Evolution of genome organizations of squirrels (Sciuridae) revealed by cross-species chromosome painting

With complete sets of chromosome-specific painting probes derived from flow-sorted chromosomes of human and grey squirrel (Sciurus carolinensis), the whole genome homologies between human and representatives of tree squirrels (Sciurus carolinensis, Callosciurus erythraeus), flying squirrels (Petaurista albiventer) and chipmunks (Tamias sibiricus) have been defined by cross-species chromosome painting. The results show that, unlike the highly rearranged karyotypes of mouse and rat, the karyotypes of squirrels are highly conserved. Two methods have been used to reconstruct the genome phylogeny of squirrels with the laboratory rabbit (Oryctolagus cuniculus) as the out-group: ( 1) phylogenetic analysis by parsimony using chromosomal characters identified by comparative cytogenetic approaches; ( 2) mapping the genome rearrangements onto recently published sequence-based molecular trees. Our chromosome painting results, in combination with molecular data, show that flying squirrels are phylogenetically close to New World tree squirrels. Chromosome painting and G-banding comparisons place chipmunks ( Tamias sibiricus), with a derived karyotype, outside the clade comprising tree and flying squirrels. The superorder Glires (order Rodentia + order Lagomorpha) is firmly supported by two conserved syntenic associations between human chromosomes 1 and 10p homologues, and between 9 and 11 homologues.

Phylogenomic study of the subfamily Caprinae by cross-species chromosome painting with Chinese muntjac paints

Chromosomal homologies have been established between the Chinese muntjac (Muntiacus reevesi, MRE, 2n = 46) and five ovine species: wild goat (Capra aegagrus, CAE, 2n = 60), argall (Ovis ammon, OAM, 2n = 56), snow sheep (Ovis nivicola, ONI, 2n = 52), red goral (Naemorhedus cranbrooki, NCR, 2n = 56) and Sumatra serow (Capricornis sumatraensis, CSU, 2n = 48) by chromosome painting with a set of chromosome-specific probes of the Chinese muntjac. In total, twenty-two Chinese muntjac autosomal painting probes detected thirty-five homologous segments in the genome of each species. The chromosome X probe hybridized to the whole X chromosomes of all ovine species while the chromosome Y probe gave no signal. Our results demonstrate that almost all homologous segments defined by comparative painting show a high degree of conservation in G-banding patterns and that each speciation event is accompanied by specific chromosomal rearrangements. The combined analysis of our results and previous cytogenetic and molecular systematic results enables us to map the chromosomal rearrangements onto a phylogenetic tree, thus providing new insights into the karyotypic evolution of these species.

Avian comparative genomics: reciprocal chromosome painting between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus, Charadriiformes) - An atypical species with low diploid number

The chicken is the most extensively studied species in birds and thus constitutes an ideal reference for comparative genomics in birds. Comparative cytogenetic studies indicate that the chicken has retained many chromosome characters of the ancestral avia

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.

DIFFERENT RATES OF MITOCHONDRIAL-DNA SEQUENCE EVOLUTION IN KIRK DIK-DIK (MADOQUA-KIRKII) POPULATIONS

We have investigated evolutionary rates of the mitochondrial genome among individuals of Madoqua kirkii using the relative rate test. Our results demonstrate that individuals of two chromosome races, East African cytotype A and Southwest African cytotype D, evolve about 2.3 times faster than East African cytotype B. Cytogenetic changes, DNA repair efficiency, mutagens, and more likely, hitherto unrecognized factors will account for the rate difference we have observed. Our results suggest additional caution when using molecular clocks in the estimation of divergence time, even within lineages of closely related taxa. Rate heterogeneity in microevolutionary timescales represents a potentially important aspect of basic evolutionary processes and may provide additional insights into factors which affect genome evolution. (C) 1995 Academic Press, Inc.

Comparative cytogenetics of bats (Chiroptera): The prevalence of Robertsonian translocations limits the power of chromosomal characters in resolving interfamily phylogenetic relationships

Although the monophyly of Chiroptera is well supported by many independent studies, higher-level systematics, e.g. the monophyly of microbats, remains disputed by morphological and molecular studies. Chromosomal rearrangements, as one type of rare genomic changes, have become increasingly popular in phylogenetic studies as alternatives to molecular and other morphological characters. Here, the representatives of families Megadermatidae and Emballonuridae are studied by comparative chromosome painting for the first time. The results have been integrated into published comparative maps, providing an opportunity to assess genome-wide chromosomal homologies between the representatives of eight bat families. Our results further substantiate the wide occurrence of Robertsonian translocations in bats, with the possible involvement of whole-arm reciprocal translocations (WARTs). In order to search for valid cytogenetic signature(s) for each family and superfamily, evolutionary chromosomal rearrangements identified by chromosomal painting and/or banding comparison are subjected to two independent analyses: (1) a cladistic analysis using parsimony and (2) the mapping of these chromosomal changes onto the molecularly defined phylogenetic tree available fromthe literature. Both analyses clearly indicate the prevalence of homoplasic events that reduce the reliability of chromosomal characters for resolving interfamily relationships in bats.

