利用DNA片段测序方法探究枳属和富民枳的分类地位

富民枳(Poncirus polyandra)属于芸香科(Rutaceae)枳属(Poncirus Raf.)。自发表以来,分类地位一直备受争议,其中在Flora of China中认为富民枳为柑橘杂交种(Poncirus polyandra),把枳属归并于柑橘属(Citrus)。该研究选取枳属的富民枳、枳(Poncirus trifoliata)及柑橘属下8个种共10个种47个个体作为研究材料,以九里香(Murraya exotica)为外类群,利用3个叶绿体片段(trn L-trn F、trn S-trn G、rbc L)、ITS片段和1个单拷贝核基因(Chr 5)数据构建系统发育树,探究枳属和富民枳的分类地位。结果表明:基于3个叶绿体片段数据构建的最大似然树(ML)和贝叶斯树(BI)的拓扑结构基本一致,10个物种聚为两大分支,即柑橘属的8个物种聚为一大分支,富民枳和枳聚为另一大分支。其中,富民枳所有个体聚为一小单系分支,枳的所有个体聚为一小单系分支,支持枳属和富民枳独立存在。2个核DNA片段数据结果显示,枳属的两个种与柑橘属的8个物种聚在一个大分支里,无法确立枳属的单系地位,但富民枳的9个个体聚在一起,暗示富民枳在遗传上是一个独立的类群。综上研究认为,无论是叶绿体DNA数据还是核DNA数据均支持富民枳是一个独立的物种,但核DNA数据不支持枳属成立。     

用SSR分子标记研究大豆属种间亲缘进化关系

利用SSR标记技术对大豆属11个种37份材料的遗传多样性进行分析,不同位点在种间的等位基因数为6－29,平均生个位点15．9个等位基因,Soja亚属的等位基因数是Glycine亚属的71．5％,并且Glycine亚属种间指纹 谱的差异大于Soja亚属种间的指纹图谱,SSR等位基因的主成分分析结果表明,大豆属中的Glycine亚属和Soja亚属的分类界限是比较明确的,利用第一主成分和第二主成分可较明显地区分开Glycine 亚属的分类界限是比较明显的,利用第一主成分和第二主成分可较明显地区分开Glycine亚属和Soja亚属,通过UPGMA方法构建了大豆属11个种的遗传进化关系,Soja亚属中G.max,G.soja和G.gracilis3个种在系统分化树上界限是比较明显的,由此看来这3个种是独立存在的。     

基于SSR标记分析小豆及其近缘植物的遗传关系

本研究利用87对SSR引物分析了80份栽培小豆(Vigna angularis)、22份野生小豆(V.angularis var.nipponensis)以及10份豇豆属(共7个种)近缘植物,旨在比较豇豆属不同种的遗传多样性,并分析种间的遗传关系.结果显示87对SSR引物在112份小豆及其近缘植物资源中共检测到667个等位变异.其中有75个、71个和82个SSR位点分别在栽培小豆、野生小豆和近缘植物中表现为多态.随机抽样分析发现,平均每SSR位点检测到的等位变异数目为近缘植物>野生小豆>栽培小豆,与多态信息含量(PIC)值一致,说明近缘植物及野生小豆中蕴含着丰富的遗传变异,是栽培小豆育种的重要基因来源.聚类分析显示,栽培小豆、野生小豆和近缘植物间的遗传分化比较明显,分别聚为三大类,其中栽培小豆的遗传背景与其生态环境相对应;近缘植物又可以分为三个亚类,亚类间的遗传距离与其亲缘关系相对应.本研究结果也说明利用SSR标记辅助豇豆属的种间分类是可行的.     

