A centromeric satellite sequence in the Pacific oyster (Crassostrea gigas Thunberg) identified by fluorescence in situ hybridization

A highly repetitive satellite sequence was previously identified in the Pacific oyster Crassostrea gigas Thunberg. The sequence has 168 bp per unit, present in tandem repeats, and accounts for 1% to 4% of the genome. We studied the chromosomal location of this satellite sequence by fluorescence in situ hybridization (FISH), A probe was made by polymerase chain reaction and incorporation of digoxigenin-11-dUTP. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. FISH signals were located at centromeric regions of 7 pairs of the Pacific oyster chromosomes. No interstitial site was found. Signals were strong and consistent on chromosomes 1, 2, 4, and 7, but weak or variable oil chromosomes 5, 8, and 10. No signal was observed on chromosomes 3, 6, and 9. Our results showed that this sequence is clearly a centromeric satellite, disputing its previous assignment to the telomeric and submetacentric regions of 2 chromosomes. No signal was detected in the American oyster (Crassostrea virginica Gmelin).

Chromosome inheritance in triploid Pacific oyster Crassostrea gigas thunberg

Reproduction and chromosome inheritance in triploid Pacific oyster (Crassostrea gigas Thunberg) were studied in diploid female x triploid male (DT) and reciprocal (TD) crosses. Relative fecundity of triploid females was 13.4% of normal diploids. Cumulative survival from fertilized eggs to spat stage was 0.007% for DT crosses and 0.314% for TD crosses. Chromosome number analysis was conducted on surviving progeny from DT and TD crosses at 1 and 4 years of age. At Year 1, oysters from DT crosses consisted of 15% diploids (2n = 20) and 85% aneuploids. In contrast, oysters from TD crosses consisted of 57.2% diploids, 30.9% triploids (3n = 30) and only 11.9% aneuploids, suggesting that triploid females produced more euploid gametes and viable progeny than triploid males. Viable aneuploid chromosome numbers included 2n + 1, 2n + 2, 2n + 3, 3n - 2 and 3n - 1. There was little change over time in the overall frequency of diploids, triploids and aneuploids. Among aneuploids, oysters with 2n + 3 and 3n-2 chromosomes were observed at Year 1, but absent at Year 4. Triploid progeny were significantly larger than diploids by 79% in whole body weight and 98% in meat weight at 4 years of age. Aneuploids were significantly smaller than normal diploids. This study suggests that triploid Pacific oyster is not completely sterile and cannot offer complete containment of cultured populations.

AFLP-based genetic linkage maps of the Pacific oyster Crassostrea gigas Thunberg

Amplified fragment length polymorphisms (AFLPs) were used for genome mapping in the Pacific Oyster Crassostrea gigas Thunberg. Seventeen selected primer combinations produced 1106 peaks, of which 384 (34.7%) were polymorphic in a backcross family. Among the polymorphic markers, 349 were segregating through either the female or the male parent. Chi-square analysis indicated that 255 (73.1%) of the markers segregated in a Mendelian ratio, and 94 (26.9%) showed significant (P < 0.05) segregation distortion. Separate genetic linkage maps were constructed for the female and male parents. The female framework map consisted of 119 markers in 11 linkage groups, spanning 1030.7 cM, with an average interval of 9.5 cM per marker. The male map contained 96 markers in 10 linkage groups, covering 758.4 cM, with 8.8 cM per marker. The estimated genome length of the Pacific oyster was 1258 cM for the female and 933 cM for the male, and the observed coverage was 82.0% for the female map and 81.3% for the male map. Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.

Chromosome segregation in fertilized eggs from triploid Pacific oysters, Crassostrea gigas (Thunberg), following inhibition of polar body 1

Chromosome segregation in fertilized eggs from triploid Pacific oysters, following inhibition of the first polar body (PB1), was studied with acetic orcein staining techniques. To block the release of PB1, fertilized eggs were treated with 0.5 mg/l of cytochalasin B (CB). Four types of segregation were observed, namely, ''tripolar segregation'' (54.5%), ''united bipolar segregation'' (12%), ''separated bipolar segregation'' (2.5%), and ''incomplete united bipolar segregation'' (4%). The remaining 23% could not be classified because of chromosome disorganization, but appeared to be variants of the above. It seemed clear that the predominant pattern that gave rise to tetraploids was united bipolar segregation, although certain separated bipolar segregations might also lead to the formation of tetraploids. The sequential events of meioses observed in CB-treated eggs are described. The asynchrony of meiotic events and possible mechanisms for the various types of chromosome segregation are discussed.

