Perspectives from agriculture: advances in livestock breeding - implications for aquaculture genetics

In this paper we present livestock breeding developments that could be taken into consideration in the genetic improvement of farmed aquaculture species, especially in freshwater fish. Firstly, the current breeding objective in aquatic species has focused almost exclusively on the improvement of body weight at harvest or on growth related traits. This is unlikely to be sufficient to meet the future needs of the aquaculture industry. To meet future demands breeding programs will most likely have to include additional traits, such as fitness related ones (survival, disease resistance), feed efficiency, or flesh quality, rather than only growth performance. In order to select for a multi-trait breeding objective, genetic variation in traits of interest and the genetic relationships among them need to be estimated. In addition, economic values for these traits will be required. Generally, there is a paucity of data on variable and fixed production costs in aquaculture, and this could be a major constraint in the further expansion of the breeding objectives. Secondly, genetic evaluation systems using the restricted maximum likelihood method (REML) and best linear unbiased prediction (BLUP) in a framework of mixed model methodology could be widely adopted to replace the more commonly used method of mass selection based on phenotypic performance. The BLUP method increases the accuracy of selection and also allows the management of inbreeding and estimation of genetic trends. BLUP is an improvement over the classic selection index approach, which was used in the success story of the genetically improved farmed tilapia (GIFT) in the Philippines, with genetic gains from 10 to 20 per cent per generation of selection. In parallel with BLUP, optimal genetic contribution theory can be applied to maximize genetic gain while constraining inbreeding in the long run in selection programs. Thirdly, by using advanced statistical methods, genetic selection can be carried out not only at the nucleus level but also in lower tiers of the pyramid breeding structure. Large scale across population genetic evaluation through genetic connectedness using cryopreserved sperm enables the comparison and ranking of genetic merit of all animals across populations, countries or years, and thus the genetically superior brood stock can be identified and widely used and exchanged to increase the rate of genetic progress in the population as a whole. It is concluded that sound genetic programs need to be established for aquaculture species. In addition to being very effective, fully pedigreed breeding programs would also enable the exploration of possibilities of integrating molecular markers (e.g., genetic tagging using DNA fingerprinting, marker (gene) assisted selection) and reproductive technologies such as in-vitro fertilization using cryopreserved spermatozoa.

Análise de marcadores moleculares do DNA mitocondrial em anuros da Mata Atlântica

A Mata Atlântica brasileira concentra uma das maiores diversidades biológicas da Terra com cerca de 7% das espécies animais e vegetais do planeta. Esse bioma abriga atualmente mais de 50% das espécies de anuros do Brasil (c.a. 500 espécies), mas sofre intensa perda e fragmentação de habitat. Um dos principais fragmentos da Mata Atlântica, a Reserva Ecológica de Guapiaçu (REGUA) abriga vasta anurofauna, com cerca de 71 espécies já descritas. Acredita-se, porém, que muitas ainda precisam ser identificadas e estudadas. A identificação de espécies baseada em caracteres moleculares vem se mostrando uma alternativa para dar suporte à identificação morfológica, e dentro deste contexto os genes de DNA mitocondrial, como o 16S, são utilizados para a realização de barcode. O objetivo deste estudo foi testar a metodologia de identificação molecular de espécies (DNA barcode) na comunidade de anfíbios anuros da REGUA utilizando o gene mitocondrial 16S. Para isso, foram coletados tecidos de 99 indivíduos, entre adultos e girinos de 23 espécies, agrupados em seis famílias distintas. Desses 99 indivíduos, 88 foram amplificados corretamente para o gene em referência e foram realizadas, com sucesso, a determinação de espécies de 84 anuros (95,45%) da REGUA. As espécies de Scinax albicans, Scinax flavoguttatus e Hylodes charadranaetes, cujas identificações haviam sido determinadas com base em critérios morfológicos, agruparam em clados de mesmo gênero, porém de espécies diferentes quando analisadas pelas metodologias neighbor-joining e maximum-likelihood. Além de altos valores de distância intraespecífica (2,18%, 3,49% e 3,77%, respectivamente) e distâncias interespecíficas nulas (0%) temos a indicação de possíveis equívocos em determinações de espécies feitas exclusivamente por critérios morfológicos. Nesse caso, as discordâncias morfológica e molecular são exclusivamente de girinos, demonstrando a dificuldade na identificação morfológica e a escassez de chaves de identificação dessas espécies em estágio larval. Os resultados mostram que o gene mitocondrial 16S teve seu uso na identificação de anuros da REGUA confirmada e apontam que, em casos de estudos com indivíduos em estágios larvais, como em girinos, a metodologia de barcode, quando complementada a identificação morfológica, suporta a correta identificação das espécies de anfíbios anuros.