4种甘草属植物EST-SSR引物开发及其亲缘关系分析

通过乌拉尔甘草表达序列标签(EST)数据库查找甘草属的SSR位点,并利用Primer 3.0软件在线设计EST-SSR引物,对来自甘草属4个种22份材料的EST-SSR指纹图谱特征和聚类结果进行分析,为探讨甘草属种间亲缘关系和疑难种的分类地位提供分子依据。结果显示:(1)去掉冗余序列后共得到441条EST序列,获得504个SSR位点,其中二核苷酸为重复单元的序列最多为350个,占69.44%,重复类型中以TC/AT、TA/AG形式的微卫星最为丰富。(2)设计的40对EST-SSR引物,均能扩增出清晰条带,其中15对引物具有多态性,在22份甘草属植物材料中共获得等位基因59个,平均每对引物检测到3.93个等位基因位点,扩增产物多态性比率为89.44%,能很好地表征甘草属种间的等位基因差异。(3)引物Primer 64对4种甘草属植物均能扩增出特异性条带,黄甘草在180和220bp 2个位点分别与光果甘草、胀果甘草基因共享,具有杂交种特征。(4)聚类分析表明,当相似性系数为0.82时,22份材料被划分为四组(与经典分类结果一致),第一组为内蒙古杭锦旗分布的乌拉尔甘草;第二组为新疆巴楚分布的光果甘草、黄甘草;第三组为新疆石河子分布的胀果甘草、黄甘草;第四组为新疆石河子分布的乌拉尔甘草;不同居群的黄甘草遗传分化较大,可能与同域分布亲本种的差异及种间的渐渗杂交有关。研究表明,开发的15对EST-SSR引物在甘草属内具有很好的适用性,可以为该属的种间亲缘关系和种内遗传分化研究及物种鉴定提供分子依据。     

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。     

松毛虫属（Dendrolimus) 部分种类亲缘关系与遗传分化的等位酶分析

采用等位酶聚丙烯酰胺凝胶电泳技术对松毛虫属5个种和亚种的野生种群进行了亲缘关系和遗传变异的研究.8种等位酶系统(乳酸脱氢酶LDH、苹果酸脱氢酶MDH、苹果酸酶ME、乙醇脱氢酶ADH、甲酸脱氢酶FDH、谷氨酸脱氢酶GDH、过氧化物酶POD、过氧化氢酶CAT)共检测到12个基因位点,其中6个位点为多态位点,检测到15个等位基因.松毛虫属5个种和亚种的总体水平多态位点比率P＝50%,平均有效基因数A = 1.917,平均期望杂合度He =0.267,平均遗传距离为0.0730～0.5701.遗传参数表明松毛虫属昆虫种间存在较高程度的遗传变异,聚类图和遗传距离数据表明赤松毛虫与马尾松毛虫亲缘关系最近,落叶松毛虫与思茅松毛虫亲缘关系最远.     

真正柑橘果树类植物基于AFLP分子标记的分类与进化研究

用AFLP分子标记技术对芸香科Rutaceae柑橘亚科Aurantioideae真正柑橘果树类(the True Citrus Fruit Trees Group) 6属植物的59个生物类型以及九里香Murraya exotica L.、蚝壳刺Severinia buxifolia Tenore和酒饼簕Atalantia buxifolia Oliv.进行了分析。从64对引物组合中筛选出15对分辨率好的引物对全部材料进行了扩增, 共获得312条带, 其中多态型带305条, 多态位点达97.8%。利用PAUP软件构建了全部材料基于AFLP数据的UPGMA距离树。结果表明, AFLP证据支持基于形态特征建立的真正柑橘果树群以及群内各属的划分, 只是金柑属Fortunella Swingle作为一个独立的分支嵌于柑橘属Citrus L.内。对于柑橘属, 我们的分子证据支持Swingle关于亚属的划分以及Scora等关于C. medica、C. grandis和C. reticulata是真正柑橘亚属subgen. Eucitrus三个基本种的观点。AFLP分子证据显示宜昌橙C. ichangensis Swingle位于真正柑橘亚属中, 与香橙C. junos Sieb. ex Tanaka的亲缘关系最近, 而与大翼橙亚属subgen. Papeda中马蜂柑C. hystrix DC.相隔甚远。另外, 莽山野橘C. mangshanensis S. W. He &; G. F. Liu在系统树上居于宽皮柑橘类的基部。最后, 对真正柑橘果树群6属植物间以及柑橘属各主要类型(种)的进化关系进行了详细的讨论。     

