改良FIASCO方法筛选砷超富集植物蜈蚣草SSR分子标记(英文)

蜈蚣草(Pteris vittata L.)是目前用于砷污染土壤修复最好的超富集植物,但其分子水平上的研究数据较少。为了开发蜈蚣草特异性SSR遗传标记,本文采用改良的FIASCO方法从蜈蚣草AG和AC微卫星富集文库中随机挑选100个克隆,分离得到51个微卫星位点,其中60%为完美型(Perfect)SSR。根据这些位点设计、合成了25对引物,并对江西庐山及湖北恩施两地蜈蚣草种群各20个个体进行了遗传多样性检测,结果发现:其中8个完美型及1个间断型(intermittent)SSR位点的引物能够扩增出清晰、稳定且具有多态性的条带。9对引物共扩增出41个等位基因,各位点等位基因数在2~7之间,平均等位基因数为4.56个;期望杂合度在0.0494~0.8169之间;没有连锁不平衡现象发生。采用大叶井栏边草(Pteris multifida Poir.)进行跨种扩增,结果发现其中6对引物能够进行种间扩增。这些SSR分子标记的开发有助于蜈蚣草生态适应性进化分析、揭示蜈蚣草地理分布格局以及探讨蜈蚣草遗传多样性,还可用于品种鉴定及选育等。     

基于磁珠富集法的绣线菊SSR分子标记分离与筛选

微卫星序列（SSR）具有多态性高、共显性遗传等特点,是一种极具价值的分子遗传标记。采用磁珠富集法从高山绣线菊基因组DNA中分离和筛选SSR标记。高山绣线菊基因组经限制性内切酶Mse I酶切后与接头连接,并与生物素标记SSR探针（AC）15和（AG）15杂交,然后通过链霉亲和素磁珠富集、洗脱、PCR扩增、克隆,完成微卫星文库构建。利用载体通用引物和探针序列引物进行PCR扩增,筛选重组克隆并测序,获得112条序列。随机挑选其中60条序列设计的引物,经初期筛选获得多态性引物16对。用所得16对引物对4个居群92个个体的蒙古绣线菊和高山绣线菊进行PCR扩增。统计分析PCR产物的毛细管电泳结果,发现4个居群的平均等位基因数、平均期望杂合度及平均观测杂合度都比较高。64个数据系列（4个居群×16个位点）中的26个显著偏离HardyWeinberg平衡,推测可能由于无效等位基因的存在所引起。分析显示研究开发的16对多态性SSR引物可以用于后续遗传多样性、物种进化与亲缘关系等方面研究,丰富了绣线菊遗传多样性研究的分子标记。     

三叶木通微卫星分子标记开发及评价

为了获得适于三叶木通遗传多样性和遗传结构研究的微卫星分子标记,该研究采用磁珠富集法构建了三叶木通微卫星富集文库。结果表明:在150个阳性克隆中发现了70个微卫星位点,富集效率为46.67%,其中含双碱基重复单元的序列占比为79.37%,三碱基和四碱基重复含有量较少。共设计引物63对,其中筛选出16对高多态引物,对1个三叶木通自然居群48个个体进行了遗传分析,结果显示位点的等位基因数为10~22个,观察杂合度和期望杂合度分别为0.370~0.792和0.724~0.936,多态性信息指数为0.725~0.919,表明以上引物均为高多态性引物。其中,12个位点偏离哈迪-温伯格平衡,呈现出纯合子过剩状态,这可能与哑等位基因和其它因素有关。综上结果表明,该研究所开发的16对引物能够用于三叶木通遗传多样性和遗传结构评价工作。     

