黑麂和费氏麂四种卫星DNA的克隆特征和染色体定位

近年来,分子细胞遗传学研究已基本证实了染色体的串联融合(端粒-着丝粒融合)是麂属动物核型演化的主要重排方式。尽管染色体串联融合的分子机制还不清楚,但通过染色体的非同源重组,着丝粒区域的卫星DNA被认为可能介导了染色体的融合。以前的研究发现在赤麂和小麂染色体的大部分假定的串联融合位点处存在着非随机分布的卫星DNA。然而在麂属的其他物种中,这些卫星DNA的组成以及在基因组中的分布情况尚未被研究。本研究从黑麂和费氏麂基因组中成功地克隆了4种卫星DNA(BMC5、BM700、BM1.1k和FM700),并分析了这些卫星克隆的特征以及在小麂、黑麂、贡山麂和费氏麂染色体上的定位情况。结果表明,卫星I和IIDNA(BMC5,BM700和FM700)的信号除了分布在这些麂属动物染色体的着丝粒区域外,也间隔地分布在这些物种的染色体臂上。其研究结果为黑麂、费氏麂和贡山麂的染色体核型也是从一个2n=70的共同祖先核型通过一系列的串联融合进化而来的假说提供了直接的证据。     

Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n=6 in the female and 7 in the male, the karyotypic evolution of which through extensive tandem fusions and several centric fusions has been well-documented by recent molecular cytogenetic studies. In an attempt to define the fusion orientations of conserved chromosomal segments and the molecular mechanisms underlying the tandem fusions, we have constructed a highly redundant (more than six times of whole genome coverage) bacterial artificial chromosome (BAC) library of Indian muntjac. The BAC library contains 124,800 clones with no chromosome bias and has an average insert DNA size of 120 kb. A total of 223 clones have been mapped by fluorescent in situ hybridization onto the chromosomes of both Indian muntjac and Chinese muntjac and a high-resolution comparative map has been established. Our mapping results demonstrate that all tandem fusions that occurred during the evolution of Indian muntjac karyotype from the acrocentric 2n=70 hypothetical ancestral karyotype are centromere–telomere (head–tail) fusions.     

Comparative gene mapping in the species Muntiacus muntjac.

An extreme case of chromosomal evolution is presented by the two muntjac species Muntiacus muntjac (Indian muntjac, 2n = 6 [females], 7 [males]) and M. reevesi (Chinese muntjac, 2n = 46). Despite disparate karyotypes, these phenotypically similar species produce viable hybrid offspring, indicating a high degree of DNA-level conservation and genetic relatedness. As a first step toward development of a comparative gene map, several Indian muntjac homologs of known human type I anchor loci were mapped. Using flow-sorted, chromosome-specific Southern hybridization techniques, homologs of the protein kinase C beta polypeptide (PRKCB1) and the DNA repair genes ERCC2 and XRCC1 have been assigned to Indian muntjac chromosome 2. The male-specific ZFY gene was presumptively mapped to Indian muntjac chromosome Y2. Ultimate generation of a comparative physical map of both Indian and Chinese muntjac chromosomes will prove invaluable in the study of mammalian karyotype evolution.     

Phylogeny ofMuntiacus (Cervidae) based on mitochondrial DNA restriction maps

Mitochondrial DNA restriction maps for 12 restriction enzymes of four species of muntjacs—Indian muntjac(M. muntjak), Gongshan muntjac(M. gongshanensis), black muntjac(M. crinifrons), and Chinese muntjac(M. reevesi)—were compared to estimate the phylogenetic relationships among them. Phylogenetic trees were constructed by both distance and parsimony methods. The two resulting trees share a similar topology, which indicates that the black muntjac and the Gongshan muntjac are closely related, followed by the Chinese muntjac; the Indian muntjac is the sister taxon to all the other muntjacs.     

