Genetic analysis of juvenile coho salmon (Oncorhynchus kisutch) off Oregon and Washington reveals few Columbia River wild fish

Little is known about the ocean distributions of wild juvenile coho salmon off the Oregon-Washington coast. In this study we report tag recoveries and genetic mixed-stock estimates of juvenile fish caught in coastal waters near the Columbia River plume. To support the genetic estimates, we report an allozyme-frequency baseline for 89 wild and hatchery-reared coho salmon spawning populations, extending from northern California to southern British Columbia. The products of 59 allozyme-encoding loci were examined with starch-gel electrophoresis. Of these, 56 loci were polymorphic, and 29 loci had P0.95 levels of polymorphism. Average heterozygosities within populations ranged from 0.021 to 0.046 and averaged 0.033. Multidimensional scaling of chord genetic distances between samples resolved nine regional groups that were sufficiently distinct for genetic mixed-stock analysis. About 2.9% of the total gene diversity was due to differences among populations within these regions, and 2.6% was due to differences among the nine regions. This allele-frequency data base was used to estimate the stock proportions of 730 juvenile coho salmon in offshore samples collected from central Oregon to northern Washington in June and September-October 1998−2000. Genetic mixed-stock analysis, together with recoveries of tagged or fin-clipped fish, indicates that about one half of the juveniles came from Columbia River hatcheries. Only 22% of the ocean-caught juveniles were wild fish, originating largely from coastal Oregon and Washington rivers (about 20%). Unlike previous studies of tagged juveniles, both tag recoveries and genetic estimates indicate the presence of fish from British Columbia and Puget Sound in southern waters. The most salient feature of genetic mixed stock estimates was the paucity of wild juveniles from natural populations in the Columbia River Basin. This result reflects the large decrease in the abundances of these populations in the last few decades.

Morphometric studies in Penaeus merguiensis from Ratnagiri waters for selection of the brood stock in genetic improvement programmes

Studies on genetic improvement of penaeid prawns for the character higher tail weight using methods of selective breeding were undertaken. Prior to the actual breeding experiments it was necessary to find out the quantum of available variability in the character tail weight amongst the natural populations of Penaeus merguiensis from the Indian waters. Thirteen morphometric variables were measured and various statistical analyses were carried out. The tail weight showed almost double values of coefficient of variation in the females than the males (C.V. 20.37 and 11.08 respectively). The combination of the characters viz. sixth segment length (SSL), sixth segment depth (SSD) and posterior abdominal circumference (PAC) gave the highest R super(2) values. These variables were easy to measure and gave maximum variation in the character tail weight without sacrificing the breeders in the brood stock. The quantitative character tail weight was influenced by both genetic and environmental factors was statistically ascertained by applying 2-Factor analysis.

Genetic structure of Chinese sucker population Myxocyprinus asiaticus in the Yangtze River based on mitochondrial DNA marker

The sequencing analysis of the mitochondrial DNA control region (mtCR DNA) was performed to assess the genetic divergence and population structure of the Chinese sucker Myxocyprinus asiaticus (Cypriniformes Catostomidae) using four sample lots from natural populations of the Yangtze River. The mtCR DNA sequences of approximately 920 base pairs were obtained. A total of 223 nucleotide positions were polymorphic, and these defined 39 haplotypes. Of the 39 haplotypes, 37 (90%) were not shared, and among the populations as a whole there was little sharing of haplotypes. The average haplotype diversity (0.958) and the average nucleotide diversity (0.052) indicated a higher level of genetic diversity of Chinese sucker through the river. Analysis of molecular variation (AMOVA) of data revealed significant partitioning of variance (P<0.001) among populations (60.29%), and within populations (39.71%). The topology according to the neighbor joining and maximum parsimony methods showed mosaic composition of the 39 haplotypes, suggesting that the populations wore not completely divergent. The pairwise F statistic values, however, indicated that the population structuring existed to some extent among the geographic populations. There was a positive relationship between the aquatic distance and the genetic distance (Fst) among the populations (P<0.05). Based on our data, it is suggested that genetic drift, gene flow, and stochastic events are the possible factors influencing the population structure and genetic variation.

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.

