Characterization of eastern oyster (Crassostrea virginica Gmelin) chromosomes by fluorescence in situ hybridization with bacteriophage P1 clones

Chromosome identification is an essential step in genomic research, which so far has not been possible in oysters. We tested bacteriophage P1 clones for chromosomal identification in the eastern oyster Crassostrea virginica, using fluorescence in situ hybridization (FISH). P1 clones were labeled with digoxigenin-11-dUTP using nick translation. Hybridization was detected with fluorescein-isothiocyanate-labeled anti-digoxigenin antibodies and amplified with 2 layers of antibodies. Nine of the 21 P1 clones tested produced clear and consistent FISH signals when Cot-1 DNA was used as a blocking agent against repetitive sequences. Karyotypic analysis and cohybridization positively assigned the 9 P1 clones to 7 chromosomes. The remaining 3 chromosomes can be separated by size and arm ratio. Five of the 9 P1 clones were sequenced at both ends, providing sequence-tagged sites that can be used to integrate linkage and cytogenetic maps. One sequence is part of the bone morphogenetic protein type 1b receptor, a member of the transforming growth factor superfamily, and mapped to the telomeric region of the long arm of chromosome 2. This study shows that large-insert clones such as P1 are useful as chromosome-specific FISH probes and for gene mapping in oysters.

Chromosomal mapping of 5S ribosomal RNA genes in the eastern oyster, Crassostrea virginica gmelin by fluorescence in situ hybridization

Chromosomal location of the 5S ribosomal RNA gene was studied in the eastern oyster, Crassostrea virginica Gmelin. using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos, and the FISH probe was made by PCR (polymerase chain reaction) amplification of the 5S rRNA gene and labeled by incorporation of digoxigenin-1 1-dUTP during PCR. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. Two pairs of FISH signals were observed on metaphase chromosomes. Karyotypic analysis showed that the 5S rRNA gene cluster is interstitially located on short arms of chromosomes 5 and 6. On chromosome 5, the 5S rRNA genes were located immediately next to the centromere, whereas on chromosome 6, they were located approximately half way between the telomere and the centromere. Chromosomes of C. virginica are difficult to identify because of their similarities in size and arm ratio, and the chromosomal location of 5S rRNA genes provides unambiguous identification of chromosomes 5 and 6. Previous studies have mapped the major rRNA gene cluster (18S-5.8S-28S) to chromosome 2. and this study shows that the 5S rRNA gene cluster is not linked to the major rRNA genes and duplicated during evolution.

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.

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).