麂属动物陈旧皮张标本的DNA提取及PCR扩增

本实验用改进的方法从保存于标本馆的动物皮张标本中提取ＤＮＡ,所得ＤＮＡ片段的分子量从１００ｂｐ到１ｋｂ以上。利用线粒体ＤＮＡ细胞色素ｂ通用引物和ＰＣＲ技术,从小麂、印度麂、贡山麂、费氏麂、黑麂ＤＮＡ中扩增出３０７ｂｐ的细胞色素ｂ特异片段。用２８种限制性内切酶对从新鲜血样和从陈旧皮张标本中所得扩增片段进行酶切分析,发现只有４个酶在这个片段上有切点,其中ＨａｅⅢ和ＨａｐⅡ的识别位点在各种麂中有所不同。     

小麂、黑麂、赤麂精母细胞联会复合体的比较研究

本工作以界面铺张——硝酸银染色技术,对小麂(Muntiacus reeuesi)、黑麂(M.crinifrons)和赤麂(M.muntjak)的精母细胞联会复合体(Syna ptonemal complex,SC)进行亚显微结构的比较研究。结果表明: 1.SC的平均相对长度和臂比指数同有丝分裂细胞相应染色体的数值有很好的一致性。根据SC的相对长度和臂比指数绘制了三种麂的SC组型图。雄性黑麂减数分裂前期形成一个复杂的易位多价体,意味着其核型的演化过程涉及两次染色体易位和一次臂间倒位。 2.在减数分裂前期,性染色体的形态和行为同常染色体的有明显差异,如性染色体嗜银性较强,配对延迟等。XY的配对起始于早粗线期,在中粗线期,Y的全长均同X配对;XY-SC开始解体于晚粗线期。 3.在粗线期,X染色体未配对区域出现自身折叠,形成“发夹”状结构。这种“发夹”结构的形成,可能是在性染色体的进化过程中,X染色体通过不对称易位得到的重复片段在减数分裂前期同源配对的一种细胞学表现。     

Localization of the repetitive telomeric sequence (TTAGGG)n in two muntjac species and implications for their karyotypic evolution

It has been suggested that the chromosome set of the Indian muntjac, Muntiacus muntjak vaginalis (female, 2n = 6; male, 2n = 7), evolved from small acrocentric chromosomes, such as those found in the complement of the Chinese muntjac, M. reevesi (2n = 46), by a series of tandem fusions and other rearrangements. The location of the highly conserved human telomeric sequence (TTAGGG)n in the metaphase chromosomes of M.m. vaginalis and its close relative, M. reevesi, was investigated by non-radioactive in situ hybridization. The (TTAGGG)n repeat was found adjacent to the centromeres in the short arm and at the telomeres in the long arm of M. reevesi acrocentric metaphase chromosomes. Tandem fusions present in the karyotype of M.m. vaginalis chromosomes were not reflected by interstitial signals of the telomere repeat, as these chromosomes displayed hybridization signals only at the ends of the chromatids. Mechanisms that might have played a role in the evolution of the reduced karyotype of the Indian muntjac are discussed.     

圈养条件下黑麂雄性标记行为与雌性化学信息存留位置的关系

化学物质被认为是哺乳动物标记行为信息传递的主要载体,而标记在鹿科动物的竞争雌性资源中具有重要的作用,因此本文重点对雄性黑麂的化学标记与雌性化学信息存留点的关系进行了行为学研究。观察发现面腺标记(利用额腺和眶下腺)是雌雄黑麂共有的化学标记行为,而行为性排尿和刨地行为(利用蹄腺)则仅为雄性具有的标记行为;躺卧不具有化学标记作用,而排粪在化学标记上的作用则难以确定。同时分析表明,雄性黑麂更倾向于在靠近雌性化学信息存留区的区域进行化学标记,且可能会因竞争压力的增大而扩大领域范围。     

Comparative genomic analysis links karyotypic evolution with genomic evolution in the Indian Muntjac (Muntiacus muntjak vaginalis)

