基于SSR标记的荷花品种遗传多样性及群体结构分析

采用SSR标记技术对42个荷花品种( Nelumbo spp.)的基因组DNA进行扩增,在此基础上,对供试品种进行UPGMA聚类分析、群体结构分析和主坐标分析( PCoA)。结果表明：采用17对SSR引物从42个荷花品种的基因组DNA中扩增出77个位点,多态性位点百分率为88.31%;每对引物可扩增出1~9个多态性位点。根据Nei's遗传距离,供试的42个荷花品种可被分成Ⅰ和Ⅱ两组,分别包含3和39个品种;在Nei's遗传距离0.150处,Ⅱ组被进一步分成Ⅱa、Ⅱb和Ⅱc 3个亚组,分别包含3、16和20个品种。群体结构分析结果表明：组分概率高于等于0.80时,供试的42个荷花品种被分成Pop1、Pop2和混合群3个亚群,分别包含17、16和9个品种。 PCoA分析结果表明：在F1水平上,供试的42个荷花品种被分成2个部分;其中,Pop1亚群的品种均分布在第二和第三象限,而Pop2亚群的品种则分布在第一和第四象限。总体来看,聚类分析、群体结构分析和PCoA分析的结果基本一致。综合分析结果表明：玉组包含美洲黄莲( N. lutea Pers.)品种‘艾江南',且与传统中国莲( N. nucifera Gaertn.)品种的亲缘关系最远,故认为该组为美洲黄莲;Ⅱ组为中国莲,其中,Ⅱc亚组以传统中国莲品种为主,而Ⅱb亚组则偏重于美洲黄莲。总体上看,供试的42个荷花品种主要被分为中国莲和美洲黄莲两组,而中美杂交莲并没有独立成组,其成因有待进一步研究。     

古莲子与现代莲子ABA含量和SOD活性的比较研究

本研究选用种龄为580±70年的古莲子──太子莲,和与之亲缘相近的种龄为8年的现代哈尔滨莲,对比测定其脱落酸的含量及超氧物歧化酶活性,以期探讨距今已有几百年种龄的古莲仍能保持其活力、萌发、开花的现象与这两项生理指标的关系。实验结果表明,两种莲子的皮（果皮和种皮）ABA含量均很高,太子莲和哈尔滨莲分别为4.18 μg/g, 5.69 μg/g。SOD活性在莲子吸胀期和幼苗期两种莲子的子叶中差异不明显,而在胚和幼芽中,太子莲比哈尔滨莲分别高出50%和27%。基于以上结果,说明其胚中具有较强的抗逆性物质,利于保持其生命力。莲皮中高含量的ABA及胚中高活性的SOD有利于莲子的休眠和增强其抗逆性,并保持胚的生命力。     

莲品种资源的SRAP遗传多样性分析

利用分子标记SRAP技术对国内广泛栽培以及新近育成的39个莲品种进行了DNA多态性分析。选取5对引物扩增基因组DNA,共获得101条带,其中88条为多态性条带,每对引物平均提供20个标记信息。由UPMGA方法得到的聚类分析结果表明了39个品种间的遗传关系,结果表明:花莲、籽莲和藕莲三大类群有明显的界限,花莲和籽莲的遗传距离较近,藕莲与它们的遗传距离较远。分子方差分析结果表明:莲品种间和品种内均存在遗传变异,藕莲品种内的遗传变异略低于品种间的遗传变异,而诱变籽莲、诱变花莲和常规籽莲品种内遗传变异均大于品种间的遗传变异,尤其是诱变籽莲、诱变花莲品种内遗传变异占总变异的分量分别超过了70%和60%,这种品种内的遗传变异,一方面对于莲的高产稳产具有重要的意义,另一方面也说明了从这些国内广泛栽培的品种以及新近育成的莲品种中可以直接进一步选育出更优良的新品种。     