菊属与亚菊属属间杂种的鉴定及其分类学意义

通过有性杂交的方法获得了栽培菊花“奥运火炬”Dendranthema×grandiflorum“Aoyunhuoju”和亚菊属Ajania矶菊A.pacifica的属间杂种。形态学、细胞学以及RAPD分子标记鉴定表明22个杂种为真正的属间杂种,成功地将矶菊的某些性状（叶色）转移到栽培菊花中,但同时也掺入了某些不良性状。杂种的叶色、叶形、花色、开花枝的分枝性和花期等性状均介于双亲之间,边缘舌状雌花大小、数量和形态不一;杂种根尖染色体数目正常（2n=72）,花粉母细胞减数分裂基本正常。矶菊与栽培菊花属间杂交的成功以及杂种减数分裂染色体行为正常表明两者亲缘关系较近,从而推测菊属Dendranthema与亚菊属之间的亲缘关系可能较近。     

基于SSR分子标记的酸浆属植物亲缘关系研究

近年来, 由于营养价值高、果实可当水果食用且具有潜在的药用价值, 酸浆属(Physalis)植物在全球范围内受到了越来越多的关注。采用SSR分子标记技术对我国范围内主要分布的4种酸浆属植物的22份样品进行了亲缘关系研究, 结果表明, 20对SSR引物共扩增出118个位点, 其中107个(90.7%)为多态性位点; 平均种间遗传相似系数为0.501。UPGMA聚类和PCoA分析结果得出相似结论, 并且将供试酸浆属植物样品分为两大类。其中, 酸浆(P. alkekengi var. francheti)的所有样品与其它酸浆属植物的遗传距离最远, 单独聚为一类, 与前人的研究结果非常吻合。研究表明, SSR标记遗传信息丰富, 可以用于酸浆属植物的亲缘关系分析。研究结果为酸浆属种质资源保护提供了有效信息, 并且为酸浆属植物的分子辅助育种奠定了重要基础。     

中国红豆杉属及其近缘植物的遗传变异和亲缘关系分析

采用水平式淀粉凝胶电泳技术,对中国产红豆杉属Taxus 3种1变种以及白豆杉Pseudotaxus chienii共12个居群的遗传多样性进行了检测。对7个酶系统9个酶位点的分析结果表明,红豆杉属和 白豆杉的种内遗传多样性水平较高,各类群的多态位点比率(P)为44.4％～77.8％,等位基因平均数 (A)为1．6～2．1,平均期望杂合度(He)和实际观察杂合度(Ho)分别为0.065～0.152和0.068～0.111。 根据对等位酶实验结果的分析,结果表明除云南红豆杉T．yunnanensis与红豆杉属其它几个种的遗传 一致度稍低(I=0.727～0.804)外,红豆杉属的其它几个种的遗传一致度较高(0.906～0.995),体现其 亲缘关系很近。笔者建议将红豆杉属的这几个种合并为一个种,并根据地理分布及不同生境发生的变 异在种下设地理宗。同时根据等位酶的实验分析结果,白豆杉和红豆杉属各种的比较有显著差异,支持白豆杉作为单独属Pseudotaxus的处理。     

Exploiting BAC-end sequences for the mining,characterization and utility of new short sequences repeat (SSR) markers in Citrus

