using primers developed for European sea bass (Dicentrarchus labrax) microsatellite loci. We found that 6 of the 13 microsatellite loci were polymorphic in DNA samples obtained from wild populations of striped bass. The number of alleles per locus varied from 3 to 12, and the observed heterozygosities ranged from 0.55 to 0.78. These results indicate that microsatellite loci provide more alleles and higher heterozygosities than other genetic markers developed for striped bass. Received November 9, 1999; accepted February 11, 2000.     

Isolation and Characterization of Four Microsatellite Loci in the Tidewater Goby (Eucyclogobius newberryi)

The first microsatellite loci for the endangered tidewater goby Eucyclogobius newberryi are described. A partial tidewater goby genomic library was constructed in pUC19 plasmid. Microsatellite-containing clones were isolated, sequenced, and amplified. Out of 12 positive clones sequenced, 3 were polymorphic and proved useful in a preliminary genetic screen of 3 well-separated populations (50 individuals each) along the central California coast. The number of alleles per locus ranged from 2 to 3. This initial study suggests a population bottleneck had occurred in one of the populations. Received May 30, 2000; accepted September 14, 2000.     

Isolation and Characterization of Twenty Microsatellite Loci in Japanese Sea Cucumber (Stichopus japonicus)

Twenty microsatellite markers were first developed from the Japanese sea cucumber Stichopus japonicus using an enrichment protocol. Of the 20 microsatellite loci, 19 loci were polymorphic in the population examined. At these polymorphic loci, the number of alleles per locus varied from 2 to 15, and the observed heterozygosities ranged from 0.03 to 0.97, which is considerably higher than those previously found for allozymes. The high variability of the microsatellite markers identified in this study will make them excellent tools for genetic analyses of S. japonicus.     

磁珠富集法筛选大弹涂鱼CA微卫星序列

以大弹涂鱼(Boleophthalmus pectinirostris)基因组DNApool为材料,构建基因组PCR文库,采用链霉亲和素包被的磁珠富集法筛选目的片段.目的片段经克隆、测序验证后获得大弹涂鱼微卫星序列.在筛选的409个菌落中共获得209个阳性克隆,其中具有144个微卫星序列(GenBank登录号为HQ852252 - HQ852395),除去重复测序和侧翼链不足的序列,可以设计引物的微卫星序列有117条.     

藏鸡微卫星文库的构建与微卫星标记筛选

通过磁珠富集法构建了藏鸡基因组微卫星富集文库,分离微卫星序列并对其进行分析.将藏鸡基因组DNA经Sau3AI酶切后纯化回收,连接特定接头.用生物素标记的(CA)12探针与藏鸡基因组酶切回收片段杂交,捕获200～900 bp片段,随后将获得的片段连接到pMD 18-T载体上,转化至JM109中,成功构建藏鸡微卫星富集文库.从1200个转化子中获得了353个阳性克隆,随机挑选53个测序,根据测序结果成功设计了18对藏鸡微卫星引物,最终筛选出6个具有多态性的微卫星标记,其PIC值均大于0.5,同时可以用于研究藏鸡遗传多样性.实验结果表明磁珠富集法能够有效提高分离微卫星标记的效率.     

Isolation and Characterization of 14 Polymorphic Microsatellite Loci in the Big-Headed Turtle (Platysternon megacephalum)

The big-headed turtle (Platysternon megacephalum) is critically endangered because of overharvesting, illegal trade, and habitat destruction. Assessment of genetic variability in existing populations becomes very important to the taxonomy and conservation of this species. Here we describe 14 microsatellite loci isolated from an enriched genomic library of the big-headed turtle, and the polymorphisms of these loci were assessed in 28 individuals from Huizhou, Heyuan, Zhaoqing, and Shaoguan of Guangdong, China. The range of polymorphism information content is 0.305–0.738, and no evidence of significant linkage disequilibrium was found among any pairs of loci. These 14 new polymorphic microsatellite loci can be used in population genetics, taxonomy, phylogeography, behavior ecology, and conservation efforts of Platysternon megacephalum.