The karyotype of Indian muntjacs (Muntiacus muntjak vaginalis) has been greatly shaped by chromosomal fusion, which leads to its lowest diploid number among the extant known mammals. We present, here, comparative results based on draft sequences of 37 bacterial artificial clones (BAC) clones selected by chromosome painting for this special muntjac species. Sequence comparison on these BAC clones uncovered sequence syntenic relationships between the muntjac genome and those of other mammals. We found that the muntjac genome has peculiar features with respect to intron size and evolutionary rates of genes. Inspection of more than 80 pairs of orthologous introns from 15 genes reveals a significant reduction in intron size in the Indian muntjac compared to that of human, mouse, and dog. Evolutionary analysis using 19 genes indicates that the muntjac genes have evolved rapidly compared to other mammals. In addition, we identified and characterized sequence composition of the first BAC clone containing a chromosomal fusion site. Our results shed new light on the genome architecture of the Indian muntjac and suggest that chromosomal rearrangements have been accompanied by other salient genomic changes. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.Qi Zhou, Ling Huang, Jianguo Zhang: these authors contributed equally to the paper.Sequence data from this article have been deposited in the GenBank Libraries under Accession No. DQ280153-DQ280188, DQ377335, DQ458964.     

黑麂Y染色体的鉴别和Sry基因的克隆及定位

以流式细胞仪分离小麂(Muntiacus reevesi)Y染色体和黑麂(Muntiacus crinifrons)Y1,Y2,X+4和1号染色体,利用DOP-PCR技术富集了分离的各单条染色体。然后,将小麂的Y染色体的DOP-PCR产物经Cy3标记后直接作为涂染探针,应用染色体涂染技术与雌雄黑麂的核型标本进行杂交,确认了黑麂真正的Y染色体为Y2染色体。再以黑麂的Y1,Y2,X+4和1号染色体的DOP-PCR产物为模板,用人的特异性的SRY（sex determining region of the Y chromosome）基因引物对其进行扩增,结果表明黑麂只有Y2染色体出现了SRY扩增片段。然后扩增产物克隆和测序,比较它与人的同源性,初步把黑麂的Sry基因定位在Y2染色体上。最后提取雄性黑麂的基因组DNA,并用同一对引物对其进行扩增,亦得到Sry基因的片段,对此扩增片段进行克隆,测序,结果表明其与Y2染色体得到的Sry基因片段完全一样,与人SRY基因的同源性均为83%。 Abstract：The single Y chromosome of Muntiacus reevesi and Y1,Y2 ,X+4,1 chromosome of Muntiacus crinifrons were obtained by flow-sorting ,then they were amplified through DOP-PCR . After that, the metaphase karyotype of Muntiacus crinifrons were painted by using the product of the DOP-PCR of the Y chromosome of Muntiacus reevesi as a special probe and the result showed that Y2 chromosome was the real Y chromosome of Muntiacus crinifrons. Secondly the product of the DOP-PCR of Y1,Y2,X+4,1 chromosome of Muntiacus crinifrons were used as the templates of the next amplification using the special primer devised according to the human SRY gene .One band was obtained only from Y2 chromosome, then it was cloned to the T-vector and sequenced. The Sry gene sequence of Muntiacus crinifrons was acquired and the conclution was that there are 83% homology between the human and Muntiacus crinifrons. It was testified that in all mammal Sry gene is consertive. On the other side the Sry gene was located to the Y2 chromosome of the Muntiacus crinifrons.     

Localization and characterization of recombinant DNA clones derived from the highly repetitive DNA sequences in the Indian muntjac cells: their presence in the Chinese muntjac

A total of seven, highly repeated, DNA recombinant M13 mp8 clones derived from a Hpa II digest of cultured cells of the Indian muntjac (Muntiacus muntjac vaginalis) were analyzed by restriction enzymes, in situ hybridization, and DNA sequencing. Two of the clones, B1 and B8, contain satellite DNA inserts which are 80% homologous in their DNA sequences. B1 contains 781 nucleotides and consist of tandem repetition of a 31 bp consensus sequence. This consensus sequence, TCCCTGACGCAACTCGAGAGGAATCCTGAGT, has only 3 bp changes, at positions 7, 24, and 27, from the consensus sequence of the 31 bp subrepeats of the bovine 1.715 satellite DNA. The satellite DNA inserts in B1 and B8 hybridize primarily but not specifically to chromosome X, and secondarily to other sites such as the centromeric regions of chromosomes 1 and 2. Under less stringent hybridization conditions, both of them hybridize to the interior of the neck region and all other chromosomes (including chromosomes 3 and Y). The other five DNA clones contain highly repetitive, interdispersed DNA inserts and are distributed throughout the genome except for the neck region of the compound chromosome X+3. Blot hybridization results demonstrate that the satellite DNA component is also present in Chinese muntjac DNA (Muntiacus reevesi) in spite of the very different karyotypes of the Chinese and Indian muntjacs.     