中国西南地区青杨(Populus cathayana Rehd)遗传多样性的ISSR研究

青杨作为一个本土树种，能较好的适应潮湿和寒冷的环境，对中国西部的人工造林有着重要的参考价值。在本实验中，选取7个中国西南地区分布的自然群体，用ISSR(inter-simple sequence repeats)作为分子标记研究其遗传多样性水平和遗传结构。通过筛选的8个ISSR引物，获得了158条清晰可重复的DNA条带，其中有156条具有多样性(占98.7%)。平均的Nei’s遗传多样性(h)为0.331；遗传分化系数(GST)为0.477，这表明有47.7％的遗传多样性发生在群体间。这种高水平的分化可能是由于当地复杂多变的地形和气候特点阻碍了基因流而引起的。此外在这7个青杨群体中，遗传距离和地理距离并未体现出有显著相关性（r=0.3122, P>0.05)。联合遗传距离和地理距离分析，鉴定出两处低水平基因交流的地区, 探讨其遗传障碍形成原因。 As a native species to China, Populus cathayana Rehd is well-adapted to the wet and cold environments where it occurs. It is considered to be an important reforestation species in western China. In the present study, we surveyed the level of genetic variation and the pattern of genetic structure in seven natural populations of P. cathayana, originating from the southeastern Qinghai-Tibetan Plateau of China, by using ISSR (inter-simple sequence repeats) markers. Based on eight primers, 158 clear and reproducible DNA fragments were generated, of which 156 (98.7%) were polymorphic. The average value of Nei's gene diversity (h) equaled 0.331. The coefficient of genetic differentiation (GST) equaled 0.477, which means that 47.7% of the total molecular variance existed among populations. Such a high level of divergence present among populations may be caused by the complex topography and variable climatic conditions present in the southeastern Qinghai-Tibetan Plateau which effectively restrict gene flow. Moreover, there is a lack of significant association between genetic and geographical distances (r=0.3122, P>0.05) in the populations of P. cathayana. The application of a novel method, which combines geographical coordinates and genetic differentiation to detect barriers for gene flow, allowed us to identify two zones of lowered gene flow.