牡蛎染色体的分子生物学分析

本研究应用显带技术和荧光原位杂交(Fluorescence in situ hybridization，FISH)技术，鉴定了牡蛎的染色体；应用FISH方法定位了一系列的重复序列和大分子的P1克隆DNA；制备了染色体特异性探针。应用FISH特异性探针成功地鉴定了长牡蛎的三体10。结果如下：1．分析了G带和C带在美洲牡蛎染色体上的分布。G带在每一条染色体上的带型不同，某些染色体间(如第1对和第4对染色体，第7对和第9对染色体)的带型差别不是很明显。G带型容易受染色体收缩程度的影响。C带型重复性较好，染色体带型较清楚，分布在染色体的端粒区域和着丝粒区域。G带和C带带型能够用来鉴定牡蛎的染色体，但是重复性低和带型差异不显著，并不适合常规的染色体鉴定。2．早期胚胎和担轮幼虫制备的染色体适合于FISH分析。染色体制备方法重复性好，可适用于其它贝类的染色体制备。3．研究了重复序列基因--rDNA的定位：1)18S-5.8S rDNA在研究的五种巨蛎属Crassostrea牡蛎均只有一个位 点。太平洋种(C.gigas，C. ariakensis和C. plicatula)中，杂交信号位于最短的染色体一第10对染色体长臂的端粒区域，在大西洋种(C. virginica和C. rhizophorae)中，同一序列定位在第2对染色体短臂的端粒区域。2)18S-28S rDNA在两种蛤中有两个位点。rDNA探针定位在侏儒蛤(Mulinis Lateralis)的第15对和第19对染色体的端粒区域，同一序列定位在硬壳蛤(Mercenaria mercenaria)的第10对染色体的长臂和第12对染色体短臂的端粒区域。信号强度在两对染色体之间有差异。 3)5s rDNA位于美洲牡蛎的第5对染色体的短臂上靠近着丝粒区域和第6 对染色体的短臂的中间区域。信号强度在两对染色体之间没有显著差异。5S rDNA探针可以作为鉴定和识别第5对和第6对染色体的特异性探针。4．研究了一些重复序列的定位1)两个短的重复序列1G8，1P2均产生很强的荧光信号分布在美洲牡蛎所有的染色体上。在低严谨条件下，这些序列均产生很强的信号散布在所有的染色体上。在高严谨条件下，信号强度大大减弱，但是信号仍散布在所有的染色体上。这些重复序列散布在美洲牡蛎的整个基因组中。2)高度重复序列Cgl70产生的信号分布在长牡蛎的7对染色体的着丝粒区域，没有发现间区信号。在第1对，第2对，第4对和第7对染色体上的荧光信号强且稳定。在第5对，第8对和第10对染色体上的信号相对弱且不稳定。在剩余的染色体上(第3对，第6对和第9对染色体)没有检测到荧光信号。结果表明此卫星序列是一个着丝粒卫星序列。在美洲牡蛎的染色体上没有检测到荧光信号，表明了这个着丝粒卫星序列在这两种牡蛎中的分布存在着显著的差异。3)脊椎动物端粒序列(TTAGGG)n的FISH信号局限在四种双壳贝类(美洲牡蛎，the mangrove oyster，硬壳蛤，侏儒蛤)所有染色体的端粒区域，没有发现间区信号的存在。研究结果与已报道的研究结果表明脊椎动物端粒序列或许存在于所有双壳贝类的染色体末端。双壳贝类是目前研究过的唯一含有脊椎动物端粒序列DNA的无脊椎动物。4)研究了RAPD探针在美洲牡蛎染色体上的定位。大多数RAPD探针产生了多个信号散布在间期细胞核和所有的染色体上。引物OPX-03，OPX-04，OPX—06，OPG-02，OPM—04，OPM-11，0PS-02制备的探针在适宜的条件下产生特异性荧光 信号，分布在牡蛎的特定的染色体上。PCR特异性带产生的探针OPX—06—310和0PG-02—300产生了特异性的荧光信号：OPX—06—310产生的信号位于第5对染色体的短臂的近端粒区域，0PG—02—300探针定位到第3对染色体的短臂上。这两个探针是鉴定美洲牡蛎单条染色体的特异性探针。5．研究了大分子Pl克隆DNA(插入片断为80～100 kb)在美洲牡蛎染色体上的定位。Pl克隆DNA通过切口平移方法标记digoxigenin—11-dUTP用作FISH的探针。Cot-1 DNA作为竞争剂有效地抑制了Pl克隆序列中的重复序列产生的信号。杂交信号用fluorescein标记的anti—digoxigenin抗体来检测，用两层抗体rabbit-anti-sheep抗体和FITC anti—rabbit抗体来扩增信号。9个P1探针成功地定位在特定的染色体上。46—1探针杂交到第1对染色体的长臂靠近着丝粒区域；47-10探针定位到第2对染色体的长臂近端粒区域；Cvpl和48-13两探针定位到第3对染色体上：Cvpl位于短臂的端粒区域，48-13探针位于长臂的近着丝粒区域；48—10探针杂交到第4对染色体的长臂上；48-1探针杂交到第5对染色体长臂的近着丝粒区域；49-11探针位于第7对染色体长臂上；探针49-10和44-11位于第8对染色体长臂上。同时我们成功地将2个P1探针杂交到同一染色体分裂相中，进一步确定了Pl探针在美洲牡蛎染色体 上的定位。6．应用18S-28S rDNA探针成功地鉴定出长牡蛎非整倍体中的三体10。经鉴定AF-35，AF-39和AF-3三体家系属于三体10家系。rDNA探针分布在三条染色体上，即多出的一条染色体为染色体10。相应地在间期细胞核上有三个信号出现。AF-34和AF-36家系不属于三体10家系。rDNA探针分布在两条染色体上，相应地在间期细胞核上有两个信号出现。FISH和染色体特异性探针为非整倍体的鉴定提供了一个快速准确可靠的方法和途径。