Territorialidade e ecologia reprodutiva de Formicivora littoralis (Thamnophilidae) na Restinga da Massambaba, RJ

A distribuição espacial dos indivíduos é decorrente da presença e ausência de microhábitats adequados, sendo aqueles que se estabelecem nas melhores manchas favorecidos pela seleção natural. A aquisição de um território permite a manutenção do indivíduo e o sucesso reprodutivo. A reprodução é considerada de alto custo energético, pois há deslocamento dos recursos para a manutenção de uma prole em vez de serem incorporados no crescimento individual. Investir em uma prole não significa alcançar o sucesso reprodutivo. O sucesso reprodutivo pode ser afetado, por exemplo, por eventos de predação, disponibilidade de alimento e cuidado parental. Este último pode ser realizado por ambos os membros do par reprodutor ou por apenas um deles. A deserção do cuidado parental por um dos sexos pode ser uma resposta à cópulas extra-par. Formicivora littoralis tem distribuição muito restrita. É a única espécie de ave considerada endêmica de restinga e se encontra ameaçada de extinção, embora seja localmente abundante. O presente estudo teve como objetivos: 1) estimar os tamanhos de territórios e compara-los entre estação reprodutiva e não reprodutiva; 2) testar a influência do tamanho dos indivíduos e quantidade de vizinhos no tamanho do território; 3) descrever ninhos, ovos, filhotes e determinar o sucesso reprodutivo; 4) quantificar o cuidado parental; 5) desenvolver marcadores moleculares de microssatélites para determinar paternidade. Para os indivíduos capturados e marcados individualmente, foram obtidas amostras de sangue e medidas morfométricas (tarso, asa, cauda, comprimento total), além do peso. Os tamanhos dos territórios foram estimados pelo método do mínimo polígono convexo (unindo pontos onde machos foram registrados vocalizando). A densidade foi estimada com base no tamanho dos territórios. Aspectos da reprodução foram acessados por meio de busca mensal por ninhos e acompanhamento destes por dois dias consecutivos. Foram obtidas as taxas de predação e a quantificação do cuidado parental. Para a paternidade foram utilizados sete marcadores de microssatélites, desenvolvidos para este fim. Formicivora littoralis possui território pequeno (0,008 a 0,32ha), que varia de acordo com a estação (menor na estação reprodutiva). O tamanho do território não foi relacionado com o tamanho do indivíduo, mas apresentou resultado significativo quando comparado com a quantidade de territórios vizinhos, mostrando ser menor quanto maior o número de vizinhos. A espécie apresentou elevada densidade (0,53 a 1,15 indivíduos/km2). Com relação à reprodução, ninhos tem o formato de cesto aberto onde foram postos no máximo dois ovos. Os filhotes nasceram sem penas. A razão sexual no ninho foi igual em ambos os sexos. A taxa de predação foi elevada na fase de incubação quando comparada à fase no ninho após a eclosão. O cuidado parental (durante a incubação e com os filhotes) foi realizado pelos dois sexos, sem diferenças na proporção do investimento realizado. Dos nove ninhos analisados, todos contiveram pelo menos um ninhego proveniente de fertilização extra-par. Um total de 81,2% dos ninhegos (13 em 16) não foram prole biológica do macho do par reprodutor que realizava o cuidado parental e que se encontrava pareado socialmente com a fêmea. Essa taxa foi a mais elevada entre os estudos já realizados nos neotrópicos