同源四倍体水稻三系亲本的遗传差异、细胞遗传学比较研究及低直链淀粉含量突变体Wx基因序列分析

同源四倍体水稻（2N=4X=48,AAAA）是由二倍体水稻（2N=2X=24,AA）通过秋水仙素诱导染色体加倍后得到的新品系，具有优良的抗病性以及较高的蛋白质含量。因此，在四倍体水平上挖掘水稻的增产潜力成为水稻育种的新手段。同源四倍体水稻具有很强的遗传可塑性和很弱的遗传保守性，利用其作为水稻远缘杂交的桥梁，从野生物种中不断地引进有益的基因，这将有助于杂交水稻的多代利用和固定水稻的杂种优势。但是迄今为止，还没有关于同源四倍体水稻遗传多样性，遗传背景的报道。目前世界关于同源四倍体水稻的研究主要集中在中国，主要研究方向为培育、筛选结实正常的亲本材料，配置和筛选结实率正常或接近正常的组合。经过几十年研究，虽然在材料构建，细胞学研究等方面取得了较大进展，但同样由于结实率低的瓶颈问题未解决，而使多倍体水稻育种未能取得实质性进展。而近年来一些关于同源四倍体水稻低结实率机理的细胞学研究也由于缺乏统计学数据而缺乏说明性。本文用SSR标记，对选取的36个结实率正常同源四倍体水稻三系亲本和14个来源二倍体亲本，分析他们的遗传差异和群体遗传结构。本文还利用我们培育的高、低结实率的同源四倍体水稻恢复系、优良保持系和杂种F1及二倍体对照为材料，进行系统深入的细胞遗传学研究，进一步探讨同源四倍体水稻有性传递后代的发育过程，探索分裂期染色体行为特征与遗传性状稳定性的关系，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%，为研究其直链淀粉含量下降的原因，本文还根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析，并根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究选取了中国科学院成都生物所培育的同源四倍体和二倍体水稻亲本，并用36个微卫星标记进行了遗传差异和种群遗传结构分析。在50个品系中，我们观察到较高水平的多态性，每基因等位基因数（Ae）分布于2至6之间（平均值3.028），多态性信息含量（PIC）分布于0.04至0.76之间（平均值0.366）；期望杂合度（He）分布于0.04至0.76之间（平均值0.370），Shannon指数（I）分布于0.098至1.613之间（平均值0.649）。同源四倍体品系的等位基因数，期望杂合性和Shannon指数都比二倍体品系高。在供试50个品系中，较多材料均发现Rare基因，根据SSR多态性指数我们构建了同源四倍体和二倍体水稻的核心指纹库。F-统计值表明遗传差异主要存在于同源四倍体品系中（Fst=0.066）。聚类分析结果表明50个品系可以分为4个组。I组包括所有的同源四倍体和二倍体籼稻保持系，以及一个同源四倍体籼稻雄性不育系及其来源二倍体。II组仅包括IR来源的品系。III组比II组和IV组更复杂，包括同源四倍体和二倍体籼稻恢复系品系。IV组包括同源四倍体和二倍体粳稻品系。此外,由于等位基因及配子的遗传差异,同源四倍体与二倍体品系中存在单位点和双位点的遗传差异。分析结果表明,二倍体和四倍体水稻基因库的不同,其中遗传变异可以区分四倍体与二倍体水稻。同源四倍体水稻具有长期而独立的遗传性，我们能够选育并得到与二倍体亲本相比有特殊优良农艺性状的品系。 本研究以高结实率的同源四倍体水稻恢复系DTP-4、D明恢63及优良保持系D46B为材料进行农艺性状及细胞遗传学比较研究。DTP-4、D明恢63及保持系D46B的的染色体组成均为2N=4X=48，花粉母细胞具有较为理想的减数分裂行为，配对染色体的比率在99%以上，这与理论染色体组构成相符。DTP-4和D明恢63PMC减数分裂各个时期单价体和三价体的比例都非常低，而在MI， PMC观察到较多的二价体和四价体且四价体多以环状形式出现，其最大频率的染色体构型分别为12II 6IV和10II 7IV。恢复系DTP-4和D明恢63在MI四价体频率分别为2.00/PMC和2.26/PMC，而保持系D46B在MI四价体频率为6.00/PMC，极显著地高于恢复系品系，表明保持系D46B具有更好的染色体配对性质；AI保持系D46B的染色体滞后频率为10.62%，远低于恢复系材料DTP－4和D明恢63的19.44％和23.14％，接近二倍体对照明恢63的7.30%水平；TI保持系D46B具有比恢复系更低频率的微核数。而在TII，D46B的正常四分小孢子比率不但高于恢复系品系甚至高于二倍体对照。对高低结实率的同源四倍体水稻恢复系和杂种F1代的花粉育性，结实率和细胞遗传学行为进行了比较研究。DTP-4, D明恢63, D46A´DTP-4和D46A´D明恢63的花粉育性和结实率比D什香和D46A´D什香显著提高。减数分裂分析的结果表明，DTP-4，D明恢63，D什香，D46A´DTP-4，D46A´D明恢63和D46A´D什香其减数分裂染色体构型分别为：0.05I +19.96 II (9.89棒状+10.07环状) +0.01III + 2.20 IV, 0.11I +19.17 II (8.90 棒状+10.37 环状) +0.09III + 2.26 IV + 0.01 VI, 1.33I +9.46 II (4.50 棒状+4.96 环状) +0.44III + 6.02 IV + 0.09VI + 0.09 VIII, 0.02I +14.36 II (6.44 棒状+7.91 环状) +0.01III + 4.80IV + 0.01VIII, 0.06 I +17.67 II (11.01 棒状+6.67 环状) +0.06 III + 3.10 IV + 0.01 VI and 1.11 I +11.31 II (5.80 棒状+5.51 环状) +0.41 III + 5.63 IV+0.03VI+0.03VIII。在同源四倍体水稻恢复系和杂种F1代材料中，最常见的染色体构型为16II +4IV和12II +6IV。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。在杂种F1代中，二价体的比例要低于其相应的恢复系亲本，同样的，单价体，三价体和多价体的比例相比其恢复系亲本也偏低。然而，在减数分裂MI，杂种F1代中四价体的比例要显著高于其恢复系亲本。在中期I，每细胞单价体的比例和花粉育性呈现出极高的负相关(-0.996)，当单价体数目升高时，花粉育性下降。其次是每细胞三价体的比例(-0.987)，之后则是每细胞多价体的比例与花粉育性的负相关(-0.948)。但是统计分析表明，二价体和四价体的比例对花粉育性和结实率没有显著影响。这一结果表明出了花粉育性和细胞减数分裂行为的相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了理论依据。 突变体是遗传学研究的基本材料。利用突变体克隆水稻基因,并进而研究基因的生物学功能是水稻功能基因组学的重要研究内容。本课题组在多年的四倍体水稻育种研究中已获得多个低直链淀粉含量突变体，其中一些突变体在直链淀粉含量下降的同时，胚乳外观也发生了显著改变，呈半透明或不透明。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因，我们根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析；同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生了碱基缺失，导致移码突变，在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致了其序列上的酶切位点的变化,对常用限制性内切酶位点分析分析结果表明同源四倍体水稻相对于籼稻和粳稻多了2个sph1酶切位点，相对于粳稻减少了6个Acc1，增加了4个Xba1，1个Xho1，1个Pst1和1个Sal1酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近，由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外，根据D4063-1Wx基因的碱基差异，我们推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因，并可能与其米饭较软等品质相关。本文还根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与我们设计的扩增普通籼稻、粳稻的Wx基因引物F5配合使用建立了识别D4063-1的显性和共显性两种检测方式的分子标记,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 研究同源四倍体水稻基因组的遗传差异，探索同源四倍体水稻的遗传规律，研究分裂期染色体行为特征与遗传性状稳定性的关系,旨在揭示四倍体水稻中同源染色体配对能力的遗传差异，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。 