中国卧龙自然保护区不同海拔川滇高山栎(Quercus aquifolioides)群体的遗传变异

海拔梯度造成的环境异质性，如崎岖的地形、复杂的植被结构以及花期延迟等可能会极大地影响到物种的形态和遗传变异格局。理解物种形态和遗传变异的海拔格局对于物种多样性的管理和保护是非常重要的。尽管植物群体遗传学是一个飞速发展的研究领域，然而与海拔相关的形态变异、遗传变异及群体间遗传差异的研究却很少。到目前为止，还不清楚遗传变异与海拔之间是否必然的相关性。 川滇高山栎是一种重要的生态和经济型树种，广泛分布于中国西南的四川、西藏、贵州和云南省的高海拔地区，在保持水土、调节气候方面起着十分重要的作用。尽管主要受阳光限制而仅分布于阳坡，但其海拔梯度范围较大，表明川滇高山栎对不同的环境具有很强的适应性。本文通过叶型及生理响应、微卫星分子标记和扩增性片段长度多态性方法，试图探索川滇高山栎叶沿海拔梯度的形态和生理响应及其沿海拔梯度的遗传变异格局，为川滇高山栎的保护和利用提供进一步的遗传学理论依据和技术指导。 对叶形、含氮量及碳同位素的试验结果表明，平均比叶面积、气孔密度、气孔长度和气孔指数等气孔参数随海拔的升高呈非线性变化。在海拔大于2800 m时，川滇高山栎的比叶面积、气孔长度和气孔指数都随海拔升高而降低，但是在海拔小于2800 m时，这些指标都随海拔的升高而增大。相对而言，单位叶面积的含氮量和碳同位素则表现出相反的变化模式。另外，比叶面积是决定碳同位素沿海拔梯度变化的最重要参数。本研究结果表明，海拔2800 m附近是川滇高山栎生长和发育的最适地带，在这里生长的植物叶片厚度更薄、气孔更大、叶碳同位素值更小。 利用六对微卫星引物对五个不同海拔川滇高山栎群体遗传多样性进行研究，结果表明，群体内表现出较高的遗传多样性，平均每位点等位基因数11.33个，平均期望杂合度达0.820。群体间差异较小，分化仅为6.6％。聚类分析也并没有显示出明显的海拔格局。然而低频率等位基因却与海拔呈显著性正相关(R2=0.97, P < 0.01)，表明在高海拔处，川滇高山栎以更多的稀有基因来适应恶劣的环境条件。本试验结果表明由海拔梯度形成的选择性压力对川滇高山栎群体的遗传变异影响并不明显。 为了进一步探讨川滇高山栎群体遗传变异与海拔之间的相互关系，我们还对其进行了扩增性片段长度多态性分析。结果表明：(1)随海拔的升高(从群体WL2到群体WL5)，群体内遗传变异降低，而群体间遗传差异增加；(2)低海拔群体WL1表现出最低的遗传变异性(HE = 0.181)，同时与其余四个群体间呈现出最大的遗传差异性(平均FST = 0.0596)；(3)在除去低海拔群体WL1后，Mantel检测表明群体间遗传距离与海拔距离之间表现出正相关性。另外，研究结果还表明，遗传变异受生境条件(过度的湿热环境)及人为干扰(火烧、砍伐和放牧)的影响，这一点至少在低海拔群体WL1上发生了作用。 通过叶形态、生理及DNA分子水平的研究，结果表明叶形态特征和碳同位素与海拔紧密相关，与海拔之间呈非线性变化，海拔2,800 m附近是川滇高山栎生长和发育的最适地带。海拔梯度在一定程度上会影响到川滇高山栎群体的遗传变异结构，但在这样一个狭窄的地理分布区域里，这种影响并不足以导致群体间较大的遗传分化。同时生境条件及人为干扰也是影响遗传变异的限制性因子，不容忽视。 Altitudinal gradients impose heterogeneous environmental conditions, such as rugged topography, a complex pattern of vegetation and flowering delay, and they likely furthermore markedly affect the morphological and genetic variation pattern of a species. Understanding altitudinal pattern of morphological and genetic variation at a species is important for the management and conservation of species diversity. Although plant population genetics is a fast growing field of research, there are only few recent investigations, which analyzed the genetic differentiation and changes of intra-population variation along altitudinal gradients. At present, it is still unclear whether there are some common patterns of morphological and genetic variation with altitude. Quercus aquifolioides Rehder & E.H. Wilson, which is an important ecological and economical endemic woody plant species, is widely distributed in the Yunnan and Sichuan provinces, Southwest China. Its large range of habitat across different altitudes implies strong adaptation to different environments, although it is mainly restricted to sunny, south facing slopes. It plays a very important role in preventing soil erosion, soil water loss and regulating climate, as well as in retaining ecological stability. In this paper, we tried to understand the altitudinal pattern of morphological and genetic variation along altitudinal gradients through the experiments of leaf morphological and physiological responses, microsatellite analysis and AFLP markers. In leaf morphological and physiological responses experiment, we measured leaf morphology, nitrogen content and carbon isotope composition (as an indicator of water use efficiency) of Q. aquifolioides along an altitudinal gradient. We found that these leaf morphological and physiological responses to altitudinal gradients were non-linear with increasing altitude. Specific leaf area, stomatal length and index increased with increasing altitude below 2,800 m, but decreased with increasing altitude above 2,800 m. In contrast, leaf nitrogen content per unit area and carbon isotope composition showed opposite change patterns. Specific leaf area seemed to be the most important parameter that determined the carbon isotope composition along the altitudinal gradient. Our results suggest that near 2,800 m in altitude