The aim of this study was to develop a large set of microsatellite markers based on publicly available BAC-end sequences (BESs), and to evaluate their transferability, discriminating capacity of genotypes and mapping ability in Citrus. A set of 1,281 simple sequence repeat (SSR) markers were developed from the 46,339 Citrus clementina BAC-end sequences (BES), of them 20.67% contained SSR longer than 20 bp, corresponding to roughly one perfect SSR per 2.04 kb. The most abundant motifs were di-nucleotide (16.82%) repeats. Among all repeat motifs (TA/AT)n is the most abundant (8.38%), followed by (AG/CT)n (4.51%). Most of the BES-SSR are located in the non-coding region, but 1.3% of BES-SSRs were found to be associated with transposable element (TE). A total of 400 novel SSR primer pairs were synthesized and their transferability and polymorphism tested on a set of 16 Citrus and Citrus relative’s species. Among these 333 (83.25%) were successfully amplified and 260 (65.00%) showed cross-species transferability with Poncirus trifoliata and Fortunella sp. These cross-species transferable markers could be useful for cultivar identification, for genomic study of Citrus, Poncirus and Fortunella sp. Utility of the developed SSR marker was demonstrated by identifying a set of 118 markers each for construction of linkage map of Citrus reticulata and Poncirus trifoliata. Genetic diversity and phylogenetic relationship among 40 Citrus and its related species were conducted with the aid of 25 randomly selected SSR primer pairs and results revealed that citrus genomic SSRs are superior to genic SSR for genetic diversity and germplasm characterization of Citrus spp.     

A Study on Glutamate Oxaloacetate Transaminase Isozymes of Citrus Germplasm Resources

The glutamate oxaloacetate transaminase isozymes (GOT) extracted from 125 biotypes of Citrus and its relatives, Fortunella, Poncirus and Microcitrus were analyzed using polyacrylamide gel electrophoresis to investigate the taxonomic relationships among citrus plants. Besides all the isozymes reported before, two new bands were detected and designated their putative alleles B and C in GOT-l. Among pummelo cultivars wide variations were found. Most of the mandarins were identical, having SS for GOI-1 and MM for GOT-2 except for Zou-Pi-Gan and Yao-Gan, which both had FS at GOT-1 and might be hybrids. This finding suggests that all of the mandarins may have originated from a common ancestor. Sour orange biotypes showed a considerable variation in GOT isozymes. Most of the sour oranges in China were assumed to be hybrids between pummelo and mandarin based on GOT isozyme patterns, but two biotypes, Xiao-Hong-Cheng and Zhu-lan, had FS at GOT-1 and MB at GOT-2, which strongly suggests that they be hybrids of pummelo and Ichang papeda because B allele of GOT-2 occurs only in Ichang papeda and its close relatives Yuzu and Ichang lemon. From this study Yuzu is assumed to be derived from hybridizationof Ichan, papeda and mandarin.     

部分柑桔属及其近缘属Giemsa C-带带型研究

本文应用Giemsa显带技术研究了枳属(Poncirus)、金柑属(Fortunella)和柑桔属(Citrus)16个分类群的染色体。枳属以末端带和着丝点带为主,金柑属与柑桔属主要以末端带为主;统计分析了各分类群每对染色体及全组染色体的异染色质含量,并列出其带型公式;探讨了金柑属的分类学地位;赞同把柚(C.grandis)作为柑桔属的基本种之一;根据异染色质含量的变化对柑桔属的带型演化进行了讨论。     

Mapping the chromosomes of Poncirus trifoliata Raf. by BAC-FISH

In spite of the importance of Citrus in agriculture and recent progress in genetic mapping and cytogenetics of this group, chromosome mapping of Citrus species is still limited to rDNA probes. In order to obtain a better chromosome characterization of one species from this group, CMA/DAPI double staining followed by in situ hybridization using 45S rDNA and 24 BACs (BAC-FISH) were used on Poncirus trifoliata. The BACs used were obtained from a genomic library of this species and were selected by membrane hybridization using genomic DNA. Four of them were isolated from the Citrus tristeza virus (Ctv) resistance gene region. The P. trifoliata karyotype is composed of two chromosome pairs with one terminal and one proximal CMA(+) band (B type chromosomes), four chromosome pairs with a single CMA(+) band (D type) and three chromosome pairs without bands (F type). In situ hybridization with 13 of the BACs gave single copy signals on seven chromosome pairs. At least one BAC was mapped on each arm of the two B chromosome pairs. Among the four D chromosome pairs, two were identified by BACs mapped on the long arms, one has a 45S rDNA site and the other had no signal. Six BACs allowed identification of the three F chromosome pairs, with one pair hybridizing with four BACs from the Ctv resistance gene region. In summary, all nine chromosome pairs could be differentiated, seven of them by BAC-FISH, while the other two chromosomes could be recognized by the CMA(+) band pattern and 45S rDNA sites. This first BAC-FISH map gives a general framework for comparative genome structure and evolutionary studies in Citrus and Poncirus, allowing the integration of genetic and physical maps when these BACs are included.     