Autotetraploid rice (2N=4X=48, AAAA) is a new germplasm developed from diploid rice (2N=2X=24, AA) through chromosomes doubling with colchicines and is an excellent resource for desirable resistance genes to the pathogens and high protein content. Therefore, heterosis utilization on polyploidy is becoming a new strategy in rice breeding. At present, the main research on autotetraploid rice centralizes in China. Breeding effort has been made to improve autotetraploid rice genetically, however, the progresses are limited due to higher degree of divergence between hybrid sterility and polygenic nature. But to date, almost nothing is reported about the genetic diversity, original and genetic background of autotetraploid rice. Despite several reports on cytological analysis of the mechanisms of low seed set in autotetraploid rice still the results are inconclusive due to lack the statistical evaluation. Therefore, the study on the mechanisms of low seed set in autotetraploid is a priority for rice breeding. Microsatellites or simple sequence repeats (SSRs) are the widely used marker for estimating genetic diversity in many species, including wild, weedy, and cultivated rice. In our research, genetic diversity and population genetic structure of autotetraploid and diploid populations collected from Chengdu Institute of Biology, Chinese Academy of Sciences were studied based on 36 microsatellite loci. For the total of 50 varieties, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus (Ae) ranging from 2 to 6 (mean 3.028) and PIC ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (He) varied from 0.04 to 0.76 with the mean of 0.370 and Shannon’s index (I) ranging from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed a slightly higher level of effective alleles, the expected heterozygosity and Shannon’s index than that of diploid populations. Rare alleles were observed at most of the SSR loci in one or more of the 50 accessions and core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed that genetic variability mainly existed among autotetraploid populations rather than among diploid populations (Fst=0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and a autotetraploid and its original diploid indica male sterile lines. Groups II contained only original of IR accessions. Group III was more diverse than either group II or IV and comprised of both autotetraploid and diploid indica restoring lines. Group IV included japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice are somewhat dissimilar, which made a variation that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some new important agricultural characters but the diploid rice has not. Cytogenetic characteristics in restorer lines DTP-4, DMinghui63 and maintainer line D46B of autotetraploid rices were studied. DTP-4, DMinghui63 and D46B showed the advantage of high seed set and biological yield. The meiotic chromosome behavior was slightly irregular in DTP-4, DMinghui63 and D46B. We observed less univalent, trivalent and multivalent at MI, but more bivalent and quadrivalent were observed. The most frequent chromosome configurations were 12II 6IVand 10II 7IV in restorer and maintainer lines, respectively. The quadrivalent frequency of DTP-4 and Dminghui63 at metaphase(MI) was respectively 2.00/PMC and 2.26/PMC. However that frequency of D46B was 6.00/PMC, which was greatly significantly higher than DTP-4 and Dminghui63. That indicates the maintainer D46B has better chromosome pairing capability in metaphase (MI). The frequency of lagging chromosomes of the maintainer D46B at anaphaseI (AI) was 10.62%, which was significantly lower than that of DTP-4(19.44%) and Dminghui63(23.14%) and nearly reaching the