川乌与贝母配伍禁忌的电喷雾质谱研究

The ESI-MS analytical method was established for studying the alkaloids content in the decoctions of Radix aconiti combined with Bulbus Fritillariae Thunbergii or bulbus fritillariae Cirrhosae.For the co-decoctions of Radix aconiti combined with Thunberg Fritillary Bulb contains more diester-alkaloids than that of co-decoctions of Radix aconiti combined with Bulbus Fritillariae Cirrhosae.When Radix aconiti was combined with Thunberg Fritillary Bulb,diester-alkaloids were mainly dissolved in water.But diester-alkaloids were mainly converted into lipo-alkaloids when Radix aconiti was combined with Bulbus Fritillariae Cirrhosae.

Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca)

Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of Crassostrea: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)-the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)-the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of Crassostrea. This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.

亚历山大藻对海产贝类胚胎和蒙古裸腹溞的影响及致毒机制研究

本文研究了典型有毒赤潮藻——亚历山大藻(Alexandrium)对海湾扇贝(Argopecten irradians Lamarck)、文蛤(Meretrix meretrix Linnaeus)和太平洋牡蛎(Ostrea gigas Thunberg)受精卵孵化的影响和致毒机制以及对蒙古裸腹溞(Moina mongolica Daday)生命活动的影响。此外，还针对我国赤潮发生特点，模拟研究了我国东海大规模赤潮对菲律宾蛤仔（Ruditapes philippinarum (Adams et Reeve)）受精卵孵化和蒙古裸腹溞种群数量的影响。 结果发现：8株产PSP毒素的亚历山大藻：塔玛亚历山大藻( ATHK, AT5-1, AT5-3, ATCI02, ATCI03)，链状亚历山大藻, A. lusitanicum、微小亚历山大藻和2株不产PSP毒素的相关亚历山藻(AC-1, AS-1)对海湾扇贝受精卵的孵化均有显著抑制作用，说明在亚历山大藻属中，这种抑制作用具有一定的普遍性，并与PSP毒素的产生无直接关系，表明存在非PSP毒素的其它毒性物质。一种PSP标准毒素STX也没有这种抑制作用，进一步证明该抑制作用与PSP毒素不直接相关。 相关亚历山大藻AC-1对海湾扇贝、文蛤和太平洋牡蛎受精卵孵化的有显著的毒害作用，其藻液、重悬液、去藻液和内容物均显著影响受精卵的孵化。相关亚历山大藻AC-1对海湾扇贝、文蛤和太平洋牡蛎担轮幼虫细胞的超微结构有显著破坏作用，破坏膜结构和胞内结构，影响细胞内的功能器官如溶酶体的稳定性，使卵黄颗粒萎缩变形；对文蛤和太平洋牡蛎的受精卵显示出极强的毒害作用： 3000cells•ml-1时，使二者胚胎完全溶掉消失；在2000cells•ml-1的藻液中培养2h后，担轮幼虫的外膜发生溶解，整个幼体呈葡萄串样。相关亚历山大藻AC-1产生的这种毒性物质可能对贝类胚胎细胞的结构和功能有影响。 亚历山大藻对蒙古裸腹溞的毒性效应与不同藻种/藻株密切有关：塔玛亚历山大藻（AT-6, ATCI02）、链状亚历山大藻、A. lusitanicum和微小亚历山大藻不影响蒙古裸腹溞的存活，而塔玛亚历山大藻（ATHK、ATCI03和AT5-1）和相关亚历山大藻(AC-1, AS-1)有显著影响。蒙古裸腹溞能摄食塔玛亚历山大藻(AT-6, ATHK, ATCI02, ATCI03, AT5-1)，链状亚历山大藻, A. lusitanicum和微小亚历山大藻，很少或基本不摄食相关亚历山大藻。亚历山大藻影响蒙古裸腹溞的RNA/DNA比值和蛋白质含量以及Na+,K+-ATP酶活性。相关亚历山大藻AC-1对蒙古裸腹溞的存活有极强的毒性作用，藻液、重悬液、内容物和碎片均有显著影响；即使与3×106cells•ml-1小球藻混合，10和50cells•ml-1的相关亚历山大藻AC-1仍能使蒙古裸腹溞的产幼数和存活时间显著下降。亚历山大藻对蒙古裸腹溞生命活动的影响不仅与PSP毒素有关，还与非PSP毒素有关；蒙古裸腹溞可能也是研究有害藻急性和慢性毒性的一种理想生物。 应用菲律宾蛤仔胚胎和蒙古裸腹溞评价我国东海特大规模赤潮对海洋生物资源的潜在危害时发现：单种链状亚历山大藻对菲律宾蛤仔受精卵的孵化和蒙古裸腹溞的种群增长均有显著不利影响；单种东海原甲藻（1~10×104cells•ml-1）对菲律宾蛤仔受精卵的孵化没有影响；较低密度的东海原甲藻能维持蒙古裸腹溞（2~5×104cells•ml-1）的种群增长；较高密度的东海原甲藻对蒙古裸腹溞（10×104cells•ml-1）种群有显著的抑制作用。两种藻以赤潮密度混合后，适当密度的东海原甲藻能在一定程度上减轻链状亚历山大藻对菲律宾蛤仔受精卵和蒙古裸腹溞的毒性。可见，东海连年爆发的大规模赤潮不仅对浮游生态系统有不利影响，若同时爆发亚历山大藻赤潮，则对海洋浮游生态系统和贝类资源的恢复产生更加不利的影响。