The review of recent advances in fish genetics and biotechnology

Great advances have been, and are being made in our knowledge of the genetics and molecular biology (including genomics, proteomics and structural biology). Global molecular profiling technologies such as microassays using DNA or oligonucleotide chip, and protein and lipid chips are being developed. The application of such biotechnological advances are inevitable in aquaculture in the areas of improvement of aquaculture stocks where many molecular markers such as RFLPs, AFLDs and RAPD are now available for genome analysis, finger printing and genetic linkage mapping. Transgenic technology has been developed in a number of fish species and research is being pursed to produce transgenic fish carrying genes that encode antimicrobial peptides such as lysozyme thereby achieving disease resistance in fish. Also it is a short cut to achieving genetic change for fast growth and other desirable traits like early sexual maturity, temperature tolerance and feed conversion efficiency. KEYWORDS: Fish genetics, transgenesis, monoploidy, diploidy, polyploidy,gynogenesis, androgenesis, cryopreservation.

Evolução da deposição da matéria orgânica na Baía da Ilha Grande, RJ

Os ambientes costeiros vem sofrendo impactos devido a ocupação humana, um dos principais efeitos está na alteração da ciclagem de matéria orgânica (MO) na zona costeira. Para investigar as principais fontes de MO e avaliar como a atividade humana tem alterado a composição da MO sedimentar na baía da Ilha Grande (BIG), quatro testemunhos sedimentares (Abraão, Saco do Céu, TEBIG e Marina Piratas) foram datados e analisados para carbono orgânico, nitrogênio total, teor de finos, esteróis e n-álcoois. Os resultados indicam maiores contribuições de matéria orgânica alóctone nos testemunhos da enseada de Abraão e principalmente do Saco do Céu, estes testemunhos são enriquecidos em carbono orgânico e sedimentos finos, em comparação com os sedimentos coletados nas proximidades do TEBIG e da Marina Piratas. A partir dos anos de 1950 algumas alterações, como o incremento da contribuição fitoplanctônica,são observadas nos testemunhos foram indicadas pela Análise de Componentes Principais (PCA), destaca-se nesta época a acentuada mudança que ocorreu nas características granulométricas do testemunho da Marina Piratas. Estas alterações são efeitos indiretos da ação do homem que devido ao elevado crescimento populacional vem poluindo corpos dágua da região e destruindo ecossistemas de elevada importância como manguezais. No entanto através da análises dos marcadores de esgoto foi observado que a influência de MO de origem fecal nos sedimentos é muito pequena e cresceu muito pouco nas últimas décadas. Em conjunto com a elevada cobertura vegetal da região, os elevados índices pluviométricos regionais auxiliam a carrear grandes quantidades de material terrígeno para os sedimentos da baía como indicado pela predomínio de n-álcoois de cadeias longas