Comparative sequence analyses reveal sites of ancestral chromosomal fusions in the Indian muntjac genome

Background

Indian muntjac (Muntiacus muntjak vaginalis) has an extreme mammalian karyotype, with only six and seven chromosomes in the female and male, respectively. Chinese muntjac (Muntiacus reevesi) has a more typical mammalian karyotype, with 46 chromosomes in both sexes. Despite this disparity, the two muntjac species are morphologically similar and can even interbreed to produce viable (albeit sterile) offspring. Previous studies have suggested that a series of telocentric chromosome fusion events involving telomeric and/or satellite repeats led to the extant Indian muntjac karyotype.

Results

We used a comparative mapping and sequencing approach to characterize the sites of ancestral chromosomal fusions in the Indian muntjac genome. Specifically, we screened an Indian muntjac bacterial artificial-chromosome library with a telomere repeat-specific probe. Isolated clones found by fluorescence in situ hybridization to map to interstitial regions on Indian muntjac chromosomes were further characterized, with a subset then subjected to shotgun sequencing. Subsequently, we isolated and sequenced overlapping clones extending from the ends of some of these initial clones; we also generated orthologous sequence from isolated Chinese muntjac clones. The generated Indian muntjac sequence has been analyzed for the juxtaposition of telomeric and satellite repeats and for synteny relationships relative to other mammalian genomes, including the Chinese muntjac.

Conclusions

The generated sequence data and comparative analyses provide a detailed genomic context for seven ancestral chromosome fusion sites in the Indian muntjac genome, which further supports the telocentric fusion model for the events leading to the unusual karyotypic differences among muntjac species.     

Chromosomal rearrangements and the first indication of an ♀X1X1X2X2/♂X1X2Y sex chromosome system in Rineloricaria fishes (Teleostei: Siluriformes)

Rineloricaria is the most diverse genus within the freshwater fish subfamily Loricariinae, and it is widely distributed in the Neotropical region. Despite limited cytogenetic data, records from southern and south-eastern Brazil suggest a high rate of chromosomal rearrangements in this genus, mirrored in remarkable inter- and intraspecific karyotype variability. In the present work, we investigated the karyotype features of Rineloricaria teffeana, an endemic representative from northern Brazil, using both conventional and molecular cytogenetic techniques. We revealed different diploid chromosome numbers (2n) between sexes (33♂/34♀), which suggests the presence of an ♀X₁X₁X₂X₂/♂X₁X₂Y multiple sex chromosome system. The male-limited Y chromosome was the largest and the only biarmed element in the karyotype, implying Y-autosome fusion as the most probable mechanism behind its origination. C-banding revealed low amounts of constitutive heterochromatin, mostly confined to the (peri)centromeric regions of most chromosomes (including the X₂ and the Y) but also occupying the distal regions of a few chromosomal pairs. The chromosomal localization of the 18S ribosomal DNA (rDNA) clusters revealed a single site on chromosome pair 4, which was adjacent to the 5S rDNA cluster. Additional 5S rDNA loci were present on the autosome pair 8, X₁ chromosome, and in the presumed fusion point on the Y chromosome. The probe for telomeric repeat motif (TTAGGG)_n revealed signals of variable intensities at the ends of all chromosomes except for the Y chromosome, where no detectable signals were evidenced. Male-to-female comparative genomic hybridization revealed no sex-specific or sex-biased repetitive DNA accumulations, suggesting a presumably low level of neo-Y chromosome differentiation. We provide evidence that rDNA sites might have played a role in the formation of this putative multiple sex chromosome system and that chromosome fusions originate through different mechanisms among different Rineloricaria species.     