长牡蛎可存活四倍体的诱发机制和异源三倍体的诱发

采用乙酸地衣红染色技术（Acetic orcein staining technique）较系统地研究了长牡蛎 Crassostrea gigas (Thunberg)三倍体产生的卵子在受精且第一极体释放受抑制后的减数分裂过及染色体的分离行为以阐明可存活四倍体的产生体的机制。用浓度为0.5 mg/L的细胞松驰素B （CB）处理受卵以抑制其第一极体的释放。在观察到个别受精卵出现第一极体时开始CB处理，持续至对照组中50％的受精卵出现的第一极体。对处理组和对照组的受精卵从受精后隔5分钟取样一次，用卡诺氏液（Carnoy's fixative, 冰醋酸和甲醇按1：3的体积比充分混合）固家样品。采用0.5%的乙酸－地衣红染料进行受精卵的染色，而后压片观察受精卵染色体行为。长牡蛎三倍体产生的卵子，中期I同源染色体构型呈现单价体（Univalents）,二价体（Bivalents）、三价体（Trivalents）以及大于三价体的多价体（Multivalents）混合出更的特征。在第一极体释放受抑制的受精卵的第二次减数分裂过程中，可确认四种染色体分离类型：三极分离（Tripolar segregation） (54.5%)、联合二极分离（United bipolar segregation） (12%)、独立二级分离（Incomplete united bipolar segregation）(4%)。其余卵子的染色体分离行为（23％）不规律，呈现不同程度的紊乱，但总体看来介于上述四种分离类型之间。此外，某些特定的独立二级也可能是四位体形成的最主要的细胞遗传学体制。此外，某些特定的独立二极分离也可能产生四倍体。轻细胞体驰素B 处理的受精卵的减数分裂过程具有显著的不同步性，表现在三个方面：第一，在两个重复组之间，即两个雌体之间，存在第二次减数分裂的时间进程的不同步性；第二，同一个雌体产生的卵子之间的发育速度不同步性，表现为不同的卵子进入第一次减数分裂的时间不同；第三，同一卵子内的染色体之间，其行为有时存在的不同步性。另外，探讨了中心类在支配第二减数分裂时各种染色体分离行为的可能机制。以长牡蛎二倍体与近江牡蛎二倍体的染交作为对照，探讨了能长牡蛎四倍体与近江牡蛎二倍体杂并诱导异源三倍体的可行性。长牡蛎Crassostrea gigas (Thunberg)四倍体和二倍体与近江牡蛎Crassostrea rivularis (Gould)二倍体的杂交以及相应的对照组共进行了三批重复实验，杂交实验采用高密度的精子。研究结果表明，自交组平均受精率依次为94％（GG）、77％ （RR）,88% （G／GG）和85％ （GG／G）。双方差分析（ANOVA）表明，各自交组之间受精率没有显著差异（F＝3.118, P=0.132）。在杂交组，直接授精后180分钟，尚未观察到受精迹象，因而无法估计受精率。授精后48小时的孵化率各组之间差异很大，并经双方差分析（ANOVA）表明存在显著性差异，（F＝3.188, P=0.018）。其中GGR和RGG组的孵化率相近似，产生的幼虫数量明显少于对照组。在四种类型的杂交实验中，二倍体C. gigas (雌体) ＊ 二倍体 C. rivularis (雌体)（ＧＲ）早最成功的。虽基ＧＲ组幼虫的生长率低于对照组，但其存活率接近于对照组。长牡蛎四倍体与近江牡蛎二倍体杂交组（GR），在授精后两天的孵化率较低，但幼虫的生长状况与对照组接近。另外两个杂交组，即近江牡蛎二倍体与长牡蛎四倍体（RGG），二倍体近江牡蛎江与二倍体长牡蛎（RG），授精后两天的孵化率很低，幼虫生长得缓慢。三个重复组的GR杂交组和一个重复组的GGR杂交组获得稚贝。聚合酶链式反应／限制性酶切片段长度的多态性（PCR／PFLP）检支分析结果证实这些稚贝均是杂交种；流式细胞术分析结果证明GGR获得的稚贝是三倍体，从而证明获得了长牡蛎与近江牡蛎的异源三倍体。有迹象表明三倍体与二倍体杂交种之间（GGR对GR）存在生长上的差异。首先，GGR的眼点幼虫大约比GR组早出现5－7天即仅次于对照组GG，G／GG，和GG／G；第二，尽管仅获得少量GGR幼贝，这些幼贝在授精后90天的大小显著大于GR组的个体。在RGG和RG组中，幼虫没能存活到眼点幼点阶段。细胞学检查结果表明，杂交组的绝大多数卵子发育停滞在第一次减数分裂中期（Metaphase I）,这一过程至少持续到授精后180分钟。仅有2％的GGR 组的卵子在授精后180分钟进入第一次减数分裂后期）（Anaphase I）. 而在此时期，GR，RGG和RG组的卵子中，仍只观察到10第二价体（Bivalents）.