野大豆和栽培大豆抗盐分子标记的研究

野大豆群体3和群体4属盐渍群体，其个体有的是抗盐的，有的是敏感的，有的是中等抗盐的，本文通过随机扩增多态性DNA (RAPD)和DNA扩增指纹(DAF)分析野大豆群体抗盐性与分子标记之间的关系，从而更好地研究野大豆群体的盐适应机理。通过12个RAPD引物和3个DAF引物扩增发现：引物OPF05，OPF19和OPH02的扩增产物中有与抗盐性可能相关的特异标记，分别是OPF05_(213);OPF19_(4361);OPF19_(1727);OPF19_(1400);OPF19_(700);OPH02_(1350)。这些特异标记在所研究的抗盐植株中都存在;在敏感型植株中都不存在;在中等抗盐植株中有的存在，有的不存在。以上表明野大豆群体的抗盐性与RAPD分子标记有一定的相关性。为进一步研究抗盐性的特异标记，本文对栽培大豆抗盐品种Morgan和文丰七号的特异DAF标记片断8-27_(240) (Zhong et al., 1997)进行了克隆测序，测序结果通过BLASTn程序与基因库中的基因序列进行同源比较，发现上的DNA序列中的19组（每组大约二十到三十个碱基）序列与基因库中的其它基因相应序列有很高的同源性，几乎全部100%同源。尤其目的序列第15个碱基到第33个碱基（共19个碱基）之间的序列与基因库中的25个基因的相应序列的同源性全部是100%，并且与之相比的基因大多来自动物和人。因而推测其有可能是保守区，而不是编码区。进一步用DNASIS软件分析其碱基组成(A/T含量是64.9%，G/C含量是35.1%)并进行翻译，结果同样表明此序列可能是一调控序列并非编码区。至于这段序列是否与抗盐紧密相关，这有待于以后把此序列转到敏感型植株中然后检测其抗性来验证。 本文还通过RAPD分析野大豆群体3和群体4的多态性，发现群体3的多态性明显高于群体4。野大豆群体3的抗盐性大于群体4早已通过生理指标的鉴定，至于多态性与抗盐性之间是否有必然联系，还需进一步研究讨论。利用RAPD数据，通过MEGA软件中的NJ计算遗传距离的方法对群体3和4进行聚类分析，研究野大豆群体间及群体内个体间的亲缘及进化关系，探讨野大豆群体盐适应机理的分子起源。

盐渍条件下野大豆群体的DNA变异

当前分子生物学的方法以惊人的速度渗透到生命科学研究的各个领域。植物对不断变化的环境逐步适应的过程中，积累了丰富的遗传多样性。与此同时，人类活动空间的不断扩大已经严重威胁到其他生命的生存和繁衍，越来越多的物种以越来越快的速度在我们还没有来得及认识它们时就已经永远地消失了。加快物种鉴定和保护的步伐就必须发展更多能充分揭示物种遗传多样性的实验技术，从具有丰富遗传多样性的野生资源中寻找到更多能够服务于人类可持续发展的基因资源。本文以杨树杂交后代过氧化物同工酶和RAPD分析为基础，论证了我们改进的RAPD方法用于遗传分析的可行性。在前期工作的基础上，进一步测定了野大豆自然群体的耐盐性变异，并且用微卫星和RAPD分析的方法研究分子标记与DXA变异、植株耐盐性之间的关系。对四个可能与抗盐性有关的RAPD片段进行克隆、测序，并进行序列比较。由此得出以下结论： 1、在本文的实验条件下，杨树同工酶和RAPD分析均表明，RAPD标记在亲本及其杂交后代中性状比例符合孟德尔遗传规律，尽管有时也会出现遗传负载等机制引起的基因分布扭曲现象。 2、初步研究了个体发育阶段和环境条件对植株耐盐性的影响。结果表明，植物耐盐性不仅仅与外界的盐度有关，而且受发育阶段和其它环境条件（如，温度）的影响。但也发现了某些个体在各种条件下都具有较高的耐盐性，而且，不易受到其它环境条件的影响。 3、微卫星标记的结果表明，10对引物中的8对引物共检测到时17个等位基因，平均每对引物2.125个等位基因。本文的实验条件下，双核苷酸和三核苷酸的引物对扩增产物都没有出现“ghosts"条带或“打滑”现象。 4、有4个RAPD标记可能与野大豆群体的耐盐性有关，分别是OPCO8460bp、OPCO8213bp、OPCO2690bp、以及OPCO5270bp。测序结果与GenBank中的序列作同源性比较，结果显示，OPCO2_(690bp)与小麦、松树等植物的吉普赛性的逆转录转座子的部分区域（24－－53）有很高的同源性（86－89％）。此外，OPCO2690bp与栽培大豆胞质谷氨酰胺合成酶（gs15）基因的启动子有高达95％的同源性。 5、本文实验条件下，RAPD扩增产物在限制性内切酶消化后，消化产物的多态性未见增大，也没有发现与耐盐性相关的多态位点。 6、野大豆自然群体DNA变异的研究中也可以应用SWAPP方法。

Connectivity of coral reefs between marine park and non-marine park islands in the Malacca Straits

This study investigates the genetic population and gene flow in the clownfish (Amphiprion ocellaris), across the Langkawi and Payar Archipelago by analysis of molecular markers in the mitochondrial region.