level of diploid CK(7.30%). In telophaseI (TI) maintainer D46B showed lower frequency of microkernel at TI and lower frequency of abnormal spores at telophaseII(TII). We also studied pollen fertility, seed set and cytogenetic characteristics of restorer lines and F1 hybrids of autotetraploid rice. DTP-4, DMinghui63, D46A´DTP-4 and D46A´DMinghui63 showed significantly higher pollen fertility and seed set than DShixiang and D46A´DShixiang. Pairing configurations in PMC of DTP-4, DMinghui63, DShixiang, D46A´DTP-4, D46A´DMinghui63 and D46A´DShixiang were 0.05 I+19.96 II (9.89 rod+10.07 ring)+0.01 III+2.20 IV, 0.11 I+19.17 II (8.90 rod+10.37 ring)+0.09 III+2.26 IV+0.01 VI, 1.33 I+9.46 II (4.50 rod+4.96 ring)+0.44 III+6.02 IV+0.09 VI+0.09 VIII, 0.02 I+14.36 II (6.44 rod+7.91 ring)+0.01 III+4.80 IV+0.01V III, 0.06 I+17.67 II (11.01 rod+6.67 ring)+0.06 III+3.10 IV+0.01 VI and 1.11 I+11.31 II (5.80 rod+5.51 ring)+0.41 III+5.63 IV+0.03 VI+0.03 VIII, respectively. Configuration 16 II+4 IV and 12 II+6 IV occurred in the highest frequency among the autotetraploid restorers and hybrids. Meiotic chromosome behaviors were less abnormal in the tetraploids with high seed set than those with low seed set. The hybrids had fewer frequencies of bivalents, univalents, trivalents and multivalents than the restorers, but higher frequency of quatrivalents than the restorers at MI. The frequency of univalents at M1 had the most impact on pollen fertility and seed set, i.e., pollen fertility decreased with the increase of univalents. The secondary impact factors were trivalents and multivalents, and bivalents and quatrivalents had no effect on pollen fertility and seed set. The correlative relationship between pollen fertility and cytogenetic behaviors could be utilized to improve seed set in autotetraploidy breeding. The amylose content of autotetraploid indica mutant Rice D4063-1 dropped by half than diploid Minghui 63, that is, its amylose content of 5.23%.The whole sequence of Waxy gene of D4063-1 is amplified and sequenced. And the discrepancy of bases is found comparing to the reported Waxy gene. The Waxy gene of autotetraploid Rice D4063-1 had a base deletion in exon sequence, which resulted frameshift mutation in exon 9 and termination codon occur early. The mutation of Wx also led to the change of some common restriction endonuclease sites. Results showed compared to indica and japonica, D4063-1 had two adding sph1 sites. Compared to japonica, D4063-1 had six decreasing Acc1, a adding Xho1, Pst1 and Sal1 restriction sites. Phylogeny analysis shows that the DNA sequence of Waxy gene of D4063-1 is closer to Indica, and we suppose that the Waxy gene of D4063-1 is origin from genotype Wxa. In addition, according to the base differences of Wx in D4063-1, we deduce that RNA processing obstacle led by base change of intron is the main cause to low the amylose content, and related to phenotype of its soft rice. Based on analysis of fragments of D4063-1, indica and japonica and according to the special point of the three species, primers as markers-AUT4063-I were designed for distinguishing the D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them., rapid and correct identification of D4063-1 from other rice could be done. The genetic analysis is important to ensure the original of autotetraploid rice, for maintaining the “distinctiveness” of autotetraploid varieties, and to differentiate between the various genetic background of autotetraploid rice. The autotetraploid breeding will benefit from detailed analysis of genetic diversity in the germplasm collections. Further investigation on mechanisms of meiotic stability should benefit polyploid breeding. These findings demonstrated opportunity to improve meiotic abnormalities as well as grain fertilities in autotetraploid rice.