could be the optimum zone for growth and development of Q. aquifolioides, and highlight the importance of the influence of altitude in research on plant physiological ecology. Genetic variation and differentiation were investigated among five natural populations of Q. aquifolioides occurring along an altitudinal gradient that varied from 2,000 to 3,600 m above sea level in the Wolong Natural Reserve of China, by analyzing variation at six microsatellite loci. The results showed that the populations were characterized by relatively high intra-population variation with the average number of alleles equaling 11.33 per locus and the average expected heterozygosity (HE) being 0.779. The amount of genetic variation varied only little among populations, which suggests that the influence of altitude factors on microsatellite variation is limited. However, there is a significantly positive correlation between altitude and the number of low-frequency alleles (R2=0.97, P < 0.01), which indicates that Q. aquifolioides from high altitudes has more unique variation, possibly enabling adaptation to severe conditions. F statistics showed the presence of a slight deficiency of heterozygosity (FIS=0.136) and a low level of differentiation among populations (FST=0.066). The result of the cluster analysis demonstrates that the grouping of populations does not correspond to the altitude of the populations. Based on the available data, it is likely that the selective forces related to altitude are not strong enough to significantly differentiate the populations of Q. aquifolioides in terms of microsatellite variation. To further elucidate genetic variation pattern of Q. aquifolioides populations under sub-alpine environments, genetic variation and differentiation were investigated along altitudinal gradients using AFLP markers. The altitudinal populations with an average altitude interval of 400 m, i.e. WL1, WL2, WL3, WL4 and WL5, correspond to the altitudes 2,000, 2,400, 2,800, 3,200 and 3,600 m, respectively. Our results were as follows: (i) decreasing genetic variation (ranging from 0.253 to 0.210) and increasing genetic differentiation with altitude were obtained from the WL2 to the WL5 population; (ii) the WL1 population showed the lowest genetic variation (HE = 0.181) and the highest genetic differentiation (average FST = 0.0596) with the other four populations; (iii) the positive correlation was obtained using Mantel tests between genetic and altitude distances except for the WL1 population. Our results suggest that altitudinal gradients may have influenced the genetic variation pattern of Q. aquifolioides populations to some extent. In addition, habitat environments (unfavorable wet and hot conditions) and human disturbances (burning, grazing and felling) were possible influencing factors, especially to the low-altitude WL1 population. The present study shows that there were close correlations between morphological features and carbon isotope composition in our data. This indicates that a coordinated plant response modified these parameters simultaneously across different altitudes. Around 2,800 m altitude there seems to be an optimum zone for growth and development of Q. aquifolioides, as indicated by thinner leaves, larger stomata and more negative d13C values. All available evidence indicates altitudinal gradients may have influenced the genetic variation pattern of Q. aquifolioides to some extent. Decreasing genetic variation and increasing genetic differentiation with altitude was obtained except for the WL1 population. And the environment of habitats and human disturbances were also contributing factors, which impact genetic variation pattern, especially to the low-altitude WL1 population.