大猿叶虫四地理种群遗传多样性的RAPD分析

以大猿叶虫Colaphellus bowringi Baly 4个地理种群的基因组DNA为材料, 进行RAPD分析。从80条引物中筛选出11条稳定性好、多态性高的引物进行扩增, 共得到65个扩增位点, 53个多态位点, Nei氏遗传多样性指数为0.1049~0.2061, Shannon多样性指数为0.1641~0.3167。结果表明所分析的大猿叶虫遗传变异很高, 其中江西龙南种群遗传变异最小, 山东泰安种群遗传变异最高。种群间的遗传距离范围为0.0636~0.3200, 其中江西龙南种群和江西修水种群间的遗传距离最小, 哈尔滨种群与江西龙南种群间的遗传距离最大, 种群遗传距离的大小与其相对地理距离的远近吻合。结果提示种群遗传距离的大小与它们生物学上的相似性有关联。     

应用SRAP标记对莲藕资源的聚类分析

利用一种新的分子标记SRAP技术对17个莲藕品种进行了DNA多态性分析。选取7对引物扩增基因组DNA,共获得168条带,其中159条为多态性条带,每对引物平均提供24个标记信息。由UPMGA方法得到的聚类分析结果表明了17个品种间的遗传关系:(1)亚洲莲与美洲莲之间有明显的差异;(2)在亚洲莲内部,藕莲和花莲有明显的遗传分化,在花莲内部亚洲莲与美洲莲的杂交后代和其它花莲品种之间存在明显的差异;(3)SRAP标记是作分子图谱的好标记,但很难区分遗传关系较近的品种。     

Reverse random amplified microsatellite polymorphism reveals enhanced polymorphisms in the 3' end of simple sequence repeats in the pepper genome

Microsatellites or simple sequence repeats (SSR) are widely distributed in eukaryotic genomes and are informative genetic markers. Despite many advantages of SSR markers such as a high degree of allelic polymorphisms, co-dominant inheritance, multi-allelism, and genome-wide coverage in various plant species, they also have shortcomings such as low polymorphic rates between genetically close lines, especially in Capsicum annuum. We developed an alternative technique to SSR by normalizing and alternating anchored primers in random amplified microsatellite polymorphisms (RAMP). This technique, designated reverse random amplified microsatellite polymorphism (rRAMP), allows the detection of nucleotide variation in the 3' region flanking an SSR using normalized anchored and random primer combinations. The reproducibility and frequency of polymorphic loci in rRAMP was vigorously enhanced by translocation of the 5' anchor of repeat sequences to the 3' end position and selective use of moderate arbitrary primers. In our study, the PCR banding pattern of rRAMP was highly dependent on the frequency of repeat motifs and primer combinations with random primers. Linkage analysis showed that rRAMP markers were well scattered on an intra-specific pepper map. Based on these results, we suggest that this technique is useful for studying genetic diversity, molecular fingerprinting, and rapidly constructing molecular maps for diverse plant species.     

Genetic relationships of the Japanese persimmon Diospyros kaki (Ebenaceae) and related species revealed by SSR analysis

Simple sequence repeat (SSR) molecular markers based on 18 primers were employed to study the genetic relationship of Japanese persimmon (Diospyros kaki) specimens. Two hundred and sixty-two bands were detected in 30 Japanese persimmon samples, including 14 Japanese and 10 Chinese genotypes of Japanese persimmon (Diospyros kaki) and six related species, D. lotus, D. glaucifolia, D. oleifera, D. rhombifolia, D. virginiana, and Jinzaoshi (unclassified - previously indicated to be D. kaki). All SSR primers developed from D. kaki were successfully employed to reveal the polymorphism in other species of Diospyros. Most of the primers were highly polymorphic, with a degree of polymorphism equal to or higher than 0.66. The results from the neighbor-joining dendrogram and the principal coordinate analysis diagram were the same; i.e., the Chinese and Japanese genotypes and related species were separated and the relationships revealed were consistent with the known pedigrees. We also concluded that 'Xiangxitianshi' from Xiangxi municipality, Hunan Province, China, is actually a sport or somaclonal variant of 'Maekawa-Jirou', and that 'Jinzaoshi' should be classified as a distinct species of Diospyros. We found that SSR markers are a valuable tool for the estimation of genetic diversity and divergence in Diospyros.     