SSR分子标记在山茶属观赏资源遗传多样性研究中的应用

采用微卫星(SSR)分子标记的方法,利用20对SSR引物对33份山茶属资源(含种和品种)的DNA样品进行了PCR扩增,从中筛选出10对扩增效果较好的SSR引物,并对33份资源进行了遗传多样性分析。结果显示:10对引物在33个植物材料中共检测出123个等位基因,每个SSR位点的等位基因数为3~22个,SSR2引物检测到的等位基因数量最多,达22个,平均每对引物含有12.3个等位基因,平均多态性信息量为0.74,变化范围为0.06~0.96。利用遗传距离矩阵按UPGMA方法进行聚类,结果表明33份植物材料可分为9个分支,很好的区分了品种间的亲缘关系,可为杂交育种的组合选择提供理论基础。不同花型的山茶属植物在10个微卫星位点上共发现42个特异等位基因,可能与不同的花型性状有关。     

苦荞SSR引物开发及其在遗传多样性分析中的应用

苦荞在中国已经成为广受欢迎的健康食品。本研究通过选择性扩增微卫星序列、体外重组扩增产物和构建SSR富集文库,开发出了一种简便的重组微卫星引物设计方法。通过这种方法,设计合成500对SSR引物,在苦荞上的有效性约50%,多态率达10.8%,PIC平均值为0.3600。利用其中的28对SSR引物,在苦荞核心种质中检测出等位基因85个,每位点的等位基因数为2～5个,平均3.0个。不同地理来源的苦荞种质资源的Shannon-Weaver多样性指数为0.3633～0.6671,来自云南、四川和西藏的苦荞材料不但遗传多样性丰富,而且亲缘关系较近,进一步证实苦荞起源于中国西南部。本研究证明重组微卫星引物设计方法开发的引物对苦荞非常有效,将有助于促进苦荞种质资源遗传多样性、有用基因发掘和分子标记辅助育种研究。     

鲮微卫星DNA分子标记的筛选与遗传多样性分析

采用磁珠富集法对已建立的生长较快的西江段野生浅色鲮群体的极大、极小群体各31尾基因组DNA进行了微卫星序列的筛选,128个阳性克隆成功测序93个,其中80个为的微卫星序列,微卫星序列完美型占85%,非完美型占6.25%,混合型占8.75%。利用微卫星序列合成了23对微卫星引物,扩增结果表明,等位基因数为1～10个,扩增片段大小为92～283bp,其中14对引物扩增条带清晰,为中度或高度多态位点,极大、极小群体的平均有效等位基因数和平均多态信息含量分别为3.0784、3.2079和0.5675、0.5974,反映出2个群体遗传多样性均较丰富;高度多态性引物4的位点b,片段大小为119bp,在极大群体中出现频率为61.3%,在极小群体占22.6%,出现频率极大群体高于极小群体近3倍,初步可作为候选差异性分子标记。     

基于全长转录组信息的枸杞SSR标记开发北大核心CSCD

枸杞是茄科枸杞属的重要经济植物,广泛分布于我国西北地区,环境适应性强。本研究分析了枸杞基因组中SSR的分布特征,并基于宁杞1号全长转录组开发SSR分子标记,对28个枸杞种质进行遗传多样性分析和亲缘关系分析。用MISA软件搜索全长转录组中SSR位点,设计并筛选多态性好的SSR引物,对206对引物经过毛细管电泳筛选后最终选出23对引物对28个不同分布区的枸杞种质进行遗传多样性分析,遗传相似系数用非加权组平均法(UPGMA)聚类。23对引物共扩增得到240个等位基因,每个位点的等位基因数范围为4~17,Shannon信息指数范围为1.177~2.487,期望杂合度和观测杂合度的范围分别为0.635~0.909和0.250~0.964,PIC的范围是0.580~0.902。遗传距离聚类分析显示28份枸杞种质在遗传相似系数为0.71时被分为4类,宁夏枸杞为Ⅰ类,中国枸杞为Ⅱ类,黑果枸杞为Ⅲ类,Ⅳ类中只有中国枸杞种的枸杞岛种质。利用全长转录组序列可以高质量实现枸杞SSR标记开发,新开发的23对引物后续可用于枸杞种质遗传多样性分析和分子标记辅助育种工作。     