性激素对抗体分泌细胞表面分子表达和抗体分泌的影响

　目的:探讨性激素调控抗体分泌细胞游走到生殖道的分子机制和对局部抗体分泌的影响。方法:以鼠源杂交 瘤细胞SG2 和PA4 为对象,检测其性激素受体、CXCR4 的mRNA 和CD31 的表达;用流式细胞术和ELISA 检测不同浓度性激素 作用下,抗体分泌细胞表面与细胞游走相关分子的表达和抗体分泌的变化。结果:SG2 和PA4 细胞表达雄激素受体和雌激素 受体β,表达CXCR4 ,但不表达CD31。性激素对抗体分泌细胞表面分子的表达和抗体分泌量并没有明显的影响。结论:性激素 对抗体分泌细胞游走的调控作用与细胞自身相关黏附分子的变化关系不大,推测可能主要是通过对内皮细胞地址素表达的 影响来实现的。

Circulating immunoglobulin A- and immunoglobulin G-secreting hybridoma cells in peripheral blood preferably migrate to female genital tracts. The role of sex hormones

Antigen-specific circulating immunoglobulin-secreting cells (ISC) migrate to various secondary and tertiary lymphoid tissues. To understand the migration of the cells into the genital tract and its regulation by sex hormones, spleen-derived SG2 hybridoma cells secreting immunoglobulin G2b (IgG2b) and Peyer's patch-derived PA4 hybridoma cells secreting polymer IgA were labelled with (3) H-TdR, and intravenously injected into syngeneic mice of both sexes. Using flow cytometry, surface molecular markers of plasma cells, CD38 and CD138, and adhesion molecules, CD49d, CD162, and CD11a were found to be positive in SG2 and PA4 cells, but CD62L, alpha4beta7 and CD44 were not expressed on these cells. The relative distribution indexes (RDIs) of the cells in genital tract and other tissues were measured. The means of RDIs of SG2 and PA4 cells in female genital tissues were 6.5 and 4.5 times as many as the means in male genital tissues, respectively. The treatment of ovariectomized mice with beta-oestradiol significantly increased the RDIs of PA4 cells in cervix and vagina, but decreased the RDIs of SG2 cells in vagina, horn of uterus, uterus and rectum (P <0.05). Progesterone treatment increased the RDIs of PA4 cells in vagina and rectum (P <0.05). The treatment with testosterone significantly increased the RDIs of SG2 and PA4 cells in epididymis and accessory sex glands (P <0.05). These results demonstrate that the female genital tract is the preferable site for the migration of circulating hybridoma cells to the male genital tract, and sex hormones play an important role in regulation of the migration of circulating ISC to genital tracts.

Microsatellite marker isolation and cultured strain identification in Carassius auratus gibelio

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.

Utility of ITS1-5.8S-ITS2 sequences for species discrimination and phylogenetic inference of two closely related bucephalid digeneans (Digenea : Bucephalidae): Dollfustrema vaneyi and Dollfustrema hefeiensis

The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from 60 specimens belonging to two closely related bucephalid digeneans (Dollfustrema vaneyi and Dollfustrema hefeiensis) from different localities, hosts, and microhabitat sites were cloned to examine the level of sequence variation and the taxonomic levels to show utility in species identification and phylogeny estimation. Our data show that these molecular markers can help to discriminate the two species, which are morphologically very close and difficult to separate by classical methods. We found 21 haplotypes defined by 44 polymorphic positions in 38 individuals of D. vaneyi, and 16 haplotypes defined by 43 polymorphic positions in 22 individuals of D. hefeiensis. There is no shared haplotypes between the two species. Haplotype rather than nucleotide diversity is similar between the two species. Phylogenetic analyses reveal two robustly supported clades, one corresponding to D. vaneyi and the other corresponding to D. hefeiensis. However, the population structures between the two species seem to be incongruent and show no geographic and host-specific structure among them, further indicating that the two species may have had a more complex evolutionary history than expected.