Chromosomal rearrangement in Pectinidae revealed by rRNA loci and implications for bivalve evolution

Karyotype and chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes were studied in two species of Pectinidae, zhikong (Chlamys farreri) and bay (Argopecten irradians irradians) scallops. using fluorescence in situ hybridization (FISH). C. farreri had a haploid number of 19 with a karyotype of 3m + 4sm + 7sm-st + 4st + 1st-t, and A. i. irradians had a haploid number of 16 with a karyotype of 5st + 11t. In C. farreri, the major and minor rRNA genes had one locus each and were mapped to the same chromosome-Chromosome 5. In A. i. irradians, the major rRNA genes had two loci, located on Chromosomes 4 and 8, and the 5S rRNA gene was found at a third chromosome-Chromosome 10. Results of this and other studies indicate that karyotype of A. i. irradians (n = 16, 21 arms) is secondary and derived from an ancestral karyotype similar to that of C. farreri (n = 19, 38 arms) through considerable chromosomal loss and rearrangements. The ability to tolerate significant chromosomal loss suggests that the modal karyotype of Pectinidae and possibly other bivalves with a haploid number of 19 is likely tetraploid; i.e., at least one genome duplication has occurred during the evolution of Bivalvia.

Classification of common oysters from North China

Oysters are commonly found on rocky shores along China's northern coast, although there is considerable confusion as to what species they are. To determine the taxonomic status of these oysters, we collected specimens from nine locations north of the Yangtze River and conducted genetic identification using DNA sequences. Fragments from three genes, mitochondrial 165 rRNA, mitochondria! cytochrome oxidase I (COI), and nuclear 285 rRNA, were sequenced in six oysters from each of the nine sites. Phylogenetic analysis of all three gene fragments clearly demonstrated that the small oysters commonly found on intertidal rocks in north China are Crassostrea gigas (Thunberg, 1793), not C. plicatula (the zhe oyster) as widely assumed. Their small size and irregular shell characteristics are reflections of the stressful intertidal environment they live in and not reliable characters for classification. Our study confirms that the oysters from Weifang, referred to as Jinjiang oysters or C. rivularis (Gould, 1861), are C. ariakensis (Wakiya, 1929). We found no evidence for the existence of C. talienwhanensis (Crosse, 1862) and other Crassostrea species in north China. Our study highlights the need for reclassifying oysters of China with molecular data.