基于RAD测序技术的黄绿卷毛菇(Floccularia luteovirens)微卫星引物的开发

黄绿卷毛菇(Floccularia luteovirens)是广泛分布于青藏高原高寒草甸的一种重要的可食用的菌根真菌。先前的研究利用EST-SSR引物已经对该物种的遗传多样性及群落结构进行了研究,但研究表明EST-SSR位点多态性较低,因此想要进一步探讨形成现有遗传分布格局的机制(该物种小尺度空间范围内基株的密度及大小)需要重新开发具有更高多态性的基于核基因的微卫星位点。本研究利用RAD测序技术使用直接测序分析的方法重新开发了12对具有多态性的引物。测序结果去接头拼装后共获得46 036条重叠群。在这些序列当中共扫描到342条含有重复单元的序列,在这当中70.47%为三碱基重复(241),8.19%为二碱基重复(28)。随机选取48对检测,12对具有多态性且测序结果良好,基于3个居群63个个体的遗传多样性研究显示,所有样品中共获得60个单倍型,每个位点的单倍型数量介于2 (GSSR26L)至9 (GSSR7L,GSSR11L)之间。Gst值介于-0.03(GSSR46L)至0.28(GSSR6L)之间、Fst值介于-0.03(GSSR33L)至0.42(GSSR6L)之间。在引物GSSR47L扩增测序中发现,其扩增出的片段不仅具有重复单元数量的变化,重复单元本身也存在突变的现象,这是之前使用聚丙烯凝胶电泳所检测不到的。本研究获得的微卫星引物将为接下来小尺度空间下研究黄绿卷毛菇基株的密度、大小及动态变化提供有力的支持。     

铲鲟微卫星引物对中华鲟的适用性研究

将21对铲鲟（Scaphirhynchus platorynchus Rafinesque)的微卫星引物在中华鲟（Acipenser sinensis Gray)基因组DNA上进行PCR扩增,14对（约占67％）引物得到了扩增产物,其中有10对（约占48％）表现为多态性,但只有2对（9．5％）引物SPl-100和Spl－168的多态性较高,且带型清晰,可直接作为分子标记应用于相关的研究,此外,对具有特异性扩增,但有Stutter band现象的4对引物的部分可分离PCR产物进行了回收和测序,并对其中的3对引物的序列进行了重新设计,最后得到两对可应用于中华鲟的引物As-100和Spl0-170b,研究结果表明,对相近种的微卫星引物进行优化设计来获得一个物种的微卫星引物是一条简捷有效的途径。     

大鼠及小鼠微卫星引物在社鼠中的跨种扩增

利用微卫星引物在同一属、科、目不同种之间具有通用性的特点,通过PCR扩增、聚丙烯凝胶电泳和银染技术对社鼠(Niviventer confucianus)近缘物种大鼠(Rattus norvegicus)、小鼠(Mus musculus)中已知的70个微卫星位点引物进行跨种扩增,筛选适合社鼠相关研究的多态微卫星引物.结果发现,40个位点引物出现扩增条带,21个位点引物能够稳定扩增,其中15个位点杂合,13个位点具有多态性;PCR扩增的Mg2+浓度主要集中在1.5及2.0 mmol/L,退火温度在50～60℃之间不等.虽然部分扩增产物有影子带的存在,但并不影响等位基因的判读.总体来看,利用大、小鼠的微卫星引物扩增社鼠的微卫星位点是可行的.     

TRAP及SSCP检测草鱼微卫星序列多态性

目的：在草鱼不同种群遗传结构比较中,应用一些来自鲤鱼的微卫星引物扩增草鱼的相关序列缺少多态性,该研究通过应用几种方法提高这些序列在遗传学研究中的价值。方法：运用TRAP随机引物与鲤鱼的微卫星引物组合,扩增出多态性好、重复性强的产物;同时采用SSCP技术对那些没有长度多态的微卫星序列进一步分析,寻找这些序列存在一些核苷酸位点的多态性。结果：TRAP可以检查到多态性好、重复性强的产物;SSCP发现一部分没有长度多态性的微卫星序列存在核苷酸位点多态性并且可以清楚分辨不同的基因型。结论：结果表明TRAP及SSCP在种群遗传学研究有良好的应用前景。     