海滨锦葵种子油脂提取及制备生物柴油研究

生物柴油是绿色清洁可再生能源，大力发展生物柴油对解决影响我国经济可持续发展的能源危机和环境危机具有重要意义。 本文就海洋滩涂能源油料植物海滨锦葵油脂的提取和制备生物柴油技术进行了研究。 1、利用超临界CO2流体萃取技术提取海滨锦葵籽油。结果表明超临界CO2流体萃取技术提取海滨锦葵籽油的最佳工艺参数为：萃取压力25MPa，萃取温度45℃，CO2流量18kg.h-1，萃取时间为120min，在该工艺条件下萃取三次，海滨锦葵籽油萃取率达到19.35%。 2、以海滨锦葵籽仁为原料，利用水酶法提取海滨锦葵籽仁油。水酶法提取海滨锦葵籽油的最佳工艺参数为：酶用量0.024ml.g-1，提取温度63℃，固液比1/6，提取时间为230min，在该工艺条件下海滨锦葵籽油提取率达到24.28%。 3、海滨锦葵油制备生物柴油的最佳工艺参数为：搅拌强度为1800r.min-1，催化剂KOH用量为海滨锦葵油质量的1%，醇油摩尔比6/1，反应时间50min，反应温度65℃，在该工艺条件下，酯交换反应三次，酯交换率达到97.8%。 4、利用固定化脂肪酶Novo435催化海滨锦葵油酯交换制备生物柴油。结果表明海滨锦葵油固定化脂肪酶催化法制备制备生物柴油的最优工艺参数为反应温度47℃，反应时间31h，催化剂用量18%，搅拌强度900r.min-1，醇油摩尔比3.2/1。在该工艺条件下酯交换率达到92.68%。 5、利用超临界法制备生物柴油，结果表明海滨锦葵油超临界法制备生物柴油的最佳工艺条件为：反应温度为300℃，反应压力为12MPa，反应时间为9min，搅拌强度为300r.min-1，醇油摩尔比为30/1。在此条件下，酯交换反应三次，酯交换率可达97.62%。 6、利用超声波辅助法制备生物柴油。结果表明海滨锦葵油超声波辅助法制备生物柴油的最佳工艺参数为：超声波功率为180W，催化剂KOH用量为海滨锦葵油质量的0.6%，反应温度65℃，醇油摩尔比7/1，在该工艺条件下酯交换反应三次，酯交换率达到99.85%。

软体动物线粒体基因组学及分子系统发生研究

本研究以双壳纲、翼形亚纲、珍珠贝目、扇贝超科的栉孔扇贝(Chlamys farreri )、海湾扇贝(Argopecten irradians)和牡蛎超科巨蛎属(Crassostrea)的长牡蛎(C. gigas)、葡萄牙牡蛎(C. angulata)、熊本牡蛎(C. sikamea)、香港巨牡蛎(C. hongkongensis)和近江牡蛎(C. ariakensis)5种牡蛎及异齿亚纲、帘蛤目、帘蛤科的紫斑文蛤(Meretrix pethechialis)为研究对象，系统的研究了以上物种的线粒体基因组全序列的特点。并以线粒体12个蛋白质编码基因的序列，在氨基酸和核苷酸水平上构建了软体动物的分子系统发生树。本研究旨在为利用线粒体基因组全序列全面构建软体动物分子系统发生树，为软体动物的系统发生和进化研究提供一种新的思路和前期基础工作，本研究主要内容分为以下三个部分： 一、栉孔扇贝和海湾扇贝线粒体基因组序列分析及分子系统发生研究 采用Long-PCR技术扩增了栉孔扇贝和海湾扇贝线粒体全基因组，利用步移法结合文库构建的测序策略获得了线粒体基因组的序列。海湾扇贝线粒体全基因组长度为16,211 bp，栉孔扇贝接近全序列长度为20,789 bp。两个基因组都编码35个基因，包括12个蛋白质编码基因，2个rRNA和21个tRNA。与典型的动物线粒体基因组相比，两个基因组都缺少一个蛋白质编码基因atp8和2个trnS, 在海湾扇贝基因组中有1个trnF的重复，而在栉孔扇贝基因组中有1个trnM的重复。基因排列比较显示，尽管海湾扇贝、栉孔扇贝和巨扇贝分类学上属于同一扇贝科，但是它们的线粒体基因排列非常不同。在四种扇贝中，虾夷扇贝与栉孔扇贝的基因排列顺序非常相似；即使排除tRNA的比较，栉孔扇贝和海湾扇贝基因组仅仅共享三个小的基因块；而海湾扇贝与巨扇贝仅有一个相同的基因块。在所有的系统发生分析中，四种扇贝稳定的系统发生关系得到强有力的支持，海湾扇贝较其他三种扇贝较早的分化出来；栉孔扇贝比其他两种扇贝与虾夷扇贝亲缘关系更近。贝叶斯法和最大似然法分析都支持扇贝超科的单系发生。 二、巨蛎属牡蛎线粒体基因组全序列分析及分子系统发生研究 采用Long-PCR扩增技术和步移法结合文库构建的技术策略获得了巨蛎属C. gigas、C. angulata、C. sikamea、C. hongkongensis和C. ariakensis 5种牡蛎线粒体全基因组序列，并于GenBank已公布的美洲牡蛎C.virginica序列进行比较研究。C. gigas、C. angulata、C. sikamea、C. hongkongensis和C. ariakensis线粒体全基因组长度分别为18,225 bp、18,225 bp、18,243 bp、18,622 bp和18,414 bp，都长于C. virginica基因组17,244 bp的长度。本研究的5种牡蛎线粒体基因组都编码39个基因，包括12个蛋白质编码基因，2个rRNA和25个tRNA。与典型的线粒体基因组相比，都缺少一个蛋白质编码基因atp8，有trnM、trnK和trnQ 3个tRNA基因的重复，更特别的是基因组中的rrnL分为两段，这在其它线粒体基因组中未见报道，有一个重复的rrnS；而C. virginica基因组编码37个基因，与其他牡蛎相比，没有trnK和trnQ重复，只有一个rrnS。基因排列比较显示，巨蛎属的5种牡蛎C. gigas、C. angulata、C. sikamea、C. hongkongensis和C. ariakensis基因排列完全一致，而与C. virginica的基因排列相比仍然有较大的差别，有多个tRNA发生易位。系统发生分析显示，C. gigas和C. angulata首先聚在一起，然后与C. sikamea聚为一支。C. hongkongensis和C. ariakensis聚成一支。C. virginica为单独的一支。系统树清楚的显示出C. gigas和C. angulata以及C. hongkongensis和C. ariakensis非常近的亲缘关系，这也是长期以来，牡蛎分类学上的经典问题，有学者认为C. gigas和C. angulata为同一物种，线粒体基因组的数据显示C. gigas和C. angulata可能达到不同物种的差异。传统分类上的“近江牡蛎”的“白蚝”和“赤蚝”，线粒体序列差别明显，完全支持两种牡蛎新种名的制定。 三、紫斑文蛤线粒体基因组全序列分析及分子系统发生研究 采用Long-PCR扩增技术和步移法结合文库构建的技术策略获得了紫斑文蛤线粒体基因组全序列。该基因组全长19,567 bp，编码36个基因，包括12个蛋白质编码基因，2个rRNA和22个tRNA。与典型的线粒体基因组相比，缺少一个蛋白质编码基因atp8和1个trnS, 有1个trnQ基因的重复。基因排列比较显示，双壳类的基因排列在低的分类阶元时相对保守。在帘蛤科中，紫斑文蛤M. petechialis和菲律宾蛤仔V. philippinarum共享四个完全一致的基因块，两个大的基因块是cox1-L1-nad1-nad2-nad4L-I 和 cox2-P-cob-rrnL-nad4-H-E-S2-atp6-nad3-nad5，另两个小基因块只包括tRNA基因。在以氨基酸序列构建的分子系统树中，帘蛤科紫斑文蛤与菲律宾蛤仔首先聚在一起，然后，它们与A. tuberculata形成一个进化枝。这一枝与H. arctica结合起来，支持异齿亚纲单系发生。

