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.     

Species identification of <Emphasis Type="Italic">Pinctada imbricata</Emphasis> using intergenic spacer of nuclear ribosomal RNA genes and mitochondrial 16S ribosomal RNA gene regions

ABSTRACT:   For pearl production, pearl oyster seeds from foreign pearl oysters as well as hybrids between native and such foreign pearl oysters are produced in Japanese hatcheries. However, it is very difficult to identify these pearl oysters and hybrids based on morphological measurements. Thus, a molecular identification method for distinguishing Atlantic pearl oysters Pinctada imbricata from the Indian-Pacific pearl oyster group including P. martensii and P. fucata , was developed. The polymerase chain reaction (PCR) products of the partial intergenic spacer (IGS) of nuclear ribosomal RNA (rRNA) genes exhibited length polymorphism between P. imbricata (590 bp) and the other two species (427 bp). The restriction fragment length polymorphism analysis of the PCR products (PCR-RFLP) cleaved with Mse  I observed in the IGS of nuclear rRNA genes also gave different profiles between P. imbricata and the other two species. The difference in PCR-RFLP using Alu  I was also detected in the mitochondrial 16S rRNA gene regions between P. imbricata and the other two species. Thus, the method developed enables the distinction of P. imbricata from P. martensii and P. fucata .     

合浦珠母贝三倍体的卵诱导四倍体

将合浦珠母贝三倍体的卵与二倍体的精子授精,用０．５μｇ／ｍＬ细胞松弛素Ｂ抑制精卵第一极体的释放诱导四倍体。研究了处理起始时间及持续时间对胚胎孵化率和四倍体诱导率的影响及幼虫的生长及存活。实验结果表明：持续时间与胚胎孵化率呈负相关,而与四倍体诱导率呈正相关,持续时间一般为１５￣１８ｍｉｎ,处理起始时间一般在第一极体出现前３￣５ｍｉｎ。在胚胎期,四倍体诱导率平均为２０％。在幼虫培养阶段,幼虫死亡严重,     

黑壳病马氏珠母贝贝肉微量元素及挥发性成分分析

采用湿法消解和索氏提取法＋水蒸气蒸馏法分别处理黑壳病马氏珠母贝（Pinctada martensii）贝肉样品。利用电感耦合等离子体质谱法（ICP—AES）检测样品中12种主要微量元素的质量分数,气质联用色谱法（GC—MS）检测其挥发性成分。结果表明,与健康贝贝肉相比,黑壳病贝贝肉中微量元素除锌（Zn）和硒（Se）的质量分数有明显变化（下降率分别为8．54％和10．98％）外,其他元素没有明显差异;病贝中含有的挥发性物质（16种）较健康贝的少（20种）,并含有金属元素、大量的苯环等。     

Studies on aneuploid pearl oyster (Pinctada martensii Dunker) produced by crossing triploid females and a diploid male following the inhibition of PB1

Viable aneuploid embryos of pearl oyster, Pinctada martensii Dunker, were produced by inhibiting the first polar body (PB1) with cytochalasin B treatment in eggs from triploids fertilized with haploid sperm. During the period of growth measurement, aneuploid showed the slowest growth compared with diploid and triploid groups. The body size and weight measurement data showed that there were no differences between aneuploids (as a group) and diploids in body size and weight (P>0.10), but that they were significantly different from triploids (P<0.01). There were no differences between aneuploids within diploid condition (2n±n) and diploids in SL (P>0.1), but significant differences in BW were found (P<0.05). Aneuploids within triploid condition (3n±n) were significantly smaller than triploids in BW (P<0.05), but not different from triploids in SL (P>0.05), and almost identical to diploids in both (P>0.1). These dada indicated that some aneuploids might be associated with growth retardation. Karyotype analysis revealed that there were metacentric, submetacentric or subtelocentric chromosomes losses or gains; aneuploid pearl oysters with the same chromosome numbers had different chromosome composition. Aneuploids are valuable research materials for genetic analysis.     

近交对马氏珠母贝生长性状、遗传多样性及矿化基因表达的影响

[目的]探讨近交对马氏珠母贝生长性状、遗传结构及矿化基因表达的影响,阐明近交引起子一代性状退化的遗传原理,为贝类品种培育提供参考依据.[方法]以两个黄壳色全同胞家系(M和N)为亲本,按照因子设计构建4个家系组合(F1:M1♀ ×M1♂;F2:M1♀ ×N1♂;F3:N1♀ ×M1♂;F4:N1♀×N1♂),比较4个家系成贝生长性状差异,利用9对SSR引物分析其遗传多样性,并以实时荧光定量PCR检测家系间矿化基因nacrein、pearlin和pif177的表达差异.[结果]4个家系间的平均壳长、平均壳高、平均壳宽、平均体重和平均壳重存在明显差异,且近交家系(F1和F4)的平均壳长、平均壳高、平均壳宽、平均体重和平均壳重均小于杂交家系(F2和F3),其生长性状退化率(Ⅲ)为11.7%~29.4%.家系F1、F2、F3和F4的平均观测杂合度分别为0.2525、0.3116、0.3178和0.2752,平均期望杂合度分别为0.3451、0.3822、0.4005和0.3549,平均多态信息含量(PIC)分别为0.4591、0.5389、0.4878和0.4810.杂交家系(F2和F3)矿化基因nacrein、pearlin和pif177的相对表达量均高于近交家系(F1和F4),且4个家系的生长性状平均值与3个矿化基因的相对表达量存在明显正相关.[结论]近交能导致马氏珠母贝生长性状、遗传多样性及矿化基因表达量明显降低,因此制定马氏珠母贝育种方案时应构建合理的交配组合系统,防止因近交导致性状退化与遗传多样性指标降低而影响育种进程.     