鸢尾属部分植物种质资源的RAPD分析

采用RAPD分子标记技术,从100个随机引物中筛选出多态性强、重复性好且稳定性高的引物18个,对38份野生鸢尾属材料进行扩增,共扩增出409条带,其中多态性带405条,多态性比率为99.0%,表明野生鸢尾属植物种间有丰富的遗传多态性;根据DNA谱带计算物种间遗传距离,聚类分析结果将鸢尾属38份材料划分为6组,其结果与传统生物学特性划分的6个亚属的分类结果基本一致;物种特有RAPD标记分析表明,利用18个引物可以较好地将鸢尾属38种植物区分开,其中9个材料得到了单一标记的扩增带,表明运用RAPD分子标记对研究鸢尾属植物特异性基因及标记的筛选等有一定的理论和实际应用价值。     

微卫星跨物种交叉PCR扩增的一个新问题:扩增非同源产物

微卫星已被广泛应用于群体遗传学、生态学和进化生物学研究。然而,一些物种微卫星尚未克隆。为了节省时间和经费,研究人员往往使用一个物种已发表的微卫星引物扩增其近缘物种的微卫星。该研究对属于3个不同科(Clariidae、Heteropneustidae 和Pimelodidae)的7个鲶鱼物种的微卫星跨物种PCR扩增产物进行了序列分析,研究发现扩增非同源（non-orthologous）产物是微卫星跨物种PCR扩增的一个新问题。该研究共采用4对胡子鲶微卫星座位引物对7个鲶鱼物种进行了跨物种PCR扩增。对获得的204个PCR产物的序列分析结果表明,两对微卫星座位引物扩增了所有7个物种的同源特异产物。而其他两个座位的引物扩增了特异但非同源的多态产物,对近缘物种的扩增也获得类似结果。另外,除胡子鲶等位基因大小异源同型(size homoplasy)的特征不明显外,其他物种在3个微卫星座位都具有这一非常明显的特征。这些数据表明,微卫星跨物种间交叉扩增能产生非同源产物;等位基因大小异源同型与微卫星座位本身有关,而与物种间的亲缘关系无明显的相关性。微卫星跨物种扩增产生的非同源产物和等位基因大小异源同型将使系统发育、群体遗传学和进化研究明显复杂化。因此,在应用微卫星跨物种交叉扩增数据以前,最好对跨物种交叉扩增产物进行测序验证。     

InDel标记的研究和应用进展

InDel是指在近缘种或同一物种不同个体之间基因组同一位点的序列发生不同大小核苷酸片段的插入或缺失(insertion-deletion), 是同源序列比对产生空位(gap)的现象。InDel在基因组中分布广泛、密度大、数目众多。InDel多态性分子标记是基于插入/缺失位点两侧的序列设计特异引物进行PCR扩增的标记, 其本质仍属于长度多态性标记, 可利用便捷的电泳平台进行分型。InDel标记准确性高、稳定性好, 避免了由于特异性和复杂性导致的后续分析模糊。此外, InDel标记能扩增混合DNA样品和高度降解的微量DNA样品, 并进行有效分型。InDel标记目前已开始应用于动植物群体遗传分析、分子辅助育种以及人类法医遗传学、医学诊断等领域。随着位于功能基因上InDel标记的开发, 结合染色体步移和基因精细定位, 可将这些标记应用于相关物种经济性状的功能基因的筛选, 有利于优良基因的进一步开发和利用。     

基于分子标记的白蜡树属椒叶梣组系统发育及谱系地理格局形成

木犀科白蜡树属椒叶梣组（Fraxinus sect. Sciadanthus）由自然分布于中国中部的对节白蜡（Fraxinus hupehensis S.Z.Qu,C.B.Shang&P.L.Su）、喜马拉雅西部的椒叶梣（Fraxinusxanthoxyloides(G.Don)DC.）和非洲北部的Fraxinus dimorpha 3个物种组成,呈现出特殊的间断分布模式,目前该组的起源和演化过程尚不清楚。利用ITS、psbA-trnH、rpl32-trnL和matK 4个标记对白蜡树属42个物种间的系统发育关系进行分析,结果表明：（1）利用ITS序列构建的贝叶斯系统树支持Wallander的分类方法,即属内再分成6个组,对节白蜡、椒叶梣和F. dimorpha都属于椒叶梣组,且该组与欧梣组（sect. Fraxinus）的亲缘关系最近,形成姐妹组;（2）椒叶梣可能是F. dimorpha与欧亚种的杂交后代;（3）BEAST分化时间图显示,白蜡树属最早起源于42.05 Ma,椒叶梣组最早分化于21.86 Ma,对节白蜡最早分化于11.87 Ma。青藏高原的快速隆起可能是导致椒叶...     