中国特有单种属血水草的微卫星分子标记开发与评价

血水草(Eomecon chionantha Hance)是中国亚热带地区特有的单种属植物。本研究采用(AG)15、(AC)15两种生物素探针及磁珠富集法从血水草基因组中开发并筛选出13个微卫星位点,且在该物种中能进行稳定的PCR扩增并表现出多态性。利用筛选出的13个SSR多态性标记对血水草4个野生居群24个个体进行PCR扩增,结果显示每个微卫星位点的等位基因数为2~7个,观测杂合度(HO)和期望杂合度(HE)分别为0.125~0.792和0.299~0.691。说明这些SSR标记可用于血水草遗传多样性的后续研究,并揭示该物种地理分布格局形成的原因,有助于保护和利用中国亚热带地区的血水草野生资源。     

麦红吸浆虫唾腺EST-SSRs的信息分析及分子标记筛选

昆虫EST资源库的扩充为开发新的分子标记提供了宝贵的资源。本研究对NCBI的EST数据库中来源于麦红吸浆虫Sitodiplosis mosellana唾腺的1 217条EST序列进行了unigene组装、 SSR信息分析和EST-SSR分子标记筛选。结果表明： 在1 047个unigenes中共找到141个SSR位点, 分布于106个（10.12%）unigenes中, 平均每3.49 kb出现一个SSR位点。在1~6碱基重复基元中, 1~3碱基是主要重复类型, 占总SSR的97.16%以上。A/T（31.21%）, AC/GT（15.60%）和AAC/GTT（9.22%）分别是单、 双和三碱基中占优势的重复基元类型。利用Primer Premier 5.0软件对查找的EST SSRs进行引物设计, 并以麦红吸浆虫基因组DNA为模板, 对从中选出的26对SSR引物进行多态性检测。结果有20对（76.92%）引物能扩增出清晰的目的条带, 并且其中9对（45%）引物表现出多态性。多态性分析结果表明, 从9对EST-SSR引物中, 共检测到51个等位基因, 平均每个位点含有等位基因数为5.67, 平均期望杂合度为0.65, 平均多态信息含量为0.60。本研究能够为今后麦红吸浆虫的种群遗传结构与遗传多样性研究提供帮助。     

中国龙虾微卫星标记的筛选及遗传多样性分析

文章以M13通用引物和重复序列(CT)₁₅、(AT)₁₅引物, 利用PCR法对中国龙虾(Panulirus stimpsoni Hoehuis)部分基因组DNA文库进行筛选。共获得78个微卫星序列, 分别分布于55个阳性重组克隆中, 其中完美型(perfect)共50个, 占64%; 非完美型(imperfect)3个, 占3.8%; 混合完美型(compound perfect)6个, 占7.7%; 混合非完美型(compound imperfect)19个, 占24.5%。根据微卫星序列, 设计并筛选出15对微卫星多态性引物, 对中国龙虾的群体进行了遗传多样性分析。获得3~12个等位基因, 等位基因大小在78~425 bp之间, 基本符合引物设计的理论长度。期望杂合度范围为0.48~0.87, 平均值为0.71, 表明中国龙虾基因组微卫星具有较高的杂合度与遗传多样性。15个微卫星位点的PIC值从0.44到0.84, 平均值为0.60, 说明这些微卫星位点在中国龙虾基因组中包含丰富的遗传信息, 合适用于中国龙虾的各种分子标记及遗传学分析和应用。     