青藏高原东缘青杨（Populus cathayana Rehd.）遗传多样性研究

青杨（Populus cathayana Rehd.）是青杨派杨树的主要树种之一，为我国特有乡土树种，其主要分布区之一是我国的青藏高原，集中分布地带在甘肃省中部及青海省东部，四川省西北部岷江上游和松潘等地区。本研究以青藏高原东缘青杨天然分布区的6个群体143个个体为材料，用AFLP、SSR和叶绿体SSR分子标记分析青杨天然群体的遗传多样性，分析其遗传结构和分化，比较6个群体间遗传多样性的高低和群体间的遗传关系。旨在为青杨基因资源评价、保护与保存、遗传改良策略制定等提供科学理论依据。通过以上研究，得出如下主要研究结果： 1 AFLP分子标记研究结果 采用4对选择性引物对6个青杨天然群体143个个体进行分析，扩增谱带分析共检测到175个位点，其中173个位点表现为多态，多态位点百分率高达98.9%。从整体上表现出较高的遗传多样性，Nei’s基因多样度(h)水平为0.306。从青杨天然群体位点分布来看，有高达20%的位点（32位点）为群体所特有，仅有9.14%的位点（16位点）在所有群体中存在。群体间的遗传分化极大，所有遗传变异中，有48.9%的遗传变异存在于群体间。在个体群丛（Individuals cluster）和主坐标（PCO analysis）分析中，青杨各群体未呈现任何地理模式，Mantel检测也显示各群体间遗传距离与地理距离无明显相关。研究认为，由于地理和空间上大尺度的隔离和地形地貌复杂使得群体间无法进行基因交流，导致群体间遗传分化极大，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 2 SSR分子标记研究结果 在SSR分析中，7个位点在6个青杨天然群体143个个体中共检测到79个等位基因，每位点检测到的等位基因数在5-16之间，平均11.3个，总体上多态位点百分率达100%。平均观察杂合度和期望杂合度分别为0.792和0.802。Hardy-Weinberg平衡检验表明青杨大部分群体都处于非平衡状态，群体大部分位点都是偏离哈迪-温伯格平衡（76.3%），只有23.7%的测验满足哈迪-温伯格平衡。分析青杨天然群体内和群体间的遗传变异，基因分化系数（GST）为0.373，即有62.7%的遗传变异存在群体内，37.3%的遗传变异存在群体间。群体内的遗传变异高于群体间水平。根据各群体遗传距离UPGMA聚类分析，有来自相临分布区、近似气候类型的群体聚在一起的趋势，但Mantel检测反映遗传距离与地理距离间并无明显相关性。 3 cpSSR分子标记研究结果 分析来自青藏高原东缘6个青杨天然群体，所用cpSSR引物中有5对cpSSR引物（CCMP2、CCMP5、SCUO01、SCU03、SCU07）都表现较高的多态性，单个引物检测的片段数都在4以上。5对cpSSR引物共检测片段数26个，组成了12种叶绿体DNA单倍型。各群体的单倍型分布和频率有较大差异，群体单倍型多样性范围为0-0.4926，TS、JZ、PW和SHY群体单倍型多样性高于QHY和LED群体水平。本研究发现，分布在青藏高原东缘的青杨天然群体，群体间不存在共享的单倍型，各群体间存在极大的遗传分化（GST=0.9223）。从青藏高原东缘地区经历的地质历史事件来看，第四纪的冰期气候变迁可能是造成青杨现今遗传结构模式的主要因素之一。根据单倍型在各群体的分布情况，进行青杨群体聚类分析结果，各群体无明显的分组现象，青杨各群体也未呈现任何清晰地理模式。 由于不同分子标记在对群体遗传多样性检测能力与效率上存在差异，所以三种标记检测的青藏高原东缘青杨天然群体遗传多性水平也不尽一致，但在与用同种方法检测其它物种或同一物种不同种源群体比较，三种分子标记方法都揭示了青藏高原东缘青杨天然群体具有中等偏上的遗传多样性水平。结果分析表明，群体间遗传分化极大，这是由于青杨天然群体分布于青藏高原东缘，既有高原又有高山峡谷，由于地理和空间上大尺度的隔离和地形地貌复杂导致了基因流物理上的阻隔。三种分子标记研究结果经Mantel分析检测，遗传距离与地理距离之间都无明显相关性。较为一致的解释是，青杨分布区域地理和空间上大尺度的隔离和和地形地貌复杂导致群体之间不存在均匀扩散现象，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 The wide geographical and climatic distribution of P. cathayana Rehd. indicates that there is a large amount of genetic diversity available, which can be exploited for conservation, breeding programs and afforestation schemes. The results are as follows: 1 Research results of AFLP genetic diversity In present study, genetic diversity was evaluated in the natural populations of P. cathayana originating from southern and eastern edge of the Qinghai-Tibetan Plateau of China by means of AFLP markers. For four primer combinations, a total of 175 bands were obtained, of which 173 (98.9%) were polymorphic. Six natural populations of P. cathayana possessed different levels of genetic diversity, high level of genetic differentiation existed among populations (GST=0.489) of P. cathayana. Individuals cluster and PCO analysis based on Jaccard’s similarity coefficient also showed evident population genetic structure with high level population genetic differentiation. The long evolutionary process coupled with genetic drift within populations, rather than contemporary gene flow, are the major forces shaping genetic structure of P. cathayana populations. Moreover, there is no correspondence between geographical and genetic distances in the populations of P. cathayana, seldom gene exchange among populations and different selection pressures may be the causes. Our finding of different levels of genetic diversity within population and high level of genetic differentiation among populations provided promising condition for further breeding or conservation programs. 2 Research results of SSR genetic diversity In this study, the genetic diversity of P. cathayana was investigated using microsatellite markers. In a total of 150 individuals collected from six natural populations in the southeastern part of the Qinghai-Tibetan Plateau in China, a high level of microsatellite polymorphism was detected. At the seven investigated microsatellite loci, the number of alleles per locus ranged from 5 to 16, with a mean of 11.3, the observed heterozygosities across populations ranged from 0.408 to 0.986, with a mean of 0.792, and the expected heterozygosities across populations ranged from 0.511 to 0.891, with a mean of 0.802. The proportion of genetic differentiation among populations accounted for 37.3% of the whole genetic diversity. The presence of such a high level of genetic diversity could be attributed to the features of the species and the habitats where the sampled populations occur: The southeastern part of the Qinghai-Tibetan Plateau is regarded as the natural distribution and variation center of the genus Populus in China. Variation in environmental conditions and selection pressures in different populations, and topographic dispersal barriers could be factors associated with the high level of genetic differentiation found among populations. The populations possessed significant heterozygosity excesses, which may be due to extensive population mixing at the local scale. The cluster analysis showed that the populations are not strictly grouped according to their geographic distances but the habitat characteristics also influence the divergence pattern. In addition, we suggest that population SHY should be regarded as an ecologically divergent species of P. cathayana. 3 Research results of cpSSR genetic diversity Genetic diversity of six natural populations of P. cathayana originating from the southeastern part of the Qinghai-Tibetan Plateau in China was studied by use of cpSSR markers. Based on 5 pairs of polymorphic primers screened from 12 pairs of primers, twenty-six different length fragments and twelve different kinds of haplotypes were reduced in 143 samples. There were significant variant haplotypes among the populations．There were no shared haplotypes found among populations, analysis of molecular variance indicated that a high proportion of the total genetic variance was attributable to variations among populations (92.23%). The pattern of genetic structure which is associated with spatial separation, variation in environmental conditions and selection pressures in different populations, is also the result of geological historical factor. A molecular phylogenetic tree based on the 12 haplotypes showed that the populations are not strictly grouped according to their geographic distances.

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.

