Ploidy variation in Trigonobalanus verticillata (Fagaceae)

Cytological studies were carried out on two populations of Trigonobalanus verticillata in Yinggeling, Hainan Province, China (YGL), and Fraser’s Hill, Malaysia (FH). In the two populations, the pattern of interphase nuclei was of the simple chromocentre type, the mitotic prophases were of the proximal interstitial type and chromosome numbers were 2n = 2x = 14 (YGL) and 2n = 6x = 42 (FH), representing diploid and hexaploid respectively. The basic chromosome number of the species was x = 7; this is a unique number in the Fagaceae and in the Fagales as well. The characteristic karyotype of the diploid population in China was 2n = 14 = 8m + 4sm + 2st. Based on the new finding of the chromosome number in this study, we clarify the previous dispute about the basic chromosome number and propose that the two Asian Trigonobalanus species have closer affinity.     

Genetic variation and relationship among and within Withania species as revealed by AFLP markers.

Withania somnifera is an important medicinal plant, and its anticancerous properties have been attributed to various classes of withanolide compounds. The objective of the present study was to investigate the inter- and intraspecific genetic variation present in 35 individuals of W. somnifera and 5 individuals of W. coagulans using AFLP (amplified fragment length polymorphism) marker technique. The information about genetic variation determined from AFLP data for 40 individuals was employed to estimate similarity matrix value based on Jaccard's coefficient. The similarity values were further used to construct a phenetic dendrogram revealing the genetic relationships. The dendrogram generated by UPGMA (unweighted pair group method of arithmetic averages) distinguished W. somnifera from W. coagulans and formed two major clusters. These two main clusters shared a similarity coefficient of 0.3, correlating with the high level of polymorphism detected. The dendrogram further separated W. somnifera into three subclasses corresponding to Kashmiri and Nagori groups and an intermediate type. The AFLP profile of Kashmiri individuals was distinct from that of the Nagori group of plants. The intermediate genotype was distinct as it shared bands with both the Kashmiri and Nagori individuals, even though it was identified as a Kashmiri morphotype. Furthermore, the intermediate type shared a similarity coefficient of 0.8 with the Kashmiri individuals. The present work revealed low levels of variation within a population though high levels of polymorphism were detected between Nagori and Kashmiri populations. The ability of AFLP markers for efficient and rapid detection of genetic variations at the species as well as intraspecific level qualifies it as an efficient tool for estimating genetic similarity in plant species and effective management of genetic resources.     

Genetic diversity in apricot revealed by AFLP markers: species and cultivar comparisons

The genetic diversity of apricot ( Prunus armeniaca; 2n = 16) was studied using AFLP markers. Forty seven apricot cultivars were selected from the following geographic regions: Europe, North America, North Africa, Turkey, Iran and China. Five EcoRI- MseI AFLP primer combinations revealed 416 legible bands, of which 379 were polymorphic markers. A similarity matrix was prepared using the simple matching coefficient of similarity. A UPGMA dendrogram demonstrated a gradient of decreasing genetic diversity of varieties from the former USSR to Southern Europe. This is coherent with the historical dissemination of apricot from its center of origin in Asia. The American cultivars were intermediate demonstrating a different genetic base than the European and/or Mediterranean cultivars. Euclidean distances from the first ten Factorial Component Analysis coordinate axes were used to generate a tree using the Ward algorithm. The results of these analyses were evaluated based on the known geographic origins and agronomic characteristics of the cultivars studied. Four cultivar groups were identified: Diversification, Geographically Adaptable, Continental Europe and Mediterranean Basin. To evaluate the relationship of the common apricot with some closely related species, one or two accessions of the following related species or sub-species from within the section Armeniaca were included in the analysis: Prunus armeniaca var. ansu, Prunus mume, Prunus brigantiaca, Prunus dasycarpa, and Prunus holosericea. A Neighbour Joining dendrogram was made using the similarity matrix. The P. holosericea accession fell well within the cultivar group, thus supporting its classification as a variant of P. armeniaca. The P. armeniaca var. ansu accession was sister to the common apricot cluster with a bootstrap value of 96%. P. mume was farther removed. P. brigantiaca was the most-distant from the common apricots. P. dasycarpa was intermediate between P. brigantiaca and P. mume, in accord with its plum-apricot hybrid origin. The results have a direct application for the selection of new breeding progenitors.     