Antioxidant responses of hyper-accumulator and sensitive fern species to arsenic

Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from the environment. This work aims to examine (i) arsenic accumulation in three fern species [Chinese brake fern (Pteris vittata L.), slender brake fern (Pteris ensiformis Burm. f.), and Boston fern (Nephrolepis exaltata L.)], which were exposed to 0, 150, or 300 muM of arsenic (Na(2)HAsO(4).7H(2)O), and (ii) the role of anti-oxidative metabolism in arsenic tolerance in these fern species. Arsenic accumulation increased with an increase in arsenic concentration in the growth medium, the most being found in P. vittata fronds showing no toxicity symptoms. In addition, accumulation was highest in the fronds, followed by the rhizome, and finally the roots, in all three fern species. Thiobarbituric acid-reacting substances, indicators of stress in plants, were found to be lowest in P. vittata, which corresponds with its observed tolerance to arsenic. All three ferns responded differentially to arsenic exposure in terms of anti-oxidative defence. Higher levels of superoxide dismutase, catalase, and ascorbate peroxidase were observed in P. vittata than in P. ensiformis and N. exaltata, showing their active involvement in the arsenic detoxification mechanism. However, no significant increase was observed in either guaiacol peroxides or glutathione reductase in arsenic-treated P. vittata. Higher activity of anti-oxidative enzymes and lower thiobarbituric acid-reacting substances in arsenic-treated P. vittata correspond with its arsenic hyper-accumulation and no symptoms of toxicity.     

Development of microsatellite loci for the endangered species Pityopsis ruthii (Asteraceae)

? Premise of the study: Microsatellite loci were developed for the endangered species Pityopsis ruthii and will permit genetic and conservation studies of the species. ? Methods and Results: A microsatellite-enriched library was used to develop 12 polymorphic microsatellite loci for P. ruthii. The loci amplified perfect and imperfect repeats with three to seven alleles per locus. Observed heterozygosity ranged from 0.05 to 0.80 and expected heterozygosity ranged from 0.23 to 0.75. ? Conclusions: These microsatellite loci provide a sufficient set of markers for further investigation of population genetics of P. ruthii.     

Calorimetric properties of water and triacylglycerols in fern spores relating to storage at cryogenic temperatures

Storing spores is a promising method to conserve genetic diversity of ferns ex situ. Inappropriate water contents or damaging effects of triacylglycerol (TAG) crystallization may cause initial damage and deterioration with time in spores placed at -15 degrees C or liquid nitrogen temperatures. We used differential scanning calorimetry (DSC) to monitor enthalpy and temperature of water and TAG phase transitions within spores of five fern species: Pteris vittata, Thelypteris palustris, Dryopteris filix-mas, Polystichum aculeatum, Polystichum setiferum. The analyses suggested that these fern spores contained between 26% and 39% TAG, and were comprised of mostly oleic (P. vittata) or linoleic acid (other species) depending on species. The water contents at which water melting events were first observable ranged from 0.06 (P. vittata) to 0.12 (P. setiferum)gH(2)Og(-1)dry weight, and were highly correlated with water affinity parameters. In spores containing more than 0.09 (P. vittata) to 0.25 (P. setiferum)gH(2)Og(-1)dry weight, some water partitioned into a near pure water fraction that melted at about 0 degrees C. These sharp peaks near 0 degrees C were associated with lethal freezing treatments. The enthalpy of water melting transitions was similar in fern spores, pollen and seeds; however, the unfrozen water content was much lower in fern spores compared to other forms of germplasm. Though there is a narrow range of water contents appropriate for low temperature storage of fern spores, water content can be precisely manipulated to avoid both desiccation and freezing damage.     

蜈蚣草砷超富集机制及其在砷污染修复中的应用

蕨类植物蜈蚣草能够从土壤中吸收砷,并储存于地上部分羽叶的液泡中。蜈蚣草具有高效的抗氧化系统,以降低砷的毒害;其砷酸还原系统和液泡区隔化是蜈蚣草进行砷解毒和砷超富集的重要机制。本文综述了目前蜈蚣草砷超富集机制研究的主要进展,并对其在修复砷污染环境的应用中进行了讨论。     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