鲫鱼微卫星DNA的分离及多态性分析

目的：用近缘物种鲤微卫星引物来分离鲫鱼微卫星标记并对其多态性进行分析。方法：以鲫鱼基因组DNA为模板,采用6对鲤微卫星引物进行PCR扩增,PCR产物经8%的非变性聚丙烯酰胺凝胶电泳和银染色检测。结果：筛选出2个以AC和TA为重复单元的鲫鱼新的微卫星标记。多态性分析表明,这2个微卫星标记的遗传杂合度分别为0.611和0.644,多态信息含量为0.536和0.572,属于高度多态性标记。结论：该研究筛选的2个微卫星标记可应用于鲫鱼遗传多样性、遗传连锁图谱构建及分子标记辅助育种等方面的研究。     

叶绿体基因infA-rpl36区域在小麦族物种中的序列变异分析

利用小麦叶绿体基因组中infA-rpl36区域的序列设计引物, 对小麦族(Triticeae)的12个二倍体和多倍体的物种进行了PCR扩增和序列测定, 获得了长度为584～603 bp的12条DNA序列。序列分析表明, 供试物种在infA-rpl36基因间隔区的核苷酸变异明显高于基因编码区。基因编码区核苷酸序列同源性高达97%, 表明了目标片段具有高度的保守性。但在5个物种的infA编码区出现了较大的插入、缺失突变, 导致推导的氨基酸序列也发生了很大的变化, 证实了infA基因是叶绿体基因组中最活跃的基因之一, 而rpl36基因的变异较小, 说明不同叶绿体基因的进化速度是不同的。基于测定序列建立的种系树分析发现, 多倍体物种中间偃麦草(Thinopyrum intermedium)具有多种不同的细胞质起源, 与核基因组一样在进化上较为复杂。     

白蜡属种间杂交子代木质素含量变异及FmPAL核苷酸多态性关联分析

研究水曲柳（Fraxinus mandshurica）×大叶白蜡（Fraxinus rhynchophylla）、水曲柳×小叶白蜡（Fraxinus sogdiana）和水曲柳×绒毛白蜡（Fraxinus velutina）的3个杂交组合与母本自由授粉子代木质素含量的遗传变异及FmPAL核苷酸多态性相关性,为水曲柳遗传育种和定向改良奠定基础。以东北林业大学帽儿山林场白蜡属种间杂交组合及母本自由授粉子代共176个无性系的水曲柳当年新生枝为材料,测定木质素的含量,进行木质素含量差异、变异参数及均值比较分析,并对木质素及其合成相关基因PAL进行分子水平的联合分析。结果表明：白蜡属各杂交组合间木质素含量差异极显著,各杂交组的木质素含量均与母本自由授粉子代水曲柳差异显著。其中大多数杂交子代水曲柳的木质素含量高于母本水曲柳木质素平均含量。本研究中的水曲柳群体的PAL基因单倍体多样性值达到了0.999 7,属高水平,证实了FmPAL基因具有较高的变异,且存在3个单核苷酸多态性（SNP）位点与木质素含量显著相关。主要结论：水曲柳各杂交组合的木质素含量存在显著性差异,FmPAL基因中SNP位点与木质素含量显著相关。     

Maternal inheritance of a chloroplast microsatellite marker in controlled hybrids between Fraxinus excelsior and Fraxinus angustifolia

Restriction fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and simple sequence repeat (SSR or microsatellites) analyses were performed to detect chloroplast DNA polymorphisms between two ash species, Fraxinus excelsior and F. angustifolia. Only one SSR locus was found to be polymorphic, confirming the very close relatedness of these species. Inheritance of this marker was studied in hybrids obtained from controlled crosses between the two tree species. Results indicated, for the first time in Oleaceae, that chloroplasts are maternally inherited. This chloroplast SSR marker is now used concomitantly with nuclear markers to analyse ash populations in sympatric areas.     