New insights into the karyotypic relationships of Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis)

To investigate the karyotypic relationships between Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis), a complete set of Chinese muntjac chromosome-specific painting probes has been assigned to G-banded chromosomes of these three species. Sixteen autosomal probes (i.e. 6-10, 12-22) of the Chinese muntjac each delineated one pair of conserved segments in the forest musk deer and gayal, respectively. The remaining six autosomal probes (1-5, and 11) each delineated two to five pairs of conserved segments. In total, the 22 autosomal painting probes of Chinese muntjac delineated 33 and 34 conserved chromosomal segments in the genomes of forest musk deer and gayal, respectively. The combined analysis of comparative chromosome painting and G-band comparison reveals that most interspecific homologous segments show a high degree of conservation in G-banding patterns. Eleven chromosome fissions and five chromosome fusions differentiate the karyotypes of Chinese muntjac and forest musk deer; twelve chromosome fissions and six fusions are required to convert the Chinese muntjac karyotype to that of gayal; one chromosome fission and one fusion separate the forest musk deer and gayal. The musk deer has retained a highly conserved karyotype that closely resembles the proposed ancestral pecoran karyotype but shares none of the rearrangements characteristic for the Cervidae and Bovidae. Our results substantiate that chromosomes 1-5 and 11 of Chinese muntjac originated through exclusive centromere-to-telomere fusions of ancestral acrocentric chromosomes.     

The Y chromosome-specific STS marker MS2 and its peripheral regions on the Y chromosome of the dioecious plant Silene latifolia.

Sex determination in Silene latifolia uses the XX/XY system. The recent evolution of dioecy in S. latifolia provides a unique opportunity to study the early stages of Y chromosome evolution. However, the current Y chromosome map still contains many large gaps with no available markers. In this study, a sequence tagged site (STS) marker, MS2, was isolated and mapped to the same locus as L8 on the Y chromosome. To investigate the peripheral regions of MS2, a bacterial artificial chromosome (BAC) library was constructed from a male plant, and the BAC clone containing MS2 (MS2-9d12F) was isolated from 32 640 clones with an average insert size of 115 kb. A 109-kb insert of the BAC clone was analyzed. BLASTX analysis showed 11 sequences similar to some known proteins, most of which are retrotransposon-like elements. The ORF Finder predicted 9 ORFs within MS2-9d12F. RT-PCR analyses revealed that only 4 of the 9 predicted ORFs are expressed in both male and female plants. These 4 ORFs are candidates for genes having counterparts on both the X and Y chromosomes. Dot-matrix plot analysis and a BLASTN search revealed LTR-like sequences close to the retrotransposon-like elements and high similarity to 3 known genomic sequences of S. latifolia. These results suggest an accumulation of retrotransposons and segmental duplications in peripheral regions of MS2 during the early stage of sex chromosome evolution.     

麂亚科动物钾离子通道基因片段及其内含子 序列的克隆与进化分析

运用PCR扩增、T-A克隆、测序等技术,获得黑麂,小麂,赤麂和毛冠鹿4种麂亚科动物基因组DNA的钾离子通道部分序列。序列分析表明:麂属动物之间的外显子区域序列差异为0.90%～1.44%,毛冠鹿与麂属动物之间的差异为0.90%～1.26%,可见这一段序列的同源性较高。而内含子部分序列差异在麂属动物之间的差异为0～1.22%,毛冠鹿与麂属动物的差异为1.83%左右。由NJ法和最大简约法（Mp法）构建的进化树表明黑麂与赤麂亲缘关系较近,小麂与他们同为一属但关系较远,而毛冠鹿与它们之间的分化程度已达到属间水平。研究表明基因组的内含子序列能够较真实反映近缘动物之间的关系,是进行分子进化比较分析的较理想标记。 Abstract: In this study, partial fragments of potassium ion channel gene were amplified using the genomic DNA of muntjak, reevesi, crinifrons, and Elaphodus cephalophus. The PCR products were ligated to the plasmid of pMD18-T Vector by the method of direct T-A cloning. The positive clones were identified by colony PCR. The sequences of the recombinant clones were determined using M13-47/RV-M universal primers and aligned by the software CLUSTALW. The nucleotide divergences of exon were 0.90%-1.44% among three species of Muntiacus, 0.90%-1.26% between E. cephalophus and each of Muntiacus deer. In the nucleotide of intron there is 0%-1.22% difference among these muntjac deers, and the divergene reached about 1.83% between E. cephalophus and the three species of Muntiacus. Using the software of MEGA to analyse molecular phylogeny, Phylogenetic trees were constructed with neighbor-joining method and maximum parsimony method. The result showed Muntiacus, crinifrons is most closely related to muntjak, with reevesi as their sister species. E. cephalophus is in the other genus.     