基于转录组测序信息的水生植物蕃菜SSR标记开发

利用软件MISA对水生植物蓄菜转录组测序所获得的79536条EST序列进行分析,共检测出12319个EST—SSR位点。在蓄菜的EST—SSR中,二核苷酸和三核苷酸重复单元是主导类型,分别占总SSR的57．31％和30．87％,其中AG／CT、AAG／C1_r分别是二、三重复单元类型的优势重复基元,分别占总SSR的29．76％和8．66％。随机挑选了130对EST-SSR引物对蓄菜两个居群进行遗传多样性检测,结果发现：78对引物能扩增出清晰可分辨的条带,其中37对能成功检测出多态性,引物多态率为47．44％。这些多态性引物共检测出114个等位基因,每个位点2—6个,平均3．08个。观测杂合度（心）及预期杂合度（He）分别在0．229～1．000和0．351-0．756之间,多态信息含量PIC值在0．286～O．698之间,平均达0．495。以上研究结果表明,通过萏菜转录组测序产生的EsT数据来开发SSR标记是一种简单而高效的途径,这些新的SSR分子标记为研究苔菜的居群遗传多样性及其遗传结构提供了工具。     

Microsatellite markers designed for tree-fern species Dicksonia sellowiana

Microsatellite markers were developed for Dicksonia sellowiana (Dicksoniaceae), an overexploited and endangered tree-fern species native to Brazil. From an enriched genomic library, 11 primer pairs were selected and used to characterize 36 D. sellowiana individuals from six Brazilian populations. Eight primer pairs amplified dinucleotide and hexanucleotide repeats with two to ten alleles per locus; three primer pairs were monomorphic. For the set of polymorphic markers, the mean observed and expected heterozygosity ranged from 0.29 to 0.44 and from 0.27 to 0.56, respectively. Eight of the primer pairs were also successfully amplified for Cyathea vestita (Cyatheaceae). These molecular markers can be useful tools for genetic studies aiming to analyze the impact of deforestation and overexploitation on the population structure and genetic diversity of fern species from various botanical families.     

Isolation of microsatellite markers for Pelophylax nigromaculata and a tentative application in detecting interspecific introgression

Twenty-seven microsatellite markers were obtained from the black-spotted frog, Pelophylax nigromaculata, using the FIASCO (Fast Isolation by AFLP of Sequences containing repeats) protocol. Genotyping of 60 individuals showed that the number of alleles ranged from 4 to 25 with an average of 12.78 alleles per locus. The observed and expected heterozygosities ranged from 0.137 to 0.956 and 0.594 to 0.948, with averages of 0.531 and 0.809, respectively. Many of these loci were successfully cross-amplified in five other ranid species. The introgression between P. nigromaculata and Pelophylax plancyi was assessed using 5 microsatellite loci on 159 individuals collected from Liaoning, northeast China. Two distinct genetic clusters (pure species) as well as seven unassigned individuals were identified by factorial correspondence analysis (FCA), NewHybrids and Structure. The unassigned individuals, which are likely the product of interspecific hybridization, indicate the usefulness of these microsatellite loci for future studies of hybridization and introgression between these two ranid species.     

Development and characterization of microsatellite markers for Arenaria grandiflora L. (Caryophyllaceae)

Genomic libraries of Arenaria grandiflora enriched for di- and trinucleotide repeats were used for the development of novel microsatellite markers. The subset of 13 polymorphic markers was characterized on 40 individuals of A. grandiflora originating from lowland locations in France. The loci amplified 3 to 10 alleles per locus and expected heterozygosities ranged from 0.46 to 0.83. The newly developed markers will be used for population genetic studies and for assessing genetic composition of a restoration experiment of lowland A. grandiflora populations that are protected in France.     

Microsatellite primers for the endangered aquatic herb, Ottelia acuminata (Hydrocharitaceae)

? Premise of the study: Microsatellite primers were developed in the endangered aquatic herb, Ottelia acuminata, to characterize its genetic diversity and understand its population structure. ? Methods and Results: Eight polymorphic microsatellite markers were developed from two populations of O. acuminata in China. The number of alleles per locus ranged from one to 15; the observed and expected heterozygosities ranged from 0 to 0.885 and from 0 to 0.888, respectively, in the two populations. Selected loci also amplified successfully in O. sinensis. ? Conclusions: These microsatellite markers will facilitate further studies on the conservation genetics and evolutionary history of O. acuminata.