水稻同源四倍体高代自交系农艺性状分析及遗传差异研究

课题组在不断地创制新的同源四倍体材料的同时，连续多年以提高结实率为目的培育、筛选自交系材料，已获得自交繁殖十二年的高代自交系材料。相对于诱导创制初期，材料表现出的结实率低，同种系单株间的差异较大；高代材料已表现出较显著的结实率提升和较一致的农艺性状表型。 本实验选取课题组多年培育的同源四倍体水稻高代自交系材料，通过形态学、农艺性状和细胞遗传学比较，研究水稻同源四倍体与二倍体之间的异同。结果显示，所有同源四倍体材料的染色体组成均为2N=4X=48，花粉母细胞(PMC)减数分裂行为较正常，99%以上的染色体都能在减数分裂中期I（MI）发生联会、配对，形成四价体和二价体，这与理论染色体组构成相符。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。统计分析表明，二价体和四价体的比例对结实率没有显著影响，但是三价体的数目对结实率有一定影响。这一结果表明了结实率和细胞减数分裂行为可能存在相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了依据。 然后，对同源四倍体水稻高代自交系材料进行农艺性状和品质性状的统计与分析。主要针对结实率、每穗实粒数、有效分蘖和穗长等主要农艺性状，以及直链淀粉含量这一重要的品质性状进行统计。将统计结果与1996年诱导加倍的初代材料的数据相对比分析，结果显示所有材料经过多代选育培养，其农艺性状已经有了较显著的提高，同时同源四倍体材料的农艺性状稳定性也有了较显著的提升。如结实率的提高幅度较大，所有材料的平均结实率均显著高于加倍初代，而同种材料不同单株间的结实率差异也显著地减少，变异系数（CV）的平均值由1996年的41.15%减少到了2008年的28.81%。其他重要农艺性状也有不同程度的改良，种内变异系数也相应地降低。此外，实验测量了同源四倍体材料和来源二倍体材料的直链淀粉含量。分析结果显示，部分材料的直链淀粉含量与二倍体亲本产生了较显著的差异，这可能是诱导加倍过程中的遗传变异造成的；同源四倍体材料的种内变异系数（CV）平均值由1996年的6%下降到了2008年的3.88%，显示出在品质性状方面，同源四倍体材料的遗传稳定性也有较显著的增加。同源四倍体材料农艺性状经过多年的选育，表现出一定的提升，同时，经过多年自交纯化，所有材料种系内的性状差异逐渐缩小，说明同源四倍体水稻的遗传稳定性随着自交纯化而增强，这为同源四倍体水稻的进一步选育打下了良好的基础。 最后，通过测量连续两年的自交系材料的遗传多态性，分析材料间遗传差异和种群遗传结构，深入研究连续两代材料间的遗传差异，研究同源四倍体水稻与二倍体材料遗传稳定性之间的差异。实验采用18对SSR微卫星标记对连续两代15个材料，共94份样本进行差异分析。通过扩增条带长度多态性分析，计算不同材料以及同种材料不同世代间的遗传距离，构建同源四倍体和二倍体水稻的分子指纹库，并绘制聚类图。结果显示，同源四倍体和二倍体不同材料间的遗传差异比较大，遗传距离处于0.4757至0.2816之间；而相同品种不同世代材料间的遗传差异较小，但也表现出一定的遗传差异。同种同源四倍体材料不同世代间的遗传差异比二倍体材料更大，两代四倍体材料间遗传距离处于0.1359至0.0485之间；而两代二倍体材料间的遗传距离处于均小于0.0388。结果表明，同源四倍体水稻高代材料具有一定的遗传稳定性，但与来源二倍体材料相比，其世代间的遗传变异性仍然较强。这种结果说明，经过多代的自交纯化培育，同源四倍体水稻材料能够建立起相对稳定的遗传结构，同时，其强于二倍体亲本的变异性有能够为新品种的选育，农艺性状、品质性状的改良提供一定的遗传基础。此外，分析结果表明通过分子标记辅助检验，水稻材料间的遗传多态性能够有效地区分不同的品种，这为水稻品种的分子鉴定提供了一定的依据。 本研究从细胞学鉴定，农艺性状统计分析以及分子标记辅助聚类分析多方面地对同源四倍体水稻高代系进行了研究，对探究同源四倍体水稻的遗传规律，进一步揭示其遗传特性、农艺性状的遗传构成，为进一步选育优质的多倍体水稻提供了一定的理论依据。 This group insists on creating new Autotetraploid Rice (Oryza sativa L.) materials, while improving the seed-setting of them for many years, cultivated and selected the inbred line materials, has obtained the high generation inbred lines after twelve years cultivation. Compared to the early induced materials, which shown the low seed setting, and the large difference between the different plants in the same germ-line; the high generation materials have shown significant improvement in seed setting and more uniform phenotype agronomic traits. The autotetraploid rice high generation inbred lines material, which has been cultivated for more than 12 years, was chose in this experiment. The similarities and differences between autotetraploid and diploid rice was studied through morphological, agronomic and cytogenetic ways. The results showed that all the chromosome of autotetraploid materials are composed of 2N=4X=48, the pollen mother cells (PMC) meiosis behavior is normal, more than 99% chromosomes in metaphase I(MI) were federated and paired to form tetravalents or bivalents, which constitutes a consistent theory of genome. In the meiosis process, the material with a higher seed setting showed less chromosome abnormal than the material whose seed setting is lower. However, statistical analysis showed that the bivalent and tetravalent rate had no significant impact on seed setting, but the number of trivalent had a certain impact on seed setting. The result shows that the seed setting may be related to the meiosis behavior, which provides a basis to cultivate new autotetraploid germ line with high seed setting through the meiotic behavior. Furthermore, the agronomic and quality traits of autotetraploid rice high generation inbred material were statistically analyzed. The statistically analysis was focused on major agronomic traits such as: seed setting, grains per panicle, effective tillers and panicle length, as well as the important quality trait amylose content. The statistic data was compared with the data in 1996, when the first induced generation of autotetraploid material, and the result shows that after a multi-generation breeding, the agronomic traits has been significantly