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides(CT)(10) and (GT)(10) served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.

Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp (Carassius auratus gibelio bloch) as revealed by RAPD assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp.

粗枝云杉( Picea asperata Mast.)天然群体的遗传变异

西南地区在我国的经济发展和生态环境建设中占重要地位，但也是我国生态环境最脆弱的地区之一，生态系统退化，生态功能减弱，严重制约着西南林业的可持续经营与发展。本项目采用DNA 分子标记SSR 研究不同生境条件下粗枝云杉群体的遗传变异及其时空分布格局，考察遗传变异与复杂的山地生态环境间的潜在联系，系统地揭示粗枝云杉天然群体与环境系统相互作用的生态适应与分子进化机制。粗枝云杉适应性强，生长迅速，在植树造林和工业用材方面占有重要地位，研究成果可为中国西南部亚高山天然林的可持续经营及退化生态系统的恢复与重建提供理论依据和科学指导。主要研究结果如下： 1. SSR 位点变异丰富，等位基因频率的分布格局多样。7 个SSR 标记全是多态位点，每位点的等位基因数变化范围为13~24，平均为19.9 个。SSR 位点的等位基因片段长度范围变化较大。73.1%的等位基因变异遵循逐步突变模型(SSM)而发生1 个重复基元的变化，22.3%和4.6%的变异分别按两阶段突变模型(TMP)发生1 个重复基元以上的变化和在SSR 位点侧翼区发生1 个碱基变化的插入-删除事件。 2. 粗枝云杉拥有中等偏高水平的遗传多样性和相对大的群体间遗传分化。通过分析代表10 个群体的250 个个体在7 个SSR位点的变化，调查了源自中国西南山区的粗枝云杉的微卫星变异。相当高的遗传多样性和强烈的群体分化发生在粗枝云杉中， 其群体平均Nei's 期望杂合度为0.707 ， 群体间遗传距离为0.121~0.224(FST)和0.100~0.537(RST)。然而，群体间遗传距离与地理距离之间无相关性，从而排除了简单的距离分离模式并暗示迁移不是影响粗枝云杉遗传变异格局的主要因素。事实上，使用私有等位基因估算的基因流数量非常低，仅等于0.753。等位基因置换检验(Allele permutation tests)揭示逐步突变及遗传漂变都对群体间分化有贡献。另外，在多数位点检测到显著的群体间遗传差异，这个结果说明自然选择，假设通过环境压力，是引起粗枝云杉微地理分化的主要因素之一。根据SSR基因型，250 个粗枝云杉个体的70%被正确地归类入其各自的来源群体，结果表明微卫星(SSR)对区分来自中国不同生态地理位点的粗枝云杉基因型是有效的。 3. 在SSR、RAPD 和AFLP 位点，显著的群体间遗传结构被发现的，但三种标记间遗传分化程度和群体遗传关系有差异。利用来自10 个群体的247 个个体，我们报告了关于样本粗枝云杉群体间遗传关系的总体看法。根据各自对评价遗传关系的信息能力和适用性，SSR、RAPD 和AFLP 标记被选用，三种技术非常有效地区别这些基因型。使用的SSR、RAPD 和AFLP 标记分别估计平均Dice 相似性系数。Mantel 检验产生显著但相对低的共表型适合度(RAPD = 0.63£AFLP = 0.60和SSR = 0.75)。比较三种标记系统，RAPD 和AFLP 共表型指数相对高地相关(r =0.59)，而RAPD 和SSR 及SSR 和AFLP 之间的相关系数分别是0.53 和0.35。所有系统树，包括不同标记资料结合获得的系统树，反映了多数群体依据它们的地理条件而成某种特定关系。结果暗示单个或结合标记系统能用来深入洞察粗枝云杉遗传研究，并且不同标记系统合并资料能提供更可靠的信息。 Southwestern region plays an important role in economic developmentand ecological construction in China. Yet, it is also one of the weak regionsof ecological environment in China with degraded ecosystem and imperfectfunction, which restricts the sustaining management and development ofsouthwestern forestry. The genetic variation and spatial distribution patternof P. asperata populations originating from different habitats wereinvestigated using SSR molecular markers in this study. The correlationsbetween genetic variation and ecological and environmental conditionswere detected, and the interaction between P. asperata populations andenvironmental system and the mechanism of ecological adaption -molecular evolution were revealed. Given the significant ecological andeconomic roles of the fast-growing and wide-adaptive species in reforestation and production of pulp wood and timber, the study couldprovide a strong theoretical evidence and scientific direction for thesustaining management of subalpine natural forest, and the afforestationand rehabilitation of degraded ecosystem. The results are as follows: 1. The genetic variation at SSR loci was abundant and the distributionof allelic frequencies was uneven. All seven loci were polymorphic, and thenumber of alleles per locus varied from 13 to 24 with a mean valueequaling 19.9. The allele sizes at SSR loci were found to vary widely.73.1% of allelic variation followed stepwise mutation model (SSM) whichresults increase or decrease by one repeat type, and 22.3% and 4.6% wereresulted from two-phase mutation model (TMP) with allele size varying bymore than one repeat type and from insertion-deletion events in theflanking regions at SSR loci with a single basepair changing, respectively. 