马氏珠母贝4种壳色选育系F3的生长特性和遗传多样性比较

以随机扩增多态性DNA (RAPD)分析为实验手段,结合形态学特征统计,对经过3代壳色及生长速度群体选育的马氏珠母贝(Pinctada martensiiDunker)黑、白、红、黄4种壳色选育系的生长及遗传多样性进行研究,为进一步选育提供理论依据。结果显示:马氏珠母贝4种壳色F₃选育系的壳色纯化率分别达到黑壳色95.83%、白壳色88.33%、红壳色100%、黄壳色95.0%;各选育系的生长速度均大于普通养殖群体,系间的生长速度也存在显著差异。4种壳色选育系平均多态性位点比率(P)分别为82.51%,81.93%,75.30%,77.03%;平均Nei’s基因多样性指数(H)分别为0.233 6,0.213 4,0.196 5,0.201 9;平均Shannon信息指数(Hi)分别为0.351 6,0.323 0,0.279 9,0.305 9;各系间的遗传分化系数(G_st)为0.214 3~0.318 6,遗传距离(Dxy)分别为黑白0.113 5,白黄0.130 3,黑黄0.134 9,白红0.154 6,红黄0.158 4,黑红0.196 8;聚类分析表明各选育系间的亲缘关系由近及远顺次为黑壳色、白壳色、黄壳色和红壳色。研究表明4种壳色选育系均具有较高的遗传多样性,选育系间遗传分化明显。研究结果为4种壳色马氏珠母贝的定向选育奠定了理论基础。     

马氏珠母贝育珠前后矿物元素的变化

采用电感耦合等离子体发射光谱法对育珠前后马氏珠母贝外套膜和珍珠囊中的Ca、Cu、Fe、Mg、Zn等8种矿物元素的含量进行分析测定,旨在通过对育珠前后外套膜和珍珠囊中8种重要的矿物元素含量的变化来探究珍珠形成机理。比较结果得出:外套膜中,Ca、Fe、Mg、Mn、Zn等5种元素在育珠后都发生了显著变化,Ca、Mg和Zn上升率分别为46.6%、30.12%和132.80%;珍珠囊中,Cd、Fe、Mg、Mn、Zn、Co等元素的含量在育珠前后存在显著差异,Zn含量变化最明显,上升达155.18%。     

马氏珠母贝TNFR27基因克隆与功能初探

肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)是一类重要的细胞因子受体,主要参与细胞凋亡、宿主免疫防御、炎症等生物过程。本研究采用cDNA末端快速扩增(RACE)技术克隆了马氏珠母贝TNFR27(PmTNFR27)的cDNA全长并进行生物信息学分析,运用实时荧光定量PCR(qPCR)技术检测了PmTNFR27 mRNA在马氏珠母贝不同组织、脂多糖(LPS)、聚肌胞苷酸(Poly(I:C))及镉胁迫后的表达模式。结果显示：PmTNFR27 cDNA全长为1 524 bp,5′UTR长为186 bp,3′UTR长为248 bp,包含28 bp的poly(A)尾巴,开放阅读框(ORF)为1 062 bp,编码353个氨基酸;结构域预测表明PmTNFR27具有一个典型的CRD结构域和一个跨膜蛋白结构域,符合肿瘤坏死因子受体超家族特征;多序列比对结果表明贝类种间的相似性不高,但功能结构域位置较保守。系统进化树结果显示马氏珠母贝与其他贝类聚为一簇。qPCR结果显示,PmTNFR27 mRNA在马氏珠母贝各组织中均有表达,在鳃中表达量最高。LPS刺激后,PmTNFR27基因在鰓中的表达量于3 h达到最高,于72 h降到最低;Poly(I:C)刺激后,PmTNFR27基因在鰓中的表达量在6 h、12 h显著上升并达到最高至96 h时降到最低。镉胁迫后,3 h表达量达到最高,24 h、48 h表达量显著下降。研究结果显示PmTNFR27可能参与了马氏珠母贝的免疫应答反应。     

海产鱼类多倍体育种的研究

为了探究不同盐度对诱导合浦珠母贝三倍体的效果,本研究首次利用低渗方法诱导合浦珠母贝三倍体,通过控制变量法从不同盐度、诱导时机以及诱导时间中探究最适诱导条件。同时对合浦珠母贝卵裂率、孵化率以及存活率、壳长、三倍体率的变化进行了探究。结果显示,在盐度14、50%受精卵释放第一极体 (PBⅠ)及诱导15 min时D形幼虫的三倍体率最高,分别为64.16%±6.92%、65.87%±6.51%以及65.14%±1.93%。三倍体幼虫并未表现出明显优势;低渗对胚胎造成的影响使幼虫存活率和三倍体率有所下降。通过对卵裂率、孵化率和15日龄存活率、壳长及三倍体率进行主成分分析,在三种诱导条件中均保留了前两个主成分,且保留的主成分的累积方差贡献率均超过了85%。盐度14、50%受精卵释放PBⅠ及诱导15 min分别在各自实验中的综合评价得分分别为第6、第1和第1。该研究表明,合浦珠母贝三倍体幼虫在生长方面的优势不明显,50%受精卵释放PB Ⅰ及诱导15 min两个诱导条件适合合浦珠母贝三倍体诱导。本研究可为合浦珠母贝三倍体育种提供一种新的思路,有助于提高生产效益。