海南鹦歌岭轮叶三棱栎(Trigonobalanus verticillata) 群落特征与保护对策

应用样方调查法,研究了中国分布新记录种--轮叶三棱栎（Trigonobalanus verticillata）种群结构及其所处森林群落特点。结果表明：轮叶三棱栎仅分布于海南鹦歌岭海拔1100-1400m近山脊处的热带山地雨林及热带山地常绿阔叶林群落中,与陆均松（Dacrydium pectinatum）、海南阿丁枫（Altingia obovata）等树种伴生。在2个面积为1500m^2的调查样方中共记录了90种乔灌木树种,均匀度和Shannon-Wiener指数分别为0.81、0.86和3.20、3.27,轮叶三棱栎的重要值在群落中排在第9-10位;种群结构分析结果显示该种群数量小且无Ⅰ龄级和Ⅲ龄级,属不稳定种群;轮叶三棱栎生态位宽度为1.69,在群落中仅排第19位,与陆均松和鸡毛松（Dacrycarpus imbricatus）的生态位重叠值分别为0.59和0.68,但与群落优势种的生态位重叠值多小于0.3。鹦歌岭在海拔1000m以上具有较大面积的台地和人为破坏较少可能是该种群得以幸存的重要因素,加强就地保护,开展该种植物的生物学特性研究,并建立国家级保护区,将海南中部山区各保护区有机地联合在一起为保护良策。     

Genetic diversity and phylogeny of Japanese sake-brewing rice as revealed by AFLP and nuclear and chloroplast SSR markers

Japanese rice (Oryza sativa L.) cultivars that are strictly used for the brewing of sake (Japanese rice wine) represent a unique and traditional group. These cultivars are characterized by common traits such as large grain size with low protein content and a large, central white-core structure. To understand the genetic diversity and phylogenetic characteristics of sake-brewing rice, we performed amplified fragment length polymorphism and simple sequence repeat analyses, using 95 cultivars of local and modern sake-brewing rice together with 76 cultivars of local and modern cooking rice. Our analysis of both nuclear and chloroplast genome polymorphisms showed that the genetic diversity in sake-brewing rice cultivars was much smaller than the diversity found in cooking rice cultivars. Interestingly, the genetic diversity within the modern sake-brewing cultivars was about twofold higher than the diversity within the local sake-brewing cultivars, which was in contrast to the cooking cultivars. This is most likely due to introgression of the modern cooking cultivars into the modern sake-brewing cultivars through breeding practices. Cluster analysis and chloroplast haplotype analysis suggested that the local sake-brewing cultivars originated monophyletically in the western regions of Japan. Analysis of variance tests showed that several markers were significantly associated with sake-brewing traits, particularly with the large white-core structure.     