Chloroplast DNA variation and postglacial recolonization of common ash (Fraxinus excelsior L.) in Europe

We used chloroplast polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and chloroplast microsatellites to assess the structure of genetic variation and postglacial history across the entire natural range of the common ash (Fraxinus excelsior L.), a broad-leaved wind-pollinated and wind-dispersed European forest tree. A low level of polymorphism was observed, with only 12 haplotypes at four polymorphic microsatellites in 201 populations, and two PCR-RFLP haplotypes in a subset of 62 populations. The clear geographical pattern displayed by the five most common haplotypes was in agreement with glacial refugia for ash being located in Iberia, Italy, the eastern Alps and the Balkan Peninsula, as had been suggested from fossil pollen data. A low chloroplast DNA mutation rate, a low effective population size in glacial refugia related to ash's life history traits, as well as features of postglacial expansion were put forward to explain the low level of polymorphism. Differentiation among populations was high (GST= 0.89), reflecting poor mixing among recolonizing lineages. Therefore, the responsible factor for the highly homogeneous genetic pattern previously identified at nuclear microsatellites throughout western and central Europe (Heuertz et al. 2004) must have been efficient postglacial pollen flow. Further comparison of variation patterns at both marker systems revealed that nuclear microsatellites identified complex differentiation patterns in south-eastern Europe which remained undetected with chloroplast microsatellites. The results suggest that data from different markers should be combined in order to capture the most important genetic patterns in a species.     

Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in Lake Malawi cichlids

Recent studies have suggested that size homoplasy is a prevalent feature of microsatellites and is expected to increase with time of divergence among populations and taxa. In this study, we performed sequence analysis of alleles from a complex microsatellite locus (Pzeb4, initially isolated from Pseudotropheus (Maylandia) zebra) from 1 midwater-feeding and 10 rock-dwelling cichlid fish species from Lake Malawi, East Africa, to investigate how widespread size homoplasy is among closely related taxa at this locus. All cichlid fishes endemic to this lake are believed to have originated within the last 700,000 years, and some species may be less than 200 years old. The number of eletromorphs found per species varied from 3 to 13. Sequence analysis of 95 cloned Pzeb4 PCR products (representing 18 electromorphs) revealed 13 new alleles. Ten of the 13 electromorphs (77%) were found to show size homoplasy due to either single nucleotide substitutions/indels or large indels. To investigate how well this locus fits the single-step mutation model (SMM), the minimum number of mutations required to explain the length differences between pairs of alleles was plotted against their size differences. Of the 300 comparisons, 166 (55.3%) corresponded to SMM expectations and 86 (28.7%) required a smaller number of mutations, and for 48 (16.0%) pairwise comparisons, a larger number of mutations were required to explain the length differences as compared with SMM expectations. Finally, a large deletion in the microsatellite sequence observed in the three rock-dwelling species Pseudotropheus lucerna, Pseudotropheus (Tropheops) 'band,' and Pseudotropheus (Tropheops) 'rust' and the midwater-feeding species Copadichromis sp. is believed to represent a shared ancestral polymorphism.     

EST-SSR标记在水曲柳雌雄鉴定中的应用

选用黑龙江省青山林木种子园成年水曲柳200棵树为实验材料,在来自NCBI中的5 423条白蜡属EST序列和该实验室测得的水曲柳转录组的179 502条序列中查找SSR位点并设计引物,通过PCR扩增筛选有效扩增和有差异片段的引物,用于水曲柳雌雄的鉴别。结果显示:(1)白蜡属EST和水曲柳转录组中的5 423条和179 502条序列中,分别查找到9 488个和3 557个SSR位点,SSR出现的频率分别是174.96%和1.98%,SSR的类型都是以二核苷酸为主。(2)232对引物中有208对引物获得有效扩增,74对引物在雌雄DNA中有差异,27对扩增片段符合预定大小片段。(3)经过3轮筛选,选出19号和56号引物用于200棵水曲柳中,雌雄差异率分别是64.5%和89.5%。研究表明,利用EST-SSR分子标记对水曲柳性别进行鉴定是一种有效可行的方法。     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Microsatellite Allelic Homoplasy Due to Variable Flanking Sequences