黑麂线粒体基因组序列分析

采用PCR产物直接测序方法测定了黑麂线粒体基因组全序列 ,初步分析了其基因组特点并定位了各基因的位置 .结果显示 :黑麂的线粒体基因组全序列长度为 1 6 35 7bp ,可编码 2 2种tRNA、2种rRNA、1 3种蛋白质 ,碱基组成及基因位置与小麂、赤麂和其它哺乳类动物的线粒体基因组相似 ;模拟电子酶切图谱与先前的报道基本一致 ;基于细胞色素b的全基因序列 ,分别以最大简约法、N J法、最大似然数法与其它 1 4种鹿类动物的相应序列进行了聚类分析 ,构建出相似的系统进化树 :初步确定了麂亚科动物在鹿科中处于与鹿亚科、北美鹿亚科并列的进化地位 .在此基础上 ,进一步以黑麂、赤麂、小麂的线粒体编码RNA和编码蛋白质的基因序列构建系统进化树 ,分析了三者的亲缘关系 .结果表明 :黑麂和赤麂亲缘关系较近 ,是较新的物种 ,而小麂是较为原始的物种     

合肥野生动物园黑麂的繁殖资料

合肥野生动物园自1978年开始进行黑麂(Muntiacus crinifrons)的饲养和繁殖,1989年第一胎圈养条件下繁殖的黑麂出生,到2001年底累计繁殖黑麂51头,繁殖种群正处迅速增长期。13年的繁殖资料统计结果表明,圈养条件下育龄母麂平均每12个月产一胎(多数在11—13个月,少数仅6—9个月),孕期240d左右,哺乳期2—3个月,少数母麂可产后发情,但极少有两年三次产仔现象。值得注意的是圈养条件下黑麂多在9—11月交配,4—7月产仔(80．39％)。圈养条件下黑麂幼年的死亡率较低(7．84％),成年黑麂多死于消化道及呼吸道感染等疾病(56％)。     

Rapid and parallel chromosomal number reductions in muntjac deer inferred from mitochondrial DNA phylogeny

Muntjac deer (Muntiacinae, Cervidae) are of great interest in evolutionary studies because of their dramatic chromosome variations and recent discoveries of several new species. In this paper, we analyze the evolution of karyotypes of muntjac deer in the context of a phylogeny which is based on 1,844-bp mitochondrial DNA sequences of seven generally recognized species in the muntjac subfamily. The phylogenetic results support the hypothesis that karyotypic evolution in muntjac deer has proceeded via reduction in diploid number. However, the reduction in number is not always linear, i.e., not strictly following the order: 46-->14/13-->8/9-->6/7. For example, Muntiacus muntjak (2n = 6/7) shares a common ancestor with Muntiacus feae (2n = 13/14), which indicates that its karyotype was derived in parallel with M. feae's from an ancestral karyotype of 2n >/= 13/14. The newly discovered giant muntjac (Muntiacus vuquangensis) may represent another parallel reduction lineage from the ancestral 2n = 46 karyotype. Our phylogenetic results indicate that the giant muntjac is relatively closer to Muntiacus reevesi than to other muntjacs and may be placed in the genus Muntiacus Analyses of sequence divergence reveal that the rate of change in chromosome number in muntjac deer is one of the fastest in vertebrates. Within the muntjac subfamily, the fastest evolutionary rate is found in the Fea's lineage, in which two species with different karyotypes diverged in around 0.5 Myr.