A Numerical Taxonomic Study of Citrus and Its Close Relatives

Fifty nine taxa of Citrus, Fortunella and Poncirus were studied by hierarchical agglomerative clustering analysis and quantas type 3 analysis using 86 morphological characters. Five affinity groups were obtained in Citrus. The first group includes C. medica, C. limon, C. limonia, C. aurantifolia and C. iambhiri; the second includes C. grandis, C. aurantium, C. sinensis and C. paradisi; the third is mandarin (C. reticulata); the forth is C. ichangensis; The last is C. hongheensis. Mandarin oranges show two subgroups: one comprise satsuma, King, Shagan, Tankan and Bendiguangju, and the other consists of the remaining typical mandrins. The two papeda oranges, honghe papeda and ichang papeda, could not be clustered into the same group, indicating that they might have evolved from two compeletly different ancestors. Pumelos(C. grandis) show some relationships to honghe papeda orange. C. limonia is assumed to be a hybrid between rough lemon and mandarin. The sweet orange(C. sinensis) is considered to be the offspring of pomelo and mandarin. Zhekiang jinju a small mandarin generally considered similar to calamondin in China, should be a true mandarin orange based on our results. Poncirus was well distinguished from the other two genera, Citrus and Fortunella, by the numerical methods used in this study. Our results show that citron (C. medica), mandarin (C. reticulata) and pumelo (C. grandis) are the original species of the subgenus Citrus, which was congruent with the re-sults obtained by Barrett and Soost (1976), Potvin (1983) and Handa (1985).     

Analysis of Microsatellites in Citrus Unigenes

Simple sequence repeats (SSRs) were investigated in the unigene sequences from expressed sequence tags (EST) of sweet orange (Citrus sinensis osbeck), trifoliate orange (Poncirus trifoliata Raf.) and other citrus species and cultivars. A total of 37 802 citrus unigene sequences corresponding to 23.29 Mb were searched, resulting in the identification of 8 218 SSRs. Among them there were 4 913 (59.8%) mono-, 1 419 (17.3%) di-, 1 709 (20.8%) tri-, 114 (1.39%) tetra-, 23 (0.28%) penta- and 40 (0.49%) hexa-nucleotide SSRs. The estimated frequency of SSRs was approximately 1/2.8 kb, which could be extrapolated to 1 SSR-containing unigene in 4.6 unigenes. The maximum length of the SSR ranged from 40 to 105 bp depending on the repeating numbers of the motif in the SSR. The overall average length of SSRs was 20.9 bp. The frequencies of different SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) were very similar between sweet orange and trifoliate orange. The mononucelotide repeats appeared to be the most abundant SSRs within sweet orange and trifoliate orange, followed by trimeric repeats. The adenine rich repeats such as A/T, AG, AT, AAG, AAAT, AAAG, AAAT, AAAAG, AAAAT etc. were predominant in each type of SSRs (mono-, di-, tri-, tetra-, and penta-), whereas the C/G, CG, CCG repeats were less abundant. Twenty-five primer pairs flanking EST-SSR loci were designed to detect the possible polymorphism of six citrus cultivars including sweet orange and trifoliate orange. The PCR result with all these 25 primer pairs revealed the existence of polymorphism within six citrus cultivars confirming that citrus EST database could be efficiently exploited for the development of gene-derived SSR markers.     