中国沙棘(Hippophae rhamnoides subsp. sinensis)天然群体的遗传变异分析

近十年，植物群体遗传学的研究飞速发展，然而与海拔相关的植物群体遗传结构和遗传变异研究却相对较少。到目前为止，还不清楚遗传变异与海拔之间是否有一个通用的格局。在山区，各种生态因子，如温度、降水、降雪、紫外线辐射强度以及土壤成分都随海拔梯度急剧变化，造成了即使在一个小的空间区域，植被类型变化显著，这种高山环境的异质性和复杂性为我们研究植物群体遗传结构和分化提供了方便。沙棘(Hippophea)属于胡颓子科(Elaeagnaceae)为多年生落叶灌木或乔木，雌雄异株，天然种群分布极为广泛。中国沙棘(H. rhamnoides subsp. sinensis)是沙棘属植物中分布较广的一个亚种，种内形态变异非常丰富，加之其具有独特的繁育系统和广泛的生态地理分布，是研究沙棘属植物遗传变异和系统分化的理想材料。本文从1,800 m 到3,400 m 分5 个海拔梯度进行取样，用RAPD 和cpSSR 分子标记研究了卧龙自然保护区中国沙棘天然群体的遗传结构和遗传变异。5 个取样群体依次标记为A、B、C、D 和E，它们分别代表分布在海拔1,800，2,200，2,600，3,000 和3,400 m 的5 个天然群体。RAPD实验用11 条寡核苷酸引物，扩增得到151 个重复性好的位点，其中143 个多态位点，多态率达94.7%。在5 个沙棘群体中，总遗传多样性值(HT)为0.289，B群体内的遗传多样性值为0.315，这完全符合沙棘这种多年生、远交的木本植物具有高遗传变异的特性。5 个群体内遗传多样性随海拔升高呈低－高－低变异趋势，在2,200 m海拔处的B群体遗传多样性达最大值0.315，3,400 m海拔处的E群体则表现最小仅0.098。5 个群体间的遗传分化值GST＝0.406，也即是说有40.6%的遗传变异存在于群体间，1,800 m海拔处的A群体与其它群体的明显分离是造成群体间遗传分化大的原因。UPGMA聚类图和PCoA散点图进一步确证了5 个群体间的关系和所有个体间的关系。最后，经过Mantel检测，遗传距离与海拔表现了明显的相关性(r = 0.646, P = 0.011)。cpSSR 实验中，经过对24 对cpSSR 通用引物筛选，11 对引物能扩增出特异性条带，只有2 对引物(ccmp2 和ARCP4)呈现多态性。4 个等位基因共组合出4 种单倍型，单倍型Ⅰ出现在A 群体的所有个体和B 群体的8 个个体中，C、D、E 三个群体均不含有，而单倍型Ⅱ出现在C、D、E 三个群体的所有个体及B 群体的18 个个体中，A 群体不含有。另外两种单倍型Ⅲ和Ⅳ为稀有类型，仅B 群体中的4 个个体拥有。这种单倍型分布模式和TFPGA 群体聚类图揭示了，C、D、E 群体可能来源于同一祖先种，而A 群体却是由另一祖先种发展起来的，B 群体则兼具了这两种起源种的信息，这可能是因为在历史上的某一时期，在中国沙棘群体高山分化的过程中，B 群体处某个或者某些个体发生了基因突变，具备了适应高海拔环境的能力，产生了高海拔沙棘群体的祖先种。 In recent ten years, studies about population genetics of plants developed rapidly,whereas their genetic structure and genetic variation along altitudinal gradients have beenstudied relatively little. So far, it is uncleared whether there is a common pattern betweengenetic variation and altitudinal gradients. In the mountain environments, importantecological factors, e.g., temperature, rainfall, snowfall, ultraviolet radiation and soil substratesetc., change rapidly with altitudes, which cause the vegetation distribution varying typically,even on a small spatial scale. The mountain environments, which are heterogeneous andcomplex, facilitate and offer a good opportunity to characterize population genetic structureand population differentiation.The species of the genus Hippophae L. (Elaeagnaceae) are perennial deciduous shrubs ortrees, which are dioecious, wind-pollinated pioneer plants. The natural genus has a widedistribution extending from Northern Europe through Central Europe and Central Asia toChina. According to the latest taxonomy, the genus Hippophae is divided into six species and12 subspecies. The subspecies H. rhamnoides ssp. sinensis shows significant morphologicalvariations, large geographic range and dominantly outcrossing mating system. Thesecharacteristics of the subspecies are favourable to elucidate genetic variation and systemevolution. To estimate genetic variation and genetic structure of H. rhamnoides ssp. sinensisat different altitudes, we surveyed five natural populations in the Wolong Natural Reserve at altitudes ranging from 1,800 to 3,400 m above sea level (a.s.l.) using random amplifiedpolymorphic DNA markers (RAPDs) and cpSSR molecular methods. The five populations A,B, C, D, and E correspond to the altitudes 1,800, 2,200, 2,600, 3,000 and 3,400 m,respectively.Based on 11 decamer primers, a total of 151 reproducible DNA loci were yielded, ofwhich 143 were polymorphic and the percentage of polymorphic loci equaled 94.7%. Amongthe five populations investigated, the total gene diversity (HT) and gene diversity within population B equaled 0.289 and 0.315, respectively, which are modest for a subspecies of H.rhamnoides, which is an outcrossing, long-lived, woody plant. The amount of geneticvariation within populations varied from 0.098 within population E (3,400 m a.s.l.) to 0.315within population B (2,200 m a.s.l.). The coefficient of gene differentiation (GST) amongpopulations equaled 0.406 and revealed that 40.6% of the genetic variance existed amongpopulations and 59.4% within populations. The population A (1,800 m a.s.l.) differed greatlyfrom the other four populations, which contributes to high genetic differentiation. A UPGMAcluster analysis and principal coordinate analyses based on Nei's genetic distances furthercorroborated the relationships among the five populations and all the sampling individuals,respectively. Mantel tests detected a significant correlation between genetic distances andaltitudinal gradients (r = 0.646, P = 0.011).Eleven of the original 24 cpSSR primer pairs tested produced good PCR products, onlytwo (ccmp2 and ARCP4) of which were polymorphic. Four total length variants (alleles) werecombined resulting in 4 haplotypes. The haplotype was present in all individuals of Ⅰpopulation A and 8 individuals of populations B, the other three populations (C, D and Epopulations) did not share. The haplotype was present in all individuals of populations C, D Ⅱand E and 18 individuals of populations B, population A did not share. The other twohaplotypes and were rare haplotypes, which were only shared in 4 individuals of Ⅲ Ⅳpopulation B. The distribution of haplotypes and TFPGA population clustering map showedthat the populations C, D and E might be origined from one ancestor seed and population Amight be from another, whereas population B owned information of the two ancestor seeds. Itwas because that gene mutation within some individual or seed in the location of population Bwas likely to happen in the history of H. rhamnoides, which was the original ancestor of thehigh-altitude populations.