猕猴桃野生居群的SSR分析初报

采用SSR分子标记技术对我国猕猴桃的2个商业栽培物种——中华猕猴桃和美味猕猴桃的9个天然居群(共221个样)的遗传多样性进行了初步分析。通过对14对猕猴桃引物的筛选,8对重现性好的引物扩增结果表现出良好的多态性。在8个多态性位点上共获得222个等位基因。居群等位基因平均数A=17．3,多态位点百分率P-100,多态信息指数PIC为0．87～0．96,显示出我国的猕猴桃野生居群具有极高的遗传多样性。中华猕猴桃和美味猕猴桃野生居群拥有高比例的共同等位基因,反映出二者的亲缘关系极近。     

荷花花蕾cDNA文库构建及质量评价

为理解荷花Nelumbo nucifera花器官转录组表达情况,分别选取不同花型的代表品种‘洪湖红莲’Nelumbo nucifera ‘Honghu Honglian’(单瓣)、‘唐招提寺莲’N. nucifera ‘Tangzhaotisi Lian’(重瓣)和‘千瓣莲’N. nucifera ‘Qianban Lian’(千瓣及全重瓣)的花蕾为材料分离mRNA,利用SMART技术合成双链cDNA,经限制性内切酶SfiI酶切后回收去掉接头和500 bp以下片段的cDNA。将cDNA与pUC19载体连接,构建荷花花蕾cDNA文库。经检测,该文库容量为1.12 × 106 pfu·mL-1,插入片段大小集中在500～2000 bp,重组率为95%。该文库的成功构建为荷花花蕾期转录组数据的开发及其花器官发育相关基因的功能研究奠定了分子基础。     

莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map

Inter-simple sequence repeat (ISSR) analysis was evaluated for its usefulness in generating markers to extend the genetic linkage map of Citrus using a backcross population previously mapped with restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and isozyme markers. ISSR markers were obtained through the simple technique of PCR followed by analysis on agarose gels, using simple sequence repeat (SSR) primers. Optimization of reaction conditions was achieved for 50% of the SSR primers screened, and the primers amplified reproducible polymorphic bands in the parents and progeny of the backcross population. Mendelian segregation of the polymorphic bands was demonstrated, with an insignificant number of skewed loci. Most of the SSR primers produced dominant loci; however co-dominance was observed with loci derived from three primers. A new genetic map was produced by combining the segregation data for the ISSR markers and data for the RFLP, RAPD and isozyme markers from the previous map and creating genetic linkages among all the markers using JoinMap 2.0 mapping software. The new map has an improved distribution of markers along the linkage groups with fewer gaps, and marker order showed partial or complete conservation in the linkage groups. The incorporation of ISSR markers into the genetic linkage map demonstrates that ISSR markers are suitable for genetic mapping in Citrus. Received: 3 February 2000 / Accepted: 12 May 2000     

Metabolic activities in germinated ancient lotus seeds

Seeds of Taizi lotus (Nelumbo nucifera Gaertn cv. Taizi) wereunearthed in a western suburb of Beijing in 1984, and determinedto be 58070-years-old by radiocarbon dating. Pretreatment withconcentrated sulphuric acid for 6 h promoted 75% of the seedstested to germinate. Over 3 weeks of incubation after the additionof water, storage starch, total soluble sugar and globulin inthe cotyledons decreased whereas albumin and soluble -aminonitrogen increased. These changes in cotyledonary componentswere similar to the changes seen in four present-day Indianlotus varieties, although the content of reducing sugar in Taizilotus was significantly lower. The polypeptide compositionsin extracts from the cotyledons and embryonic axis of Taiziseeds, as analysed by SDS-PAGE, were similar to those of present-dayJiangxi seeds. Taizi lotus also showed the ability to incorporateradioactive leucine into the trichloroacetic acid (TCA)-insolublefraction of a cotyledonary extract during the 24 h post-imbibitionperiod. The incorporation was inhibited when seeds of Taizilotus were allowed to rehydrate for 24 h in the presence of-amanitin or cycloheximide. Key words: Ancient lotus, Nelumbo nucifera, protein synthesis, seed germination, seed viability     