Microsatellite DNA sequences have become the dominant source of nuclear genetic markers for most applications. It is important to investigate the basis of variation between alleles and to know if current assumptions about the mechanisms of microsatellite mutation (that is to say, variations involving simple changes in the number of repeat) are correct. We have characterized, by DNA sequencing, the human alleles of a new highly informative (CA)n repeat localized approximately 20 kb centromeric to the HLA-B gene. Although 12 alleles were identified based on conventional length criteria, sequencing of the alleles demonstrated that differences between alleles were found to be more complex than previously assumed: A high degree of microsatellite variability is due to variation in the region immediately flanking the repeat. These data indicate that the mutational process which generates polymorphism in this region has involved not only simple changes in the number of dinucleotide CA repeats but also perturbations in the nonrepeated 5′ and 3′ flanking sequences. Three families of alleles (not visible from the overall length of the alleles), with presumably separate evolutionary histories, exist and can yield to homoplasy of size. Effectively, we can observe alleles of the same size with different internal structures which are separated by a significant amount of variation. Although allelic homoplasy for noninterrupted microsatellite loci has been suggested between different species, it has not been unequivocally demonstrated within species. A strong association is noted between alleles defined at the sequence level and HLA-B alleles. The observation of several families of alleles at the population level provides information about the evolutionary history and mutation processes of microsatellites and may have implications for the use of these markers in phylogenetic, linkage disequilibrium studies, and gene mapping. Received: 14 May 1996 / Accepted: 9 September 1996     

Size homoplasy and mutational processes of interrupted microsatellites in two bee species, Apis mellifera and Bombus terrestris (Apidae)

Similar microsatellite electromorphs (PCR products of the same size) can arise from independent mutational events. Such alleles are not identical by descent. This phenomenon, termed size homoplasy, was studied by sequencing electromorphs of two microsatellite loci in which the stretch of basic repeats is interrupted by different short (1-2 bp) DNA motifs. The number and position of these interruptions were established for electromorphs from closely and distantly related populations of honeybees and bumblebees. No sequence difference was found when electromorphs came from the same subspecies or from closely related subspecies, suggesting that they were probably identical by descent. In contrast, sequence differences were often detected in distantly related subspecies, showing that size homoplasy frequently occurs at this level of population differentiation. Size homoplasy is increased by limits to free length variation of alleles, a phenomenon that seems to act on interrupted microsatellites when comparing distantly related taxa, that is, honeybee subspecies from different evolutionary lineages. Electromorph sequences suggest that, within the scope of these limits, large mutation events have occurred frequently at both interrupted loci studied. In good agreement with the molecular data, computations based on the observed heterozygosity and number of electromorphs and simulation studies showed that neither locus fits the one-step stepwise mutant model (SMM). We speculate that interrupted microsatellites in general could be characterized by a higher variance in repeat number and consequently a lower homoplasy rate than pure ones. Hence, interrupted microsatellites should be most appropriate for investigating population differentiation and evolutionary relationship between relatively distant populations.      

Heterologous nuclear and chloroplast microsatellite amplification and variation in tea, Camellia sinensis.

The advantage of the cross transferability of heterologous chloroplast and nuclear microsatellite primers was taken to detect polymorphism among 24 tea (Camellia sinensis (L.) O. Kuntze) genotypes, including both the assamica and the sinensis varieties. Primer information was obtained from the closely related Camellia japonica species for four nuclear microsatellites, and from Nicotiana tabaccum for seven universal chloroplast microsatellites. All of the nuclear microsatellite loci tested generated an expected DNA fragment in tea, revealing between three and five alleles per locus. Four out of the seven chloroplast microsatellites primers amplified positively, and of these only one was polymorphic with three alleles, which is in agreement with the conserved nature of chloroplast microsatellites at the intraspecific level. A factorial correspondence analysis carried out on all genotypes and nuclear microsatellite alleles separated the assamica and sinensis genotypes into two groups, thus demonstrating the value of these markers in establishing the genetic relationship between tea varieties. Genetic diversity measured with nuclear microsatellites was higher than that measured with other types of molecular markers, offering prospects for their use in fingerprinting, mapping, and population genetic studies, whereas polymorphisms detected at a cpSSR locus will allow the determination of plastid inheritance in the species.