improved in all the materials, while the stability of agronomic traits also significant upgraded. For instant, the seed setting increased significantly, the average seed setting of all materials was significantly higher than the first induced generation, and the differences between different plants in the same species also significantly reduced, the average of the coefficient of variation (CV) was reduced from 41.15% in 1996 to 28.81% in 2008. Other important agronomic traits had improved in different degrees; the coefficient of variation within species is also reduced accordingly. In addition, the amylose content of autotetraploid and diploid materials was measured in this experiment. The results shows that the amylose content of some of the material differed from diploid parents significantly, it may caused by the genetic change during the inducing, autotetraploid materials intra-specific coefficient of variation (CV) average reduced from 6% in 1996 to 3.88% in 2008, shows that this is a significant increase of quality traits stability in autotetraploid rice. Agronomic traits of autotetraploid material shows some improvement after years of breeding, at the same time, after years of purification, all material within the germ-line gradually narrow the differences in traits indicates that autotetraploid rice genetic stability was enhanced, which laid a good foundation for the further autotetraploid rice breeding. Finally, this experiment studied the genetic differences between materials of two generations and researched the difference of genetic stability between diploid and autotetraploid rice materials through investigating the genetic polymorphism, genetic differences between materials and population genetic structure of inbred line materials of two consecutive years.18 pairs of SSR microsatellite markers for 15 materials of two generations were used in this experiment, and the total of 94 samples were analyzed. Through the amplification length polymorphism analysis of different materials and materials in different generations, the genetic distance between materials and generations was analyzed, a diploid and autotetraploid rice molecular fingerprint database and map rendering cluster were constructed. The result shows that the genetic distance is between 0.4757 to 0.2816 among different autotetraploid and diploid materials; the genetic distance between different generations of same species was less, but also shows a certain degree of genetic differences. The inter-generational genetic differences of autotetraploid materials were greater than of the diploid materials, which are 0.1359 to 0.0485 as the genetic distance; comparing with the 0.0388 of diploid materials. The result shows that high generation inbred autotetraploid rice material has a certain genetic stability, but the genetic variation between generations is still strong comparing with the source diploid materials. It indicates that, after many generations of purification cultivation, autotetraploid rice materials established a relatively stable genetic structure, at the same time, stronger variability than its diploid parents are useful in the breeding of new varieties, provides a genetic foundation to the agronomic and quality traits improvement. In addition, the analysis result shows that the through the molecular marker-assisted testing, rice genetic polymorphism between materials can effectively distinguish different species, provides a certain basis for molecular identification of varieties of rice. A series of investigation such as cytological identification, statistical analysis of agronomic traits, molecular marker-assisted cluster analysis was applied in this experiment to research genetic pattern of autotetraploid rice high generation inbred lines, revealed the genetic characteristics and the genetic composition of agronomic traits, provides a theoretical basis for the further selection of high quality autotetraploid rice.