川西云杉(Picea balfouriana)天然群体的遗传变异

青藏高原东南缘由于特殊的生态地理条件，有着丰富的森林资源，这些资源是长江上游涵养水源、保持水土的生态屏障，是生物多样性的资源宝库。但随着过量的森林采伐，使该区曾经丰富的生物多样性资源遭受了前所未有的破坏，天然林的质量严重下降，生态系统退化，功能减弱。与此同时，许多物种的种群规模正在锐减，物种的遗传多样性也严重丧失。川西云杉是西部地区分布最广的云杉树种之一，在较高海拔的地区有着重要的生态学功能，是一种适应性很强的乡土树种。本项目采用简单序列重复标记(SSR)和特定序列位点(STS)研究不同生境条件下川西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，系统地揭示川西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。研究成果能有效地为该树种遗传资源的科学保护与合理利用提供理论依据和科学指导，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依据。主要研究结果如下： 1 STS和SSR两种分子标记的研究结果表明：川西云杉群体拥有中等水平的遗传多样性(基于SSR标记，平均He = 0.640；基于STS标记，平均He = 0.553)。造成这种中等水平的遗传多样性，可能是由于历史原因，川西云杉天然林被大量采伐，导致了遗传多样性的丧失。两种方法都检测出群体BT有着最高水平的遗传多样性。 2 两种标记的结果都一致说明：检测的10个川西云杉群体间遗传分化比较高，其存在群体间的遗传变异比例要明显地高于广泛分布的挪威云杉、横贯大陆的黑云杉以及兼有连续分布和不连续分布的西加云杉等，但要低于分布范围狭窄且呈不连续分布的粗枝云杉。青藏高原东南部的片段化生境可能是导致高水平遗传分化的主要原因。 3 基于两种标记的UPGMA 聚类和PCA分析结果，以及基于SSR标记的FCA分析结果都显示：群体BY遗传上明显区别于其它群体，其可能原因是青藏高原东南缘山脉阻隔而导致的生殖隔离的结果。 4 根据软件Bottleneck 1.2.02的检测以及不依赖哈迪一温伯格平衡的M比率检测结果：川西云杉种群很有可能经历了近期的遗传瓶颈效应。在本研究中遗传瓶颈效应并没有显著影响到物种的遗传多样性。然而，在这些片断化群体未来的子代群体中很可能出现遗传瓶颈导致的遗传多样性下降的效应。 Due to the extremely complex topography and climatic conditions, the southeast of the Qinghai-Tibet Plateau is region abundant in forest resource, which is benefit to the upper reaches of the Yangtze River water conservation, and protecting natural environment and biodiversity resources. However, by the reason of a plenty of trees were cut in these yeas, the biodiversity resource was destroy with degraded ecosystem and imperfect funcation. Picea balfouriana is one of the regionally distributed conifer species in the southeast of the Qinghai-Tibet Plateau and considered as a constructive species within its distribution area. It is an optimal species for the production of biomass. Moreover, it is well adapted to stressful environments at high altitude, especially to cold and drought conditions which are generally harsh for other trees. In our study, two types of molecular markers (SSR and STS) were used to estimate the genetic diversity and genetic structure of P. balfouriana populations originating from the southeast of the Qinghai-Tibet Plateau with varying climatic and geographical conditions. The results will not only provide a deep insight into its genetic diversity and population genetic structure but also valuable information for further management and breeding programs in P. balfouriana. In summary, the results obtained in this study revealed that: 1 A moderate degree of genetic variation is present in P. balfouriana in the southeast of the Qinghai-Tibet Plateau and it may caused by many trees were cut in the past. Population BT owns the highest level of genetic diversity by both two types of markers. 2 Considerable population differentiation exists among the ten P. balfouriana populations based on both SSR and STS markers, possibly caused by habitat fragmentation and heterogeneous environments. 3 The UPGMA clustering and PCA analyses based on SSR and STS marker, and FCA analyses based on SSR marker congruously separated population BY from other populations, which is likely due to the presence of mountain barriers. 4 The results of bottleneck analysis indicated that P. balfouriana populations that have undergone recent declines. In our research, the bottleneck effect don’t have a significant impact on the genetic diversity of species, however, the level of genetic diversity of P. balfouriana offspring populations growing in the fragmented habitats will decline in the future.