Phylogenetic relationships among Oryza species revealed by AFLP markers

 The genus Oryza to which cultivated rice belongs has 22 wild species. Seventy-seven accessions of 23 Oryza species, five related genera, and three outgroup taxa were fingerprinted using amplified fragment length polymorphism (AFLP). A total of 1191 polymorphic markers were obtained using five AFLP primer combinations. AFLP data were analyzed to study species relationships using different clustering algorithms, and the resulting phenograms were tested for stability and robustness. The findings suggest a common ancestry to the genus Oryza. Moreover, the results demonstrate that: (1) evolution in Oryza has followed a polyphyletic path wherein multiple lineages underwent independent divergence after separation early in the evolution from a common ancestor/pool of related taxa; (2) newly assigned genomes, GG for O. meyeriana and HHJJ for O. ridleyi complexes, are among the most diverged in the genus; (3) CCDD tetraploids have a relatively ancient origin among the Officinalis complex; (4) O. malampuzhaensis, O. indandamanica, O. alta, and O. grandiglumis are diverged enough to deserve species status; (5) O. officinalis and O. eichingeri (CC) are putative progenitors of O. minuta ^* O. malampuzhaensis and tetraploid O. punctata, respectively, (6) O. brachyantha is most diverged species in the genus. AFLP is reliable molecular technique and provides one of the most informative approaches to ascertain genetic relationships in Oryza, which may also be true for other related species/organisms. Received: 1 July 1998 / Accepted: 2 November 1998     

Genetic diversity of Carica papaya as revealed by AFLP markers.

Genetic relationships among Carica papaya cultivars, breeding lines, unimproved germplasm, and related species were established using amplified fragment length polymorphism (AFLP) markers. Seventy-one papaya accessions and related species were analyzed with nine EcoRI-MseI primer combinations. A total of 186 informative AFLP markers was generated and analyzed. Cluster analysis suggested limited genetic variation in papaya, with an average genetic similarity among 63 papaya accessions of 0.880. Genetic diversity among cultivars derived from the same or similar gene pools was smaller, such as Hawaiian Solo hermaphrodite cultivars and Australian dioecious cultivars with genetic similarity at 0.921 and 0.912, respectively. The results indicated that self-pollinated hermaphrodite cultivars were as variable as open-pollinated dioecious cultivars. Genetic diversity between C. papaya and six other Carica species was also evaluated. Carica papaya shared the least genetic similarity with these species, with an average genetic similarity of 0.432; the average genetic similarity among the six other species was 0.729. The results from AFLP markers provided detailed estimates of the genetic variation within and among papaya cultivars, and supported the notion that C. papaya diverged from the rest of Carica species early in the evolution of this genus.     

濒危植物三棱栎遗传多样性的RAPD分析

用随机扩增多态DNA (RAPD)标记对 5个三棱栎 (Trigonobalanusdoichangensis)居群共 99个个体进行遗传多样性和居群遗传结构分析。 16个引物共检测到 15 7个位点 ,其中多态位点 83个 ,占 5 2 87%。物种水平Shannon多样性指数I =0 2 4 31,Nei基因多样度h =0 15 95 ,种内总遗传变异量Ht=0 16 0 0 ,居群内遗传变异量Hs =0 0 74 9,居群间变异量大于居群内变异量 ,表明三棱栎的遗传变异主要存在于居群之间。与同科植物相比 ,三棱栎遗传多样性较低 ,遗传分化系数Gst =0 5 32 0 ,说明居群间的遗传变异占 5 3 2 0 % ,居群间已出现强烈的遗传分化。当地人的强烈活动造成的生境破碎化和居群隔离 ,以及三棱栎演化过程中的地史变化对其种群发展的影响等 ,可能是造成其居群间强烈的遗传分化和较低遗传多样性的原因。基于本研究结果 ,提出了三棱栎遗传多样性的保护策略。     

壳斗科原始植物轮叶三棱栎在云南西双版纳的发现及其生物地理意义

于云南西双版纳发现了壳斗科植物的原始类群——轮叶三棱栎（Trigonobalanus verticillata Forman）。轮叶三棱栎被认为是栎属（Quercus L.）和水青冈属（Fagus L.）植物的祖先之一,原纪录分布于印度尼西亚和马来西亚,后来在我国海南岛被发现,其分布格局的形成一直未能合理解释。本次在云南新发现轮叶三棱栎,不仅可以解释它在海南岛的分布,也为海南岛历史上可能曾经连接着越南北部和广西西南部的假设提供了科学支持,具有重要的科研价值和生物地理学意义。     

Genetic variation and species identification among selected leeches (Hirudinea) revealed by RAPD markers