Chromosomal mapping of major ribosomal rRNA genes in the hard clam (Mercenaria mercenaria) using fluorescence in situ hybridization

Karyotype and chromosomal location of the major ribosomal RNA genes were studied in the hard clam (Mercenaria mercenaria Linnaeus) using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos. Internal transcribed spacers (ITS) between major RNA genes were amplified and used as FISH probes. The probes were labeled with digoxigenin-11-dUTP by polymerase chain reaction and detected with fluorescein-labeled anti-digoxigenin antibodies. FISH with the ITS probes produced two to four signals per nucleus or metaphase. M. mercenaria had a haploid number of 19 chromosomes with a karyotype of seven metacentric, four metacentric or submetacentric, seven submetacentric, and one submetacentric or subtelocentric chromosomes (7M + 4M/SM + 7SM + 1SM/ST). Two ITS loci were observed: one located near the centromere on the long arm of Chromosome 10 and the other at the telomere of the short arm of Chromosome 12. FISH signals on Chromosome 10 are strong and consistent, while signals on Chromosome 12 are variable. This study provides the first karyotype and chromosomal assignment of the major RNA genes in M. mercenaria. Similar studies in a wide range of species are needed to understand the role of chromosomal changes in bivalve evolution.

Its length polymorphism in oysters and its use in species identification

In an effort to develop genetic markers for oyster identification, we studied length polymorphism in internal transcribed spacers (ITS) between major ribosomal RNA genes in 12 common species of Ostreidae: Crassostrea virginica, C. rhizophorae, C. gigas, C. angulata, C. sikamea, C. ariakensis, C. hongkongensis, Saccostrea echinata, S. glomerata, Ostrea angasi, O. edulis, and O. conchaphila. We designed two pairs of primers and optimized PCR conditions for simultaneous amplification of ITS 1 and ITS2 in a single PCR. Amplification was successful in all 12 species, and PCR products were visualized on high-resolution agarose gels. ITS2 was longer than ITS 1 in all Crassostrea and Saccostrea species, whereas they were about the same size in the three Ostrea species. No intraspecific variation in ITS length was detected. Among species, the length of ITS I and ITS2 was polymorphic and provided unique identification of 8 species or species pairs: C. ariakensis, C. hongkongensis, C. sikamea, O. conchaphila, C. virginica/C. rhizophorae, C. gigas/C. angulata, S. echinata/S. glonzerata, and O. angasi/O. edulis. The ITS assay provides simple, rapid and effective identification of C. ariakensis and several other oyster species. Because the primer sequences are conserved, the ITS assay may be useful in the identification of other bivalve species.