柑橘EST-SSR分子标记分析

对来源于甜橙（Citrus sinensis Osbeck）、枳壳（Poncirus trifoliata Raf．）和其他柑橘非冗余EST数据库的38124条单-基因（Unigene）序列进行了简单重复序列SSRs（Simple Sequence Repeat）搜索,所分析的柑橘非冗余核酸序列总长23．29Mb,从中获得了8218条SSR,其中包括单碱基重复4913条（59．8％）,2碱基重复1419条（17．3％）,3碱基重复1709条（20．8％）,4碱基重复114条（1．39％）,5碱基重复23条（0．28％）,6碱基重复40条（0．49％）。大约每2．8kb长度的单-基因序列中即存在1个SSR,即平均4．6个单-基因中存在1个SSR。随碱基重复单元（motif）的不同,SSR的最大长度在40-105之间,全部重复序列的平均长度为20．9bp。各种SSR（1-,2-,3-,4-,5-,6-核苷酸重复）的发生频率在甜橙和枳壳间非常接近。其中单碱基重复序列是最丰富的重复单元,其次为3碱基重复。在所得的SSR的重复单元中,富含A碱基的重复单元的分布占据优势地位,出现的频率与密度均较高,而富含CG碱基的重复单元出现频率和密度较低。用25对EST-SSR引物对6个柑橘品种的多样性进行了PCR检测,结果表明,所有25对引物在6个柑橘品种间均扩增到多样性条带,证实通过柑橘EST数据库的发掘能够高效地筛选到基因水平的SSR标记。     

Heterochromatin banding patterns in Rutaceae-Aurantioideae--a case of parallel chromosomal evolution

The heterochromatin banding patterns in the karyotypes of 17 species belonging to 15 genera of Rutaceae subfamily Aurantioideae (= Citroideae) were analyzed with the fluorochromes chromomycin (CMA) and 4'-6-diamidino-2-phenylindole-2HCl (DAPI). All species were diploids, except one tetraploid (Clausena excavata) and two hexaploids [Glycosmis parviflora agg. (aggregate) and G. pentaphylla agg.]. There are only CMA/DAPI bands, including those associated with the nucleolus. Using recent cpDNA (chloroplast DNA) sequence data as a phylogenetic background, it becomes evident that generally more basal genera with rather plesiomorphic traits in their morphology, anatomy, and phytochemistry exhibit very small amounts of heterochromatin (e.g., Glycosmis, Severinia, Swinglea), whereas relatively advanced genera from different clades with more apomorphic characters display numerous large CMA bands (e.g., Merrillia, Feroniella, Fortunella). Heterochromatin increase (from 0.7 to 13.7%) is interpreted as apomorphic. The bands are mostly located in the larger chromosomes and at telomeric regions of larger arms. However, one of the largest chromosome pair has been conserved throughout the subfamily with only very little heterochromatin. The heterochromatin-rich patterns observed in different clades of Aurantioideae appear quite similar, suggesting a kind of parallel chromosomal evolution. In respect to the current classification of the subfamily, it is proposed to divide Murraya s.l. (sensu lato) into Bergera and Murraya s.s. (sensu stricto) and to place the former near Clausena into Clauseneae s.s. and the latter together with Merrillia into Citreae s.l. The subtribes recognized within Clauseneae s.s. and Citreae s.l. appear heterogeneous and should be abandoned. On the other hand, the monophyletic nature of the core group of Citrinae, i.e., the Citrus clade with Eremocitrus, Microcitrus, Clymenia, Poncirus, Fortunella, and Citrus, is well supported.     

用SSR标记分析辣椒属种质资源的遗传多样性

利用21对引物在33个辣椒材料中共检测到54个等位基因,每对SSR引物检测到2~4个等位基因变异,平均为2.6个,说明辣椒种质资源的遗传多样性相对较少。通过MVSP3.13f软件对SSR数据进行聚类分析,把33个辣椒材料分为五类,同个种的辣椒种质资源基本聚在一起,说明用SSR标记来区分辣椒种质是可行的,是研究辣椒种质资源遗传多样性的有效手段。