中国翼手目六个类群的比较分子细胞遗传学研究

翼手类(Order Chiroptera)是哺乳动物中最为神秘的类群之一。它们也是哺乳动物中唯一真正具有空中飞翔能力的动物，而且多数物种还具有回声定位功能。翼手目分布十分广泛，除南极洲之外全球几乎任何地方都有分布，其数量约占整个哺乳动物种类的20％。然而，有关该类群的许多系统发育假说仍存有争议，至今其进化历史还不是很清楚，并且形态和分子研究所得出的结论经常不一致。近年来分子细胞遗传学的发展,特别是比较染色体涂色及其相关技术的应用，使人们可以快速、准确地鉴定种间的同源染色体（或同源染色体片段）。根据构建的染色体同源图谱及外群比较可确定一些细胞遗传学特征，并将其用于系统发育研究。虽然比较染色体涂色技术已广泛应用在哺乳动物的核型进化和系统发育研究中，但是在翼手目中却相当滞后。到目前为止，仅有少数蝙蝠物种有染色体涂色数据。 本研究分别利用三叶小蹄蝠（蹄蝠科）、大鼠耳蝠（蝙蝠科）及人的染色体特异性涂色探针，通过种间染色体涂色，首次比较系统的构建了狐蝠科（3种）、菊头蝠科（6种）、蹄蝠科（4种）、假吸血蝠科（1种）、鞘尾蝠科（1种）和犬吻蝠科（2种）共6个科17种蝙蝠间的染色体同源图谱，并将它们与已发表的染色体图谱进行整合。我们的结果不仅揭示了这些物种间的核型进化关系及其核型进化的主要机制，而且加深了我们对整个翼手目系统发育关系的认识。研究结果表明： 1） 长舌果蝠亚科和狐蝠亚科的果蝠部分物种的核型（2n = 36）可能代表了整个狐蝠科的祖先形式，而狐蝠亚科的犬蝠部分物种的核型属于衍生形式。2） 整臂保守性广泛存在于菊头蝠属物种的核型进化中，罗伯逊易位是其核型进化的主要方式。此外，整臂的相互易位也可能在其中起一定的作用。 3） 蹄蝠科核型进化的主要机制为罗伯逊易位和臂内倒位。而臂内倒位和异染色质扩增在蹄蝠属的物种形成中可能起了非常重要的作用。 4） 与蹄蝠属相比，三叶蹄蝠属物种的核型中可能包含更多的祖先染色体或染色体片段；中蹄蝠与大蹄蝠具有较近的亲缘关系；与中蹄蝠和大蹄蝠相比，小蹄蝠的核型较为原始。 5） 犬吻蝠科内来自不同属但具有相同二倍染色体数目的物种间核型非常保守；该科物种核型间的差异主要是由倒位和异染色质的分布引起的。 6） 菊头蝠超科的菊头蝠科、蹄蝠科和假吸血蝠科具有较近的亲缘关系，并且它们与大蝙蝠亚目的狐蝠科共有一些细胞遗传学特征，进一步支持了小蝙蝠亚目双系起源的观点。