2. P. asperata possessed a moderate to high level of genetic diversityand considerable genetic differentiation. Microsatellite variation of P.asperata. originating from the mountains of southwestern China wasinvestigated by analyzing variation at seven SSR loci in 250 individualsrepresenting ten populations. A fair degree of genetic diversity and strongpopulation subdivision occurred with the mean gene diversity (H) of 0.707,and genetic distances among populations varying between 0.121 and 0.224(FST) and between 0.100 and 0.537 (RST). However, inter-populationgenetic distances showed no correlation with geographic distances between the population sites. This ruled out a simple isolation by distance modeland suggested that migration does not have a great impact. In fact, theamount of gene flow, detected using private alleles, was very low, equalingonly 0.753. Allele permutation tests revealed that stepwise-like mutations,coupled with genetic drift, could contribute to population differentiation.Moreover, significant genetic differences between populations weredetected at most loci. The results indicate that natural selection, presumablythrough environmental stress, may be one of the main factors causingmicro-geographical differentiation in the genetic structure of P. asperata.Based on SSR genotypes, 70% of the 250 individuals were correctlyclassified into their sites of origin. This suggests that microsatellites (SSRs) are effective in distinguishing genotypes of P. asperata originating fromdiverse eco-geographical sites in China. 3. Using a set of 247 individuals from ten P. asperata populations wereport an overview on the genetic relationship among the sampled P.asperata populations. RAPD, AFLP and SSR were used in terms of theirinformativeness and applicability for evaluate relationship and all threetechniques discriminated the genotypes very effectively. Mean Dicesimilarities coefficient were estimated using RAPD, AFLP and SSR,respectively. The Mantel test resulted in a significant but relatively low fit(RAPD = 0.63, AFLP = 0.60 and SSR = 0.75) of cophenetic values.Comparing the three marker systems to each other, RAPD and AFLP cophenetic indices were highly correlated (r = 0.59), while correlationcoefficient between RAPD and SSR was r = 0.53 and between SSR andAFLP was r = 0.35. For all markers a relatively high similarity indendrogram topologies was obtained although some differences wereobserved. All the dendrograms, including that obtained by the combineduse of all the marker data, reflect some relationships for most of thepopulations according to their geographic conditions. The results indicatethat single or combined marker system could be used to insight into geneticstudy in P. asperata and the combined data of different marker systems canprovide more reliable information.