Development of mapped simple sequence repeat markers from common bean (Phaseolus vulgaris L.) based on genome sequences of a Chinese landrace and diversity evaluation

Microsatellite or single sequence repeat (SSR) markers have been commonly used in genetic research in many crop species, including common bean (Phaseolus vulgaris L.). A limited number of existing SSR markers have been designed from high-throughput sequencing of the genome, warranting the exploitation of new SSR markers from genomic regions. In this paper, we sequenced total DNA from the genotype Hong Yundou with a 454-FLX pyrosequencer and found numerous SSR loci. Based on these, a large number of SSR markers were developed and 90 genomic-SSR markers with clear bands were tested for mapping and diversity detection. The new SSR markers proved to be highly polymorphic for molecular polymorphism, with an average polymorphism information content value of 0.44 in 131 Chinese genotypes and breeding lines, effective for distinguishing Andean and Mesoamerican genotypes. In addition, we integrated 85 primers of the 90 polymorphism markers into the bean map using an F2 segregating population derived from Hong Yundou crossed with Jingdou. The distribution of SSR markers among 11 chromosomes was not random and tended to cluster on the linkage map, with 14 new markers mapped on chromosome Pv01, whereas only four loci were located on chromosome Pv04. Overall, these new markers have potential for genetic mapping, genetic diversity studies and map-based cloning in common bean.     

基于磁珠富集法开发并筛选西川红景天的微卫星标记

利用FIASCO磁珠富集法,开发和筛选青藏高原特有珍贵植物西川红景天(Rhodiola alsia)多态性微卫星标记。结果表明:用(AG)15和(AC)15两种微卫星标记探针构建富集文库,共获得阳性克隆约2500个;从中随机挑取1200检测,发现具有多态性的阳性克隆达400个;随机挑取200多态性的阳性克隆进行测序,从中获得105个SSR位点,用在线软件primer3-2.3.4成功设计得SSR引物105对;其中45对可以成功扩增,而13对所扩增的片段在相距较远的4个自然居群的24个个体中显示较高的遗传多态性。用4个居群的80个个体检测这13对引物发现,平均等位基因数(A)约为9.192,观测杂合度(Ho)和期望杂合度(He)均值分别约为0.712和0.734,如此高的多态性已经满足后期研究的需要;数个位点在某些居群中显著偏离哈迪温伯格平衡(P0.01),这可能是实际研究的居群并不能达到哈迪—温伯格定律所假设的无限大等理想状态所致。结合此前基于表达序列标签(Expressed Sequence Tag,EST)序列开发的SSR多态性位点,该研究结果为今后利用SSR位点开展西川红景天的居群遗传结构分析和其他研究提供了一组有效工具。     

基于转录组测序的枫香EST-SSR引物开发及有效性评价

枫香是广西重要的乡土树种之一，具有较高的用材、观赏及药用价值。为验证枫香SSR引物的实际应用效果，该研究基于转录组测序技术，检测枫香SSR位点并设计引物，通过PCR扩增和聚丙烯酰胺凝胶电泳筛选出具有较高多态性的枫香EST-SSR引物，并对1个枫香天然群体进行遗传多样性分析。结果表明：(1)共发掘到23 777个SSR位点，单核苷酸重复类型SSR位点占总位点比例最高(46.54%),在重复次数上5～12次之间的SSR位点占比最高(72.36%)。(2)共开发出262对SSR引物，有效扩增率为53.1%,最终筛选出扩增稳定、条带清晰的引物18对。(3)多态性检测结果显示所有位点均具有多态性，天然群体遗传多样性结果显示该天然群体中等位基因数量(Na)、有效等位基因数量(Ne)、Shannon多样性指数(I)、观测杂合度(Ho)变化范围分别为2～4、1.112 8～2.609 6、0.208 9～1.112 7和0.275 9～1.000 0,平均值分别为2.333 3、1.957 4、0.708 5和0.722 6。综上认为，枫香中占优势的SSR位点重复类型和重复基序与其他物种基本相同，所开...     