三株肺癌细胞株的分子细胞遗传学研究

本课题采用流式细胞分选和双向及多色的染色体涂色等技术，详细地分析了三株非小细胞肺癌细胞系（YTMLC-90、GLC-82和A549）核型的异同，以揭示其核型特点，并希望能够找到三株细胞系共有的染色体断裂位点和可能发生的基因重组。染色体涂色技术揭示了三株细胞复杂的染色体数目和结构畸变。引人注目的是YTMLC-90和GLC-82具有相似且保守的核型，其中GLC-82的核型与其刚建立时的核型相比几乎没有发生改变。这与A549 的核型特点差别迥异。YTMLC-90 和GLC-82都有23 条异常染色体，除了i (5p) 有3个拷贝外，其余均为单拷贝；A549 有13条衍生染色体。在这三株细胞中，含有两个共有的断裂位点：HSA12q13 和 8q24.1。通过对NCBI的CancerChromosome数据库进行数据挖掘，发现8个可能的肿瘤相关基因位于这两个位点，其中3个已经被证实是肿瘤相关基因，其它5个基因可能是潜在的肿瘤抑制基因，需要进一步的实验来验证。

翼手目四个类群的比较分子细胞遗传学研究

翼手类(Order Chiroptera)是现生哺乳动物中唯一真正具有空中飞翔能力的动物；同时，它也是哺乳动物中为数不多的具有回声定位功能的几个分类类群之一。现存的翼手目动物约有1100种，占整个哺乳动物种类的20％以上，是哺乳动物中仅次于啮齿目的第二大目。尽管对于翼手目系统发育已经进行了大量的形态学和分子系统学等方面研究，但是翼手目中主要类群的系统发育关系尚存在着许多争议。进化过程中发生的染色体重排在揭示物种的系统发育关系中起到非常重要的作用。种间染色体涂色可以准确、快速、直观地鉴定物种进化过程中发生的大规模染色体重排，已成为在全基因组水平上比较研究哺乳动物核型多样性及其起源和演化的首选方法。利用染色体涂色可以构建不同物种间的比较染色体图谱，通过分析进化过程中保守的染色体片段在不同类群物种核型中的分布和排列方式，可以推导各类群可能的祖先核型，并重建伴随物种形成所发生的基因组变化历史，包括染色体重排的类型、速率和核型演化的趋势。但是，目前关于翼手目染色体涂色研究还很少，至今仍不能为翼手目各级分类水平上的系统发育关系研究提供系统完整的细胞遗传学证据。本研究利用大鼠耳蝠染色体特异涂色探针，通过种间染色体涂色，首次构建了狐蝠科、菊头蝠科、蹄蝠科和蝙蝠科代表物种间的比较染色体同源图谱，并且将它们与之前发表的用人的染色体探针构建的比较染色体图谱进行整合。通过分析这些物种之间保守染色体片段的分布，推测翼手目动物在核型进化过程中所发生的染色体重排的类型和数量，以期为研究翼手目的系统发育关系提供细胞遗传学证据。研究结果表明： 1. 翼手目动物的大多数染色体臂具有高度保守性，罗伯逊易位是其核型进化的主要机制。此外，倒位也是翼手目染色体进化过程中常见的染色体重排方式。 2. 小蝙蝠亚目的菊头蝠科和蹄蝠科与大蝙蝠亚目的狐蝠科亲缘关系更为接近，而与小蝙蝠亚目其它科的亲缘关系较远。 3. 菊头蝠科和蹄蝠科应该各自作为独立的科，但这两个科拥有一个共同的细胞遗传学衍生特征，这一特征在翼手目其它科中并未发现，表明它们之间有非常紧密的系统发育关系。 4. 菊头蝠科的祖先核型可能并不是前人所推测的全由端着丝粒染色体构成的2n＝62的核型；很有可能是包含有双臂染色体的，2n低于62的核型。 5. 三叶小蹄蝠比中蹄蝠保留更多的祖先染色体特征，其核型蹄蝠科中较为原始的核型。 6. 着丝粒融合是蝙蝠科物种核型演化的主要机制。蝙蝠科的祖先核型并不是类似于2n=44鼠耳蝠的核型，而可能更类似于棕蝠的2n＝50核型，由全端着丝粒染色体构成。 7. 除着丝粒融合外，异染色质的扩增也是蝙蝠科扁颅蝠属和山蝠属的染色体进化方式之一。 8. 绒山蝠与三种亚洲的伏翼共有两个同源染色体片段联合（MMY 8＋11和9＋13）,提示蝙蝠科的山蝠属和伏翼属有非常紧密的亲缘关系。 9. MMY 9 + 23和MMY 18＋19 这两个保守的同源染色体片段联合是蝙蝠科扁颅蝠属的细胞遗传学鉴定特征。 通过翼手目这些动物的染色体涂色研究，我们不但在全基因组的水平上揭示了翼手目几个主要类群代表动物的核型系统发育关系，而且所构建的比较染色体图谱将有助于基因组的序列信息从已测序物种（人）向序列信息较少的翼手目物种转移，这些研究结果也可以为翼手目的分类、物种鉴定、系统发育关系和生物医学研究（如SARS病毒）等提供基础性资料。

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.