THE COMPOUND EYE AS AN INDICATOR OF AGE AND SHRINKAGE IN ANTARCTIC KRILL

Laboratory studies have shown that Antarctic krill (Euphausia superba) shrink if maintained in conditions of low food availability. Recent studies have also demonstrated that E. superba individuals may be shrinking in the field during winter. If krill shrink during the winter, conclusions reached by length-frequency analysis may be unreliable because smaller animals may not necessarily be younger animals. In this study, the correlation between the body-length and the crystalline cone number of the compound eye was examined. Samples collected in the late summer show an apparent linear relationship between crystalline cone number and body-length. From a laboratory population, it appears that when krill shrink the crystalline cone number remains relatively unchanged. If crystalline cone number is little affected by shrinking, then the crystalline cone number may be a more reliable indicator of age than body-length alone. The ratio of crystalline cone number to body-length offers a method for detecting the effect of shrinking in natural populations of krill. On the basis of the crystalline cone number count, it appears from a field collection in early spring that E. superba do shrink during winter.

Identification and mapping of amplified fragment length polymorphism markers linked to shell color in bay scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F-1 families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.

Phylogeography of Pinus tabulaeformis Carr. (Pinaceae), a dominant species of coniferous forest in northern China

How coniferous trees in northern China changed their distribution ranges in response to Quaternary climatic oscillations remains largely unknown. Here we report a study of the phylogeography of Pinus tabulaeformis, an endemic and dominant species of coniferous forest in northern China. We examined sequence variation of maternally inherited, seed-dispersed mitochondrial DNA (mtDNA) (nad5 intron 1 and nad4/3-4) and paternally inherited, pollen- and seed-dispersed chloroplast DNA (cpDNA) (rpl16 and trnS-trnG) within and among 30 natural populations across the entire range of the species. Six mitotypes and five chlorotypes were recovered among 291 trees surveyed. Population divergence was high for mtDNA variation (G(ST) = 0.738, N-ST = 0.771) indicating low levels of seed-based gene flow and significant phylogeographical structure (N-ST > G(ST), P < 0.05). The spatial distribution of mitotypes suggests that five distinct population groups exist in the species: one in the west comprising seven populations, a second with a north-central distribution comprising 15 populations, a third with a southern and easterly distribution comprising five populations, a fourth comprising one central and one western population, and a fifth comprising a single population located in the north-central part of the species' range. Each group apart from the fourth group is characterized by a distinct mitotype, with other mitotypes, if present, occurring at low frequency. It is suggested, therefore, that most members of each group apart from Group 4 are derived from ancestors that occupied different isolated refugia in a previous period of range fragmentation of the species, possibly at the time of the Last Glacial Maximum. Possible locations for these refugia are suggested. A comparison of mitotype diversity between northern and southern subgroups within the north-central group of populations (Group 2) showed much greater uniformity in the northern part of the range both within and between populations. This could indicate a northward migration of the species from a southern refugium in this region during the postglacial period, although alternative explanations cannot be ruled out. Two chlorotypes were distributed across the geographical range of the species, resulting in lower levels of among-population chlorotype variation. The geographical pattern of variation for all five chlorotypes provided some indication of the species surviving past glaciations in more than one refugium, although differentiation was much less marked, presumably due to the greater dispersal of cpDNA via pollen.

The ecological effects of exotic disease resistance genes introgressed into British gooseberries

Warren, J. and James, P. (2006). The ecological effects of exotic disease resistance genes introgressed into British gooseberries. Oecologia 147(1),69-75. RAE2008