藕莲种质资源形态与农艺性状的遗传变异分析

藕莲是莲（Nelumbo nucifera Gaertn.ssp.nucifera）的3种类型之一,在我国作为水生蔬菜栽培的历史愈两千年。对国家种质资源圃中来自17个省和直辖市的68份藕莲资源的19个形态和农艺性状进行了观察,并对其遗传变异进行了分析。结果发现整藕重与主藕重、主藕长和第三节粗成极显著正相关。通径分析结果表明主藕重对整藕重的正向效应最高（0.982）。主成分分析结果显示前4个成分占总变异的77.33%。结果还显示对整藕重负效应最高的是主藕重/整藕重（-0.296）。聚类分析发现所有藕莲资源可分为5组。     

从CIMMYT引进的人工合成六倍体小麦D染色体组微卫星分子标记的遗传差异

采用微卫星（SSR）分子标记技术,选用23个D染色体组特异性引的对来自CIMMYT的26份人工合成六倍体小麦D染色体组的遗传多样性进行了分析。研究发现,26份材料在D染色体组上存在丰富的等位基因变异（92个）,平均每个基因座为4个。遗传距离计算结果也显示,26份材料D染色体组之间具有较大的遗传差异,平均遗传距离高达0．4955。因此,人工合成六倍体小麦D染色体组中存在丰富的遗传多样性,可以作为拓宽普通小麦遗传基础的新的遗传变异来源。研究还发现,由同一个粗山羊草基因型与不同硬粒小麦杂交合成的人工合成六倍体小麦（如合成种17和18）在所用检测的23个基因座中有3个存在差异,说明小麦在多倍化后,供体基因组在重复序列区域会发生遗传分化。     

中国35个苦荞审定品种EST-SSR指纹图谱构建与遗传多样性分析

核心引物对种质资源遗传多样性分析、品种鉴定、指纹图谱构建等研究具有重要价值。本研究以35个苦荞（Fagopyrum tataricum（L.） Gaertn）审定品种为材料,从91对苦荞EST-SSR引物中筛选出50对多态性引物。综合考虑引物多态性信息量（PIC）大小、鉴别力（DP）,筛选出等位变异位点数在2~4,PIC值在0.60~0.78之间的6对引物（SSR9007、SSR6873、SSR7642、SSR2234、SSR6789、SSR68216）构建了供试品种的分子指纹图谱。遗传多样性聚类分析结果表明,供试品种的相似系数为0.50~0.99。当遗传相似系数为0.60时,可将供试品种分为4大类群,其中54.3%的供试品种被聚为一类,表明苦荞审定品种遗传组成差异较小,遗传基础狭窄。聚类结果表明各类群间没有明显的地域分布趋势,但能较好的反映供试品种间的亲缘关系。     

Development and characterization of EST-derived simple sequence repeat (SSR) markers for pasture grass endophytes.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichlo? isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichlo? species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.     

基于EST-SSR标记的南方型黑杨主栽无性系鉴定及亲缘关系分析

以南方型黑杨引进后主栽区重点推广栽植的12个无性系为研究对象,利用EST-SSR分子标记进行多态性分析,并建立无性系鉴定的指纹图谱,为优良黑杨无性系的推广提供依据。用EST-trimmer对NCBI数据库中的EST序列进行分析、MISA软件找出SSR位点后运用primer3在线设计获得的30对EST-SSR引物,对12个黑杨无性系进行PCR扩增。经过筛选获得18对多态性引物,对12个黑杨无性系进行扩增获得88个等位基因,多态率为70.5%,平均观察等位基因数为2.1463,平均Shannon’s多样性指数为0.5927。通过5对引物组合(EU147、EU43、EU11、EU164和EU81)可以将12个黑杨无性系进行区分,并在此基础上构建指纹图谱。亲缘关系分析发现无性系间的遗传相似系数为0.6020~0.9040,平均值为0.7691;无性系间的遗传距离较近,与其均来源于美洲黑杨和欧美杨的相似遗传背景有关。总之,EST-SSR标记可以对南方型黑杨主栽无性系进行有效鉴定。