Leech taxonomy is based on unstable morphological characters. The overall level of genetic variability and species differentiation is unknown. Using the RAPD assays genetic diversities and genetic similarities were estimated in twelve species collected in North-Eastern Poland and representing three families and five subfamilies. Ten primers revealed 204 reproducible bands. Genetic diversities varied from 0.099 to 0.219 classifying studied species among variable invertebrates. Total 45 markers comprised 22% of all amplified bands were unique for species thus enabling their identification. Genetic similarities among species (0.528–0.811) evidenced several stages of differentiation, which is mirrored in the current taxonomy. The UPGMA and multidimensional scaling (nMDS) based on our RAPDs are congruent and reflected traditional division into “Rhynchobdellida” and Arhynchobdellida. The RAPD approach proved to be an effective tool in population and evolutionary studies of leeches. For the first time, genetic parameters were estimated enabling to compare leeches with outcomes from other animals.     

Genetic relationships among Aster species by multivariate analysis and AFLP markers

Fourteen species of Aster were characterized for morphological traits of commercial interest, chromosome number and genetic diversity by AFLP markers to exploit this material not only for breeding purposes but also as source of pharmaceuticals. The variation observed among the Aster species for five morphological traits was summarized by means of discriminant analysis. Three significant canonical variables, accounting for about 96% of total variance, were mainly correlated with capitulum diameter, number of ligulae and leaf length. The morphological variation appeared related to ploidy level of the species. A diploid chromosome number ranging from 10 to 18 was evident in seven species whereas the other species are polyploids with variable chromosome numbers up to 52. The pollen production and stainability were quite variable among Aster species. Furthermore, seven species produced big pollen grains besides normally-sized ones, at a frequency ranging from 1 to 12%, possibly due to 2n pollen production. The possibility that evolution of Aster genus could be driven by 2n gametes and sexual polyploidization is discussed. Implications of 2n gametes for Aster breeding are also mentioned. AFLP analysis of the fourteen Aster species based on six primer combinations revealed a total number of 421 polymorphic amplified DNA fragments. The genetic similarities estimated between the Aster species, based on both shared and unique amplification products ranged from 0.335 to 0.604 across all species and revealed a geographically based clustering within the genus. The highest similarity was detected between A. alpinus and A. amellus with Eurasian origin.     

Genetic relationships among ten endod types as revealed by a combination of morphological,RAPD and AFLP markers

The genetic relationships among ten types of endod (Phytolacca dodecandra) cultivated by the Institute of Pathobiology of the Addis Ababa University to combat the disease bilharzia in Ethiopia were studied using morphology and molecular markers. A total of 18 morphological characters, 194 amplified fragment length polymorphism (AFLP) and 42 random amplified polymorphic DNA (RAPD) markers were used to determine genetic proximity between types. Genetic distance and cluster analysis of the AFLP data revealed the lack of genetic difference between E47 and E48 but relatively wider genetic difference among the other endod types. Cluster and principal component analyses performed on the AFLP and RAPD markers demonstrated the presence of distinct separation of E56 but not that of E44 from the others. The AFLP and RAPD data, thcrefore, did not support the hypothesis that the superiority of E44 in agronomic traits and molluscicidal potency is linked to its distinct genetic difference from the other endod types. Matrices correspondence tests demonstrated the presence of greater correspondence between AFLP and RAPD data (r = 0.842) but not between the morphology and that of AFLP and RAPD. This indicates the correspondence more between the two DNA markers systems than either of them with morphological traits. The cophenetic correlation coefficients also revealed poor fit for morphology (r = 0.716), good fit for RAPD (r = 0.872) and very good fit for AFLP (r = 0.975), reflecting the hyper-variability and higher resolving power of AFLP.     

Genetic variation of Avicennia marina (Forsk.) Vierh. (Avicenniaceae) in Vietnam revealed by microsatellite and AFLP markers

Genetic variation of Avicennia marina in the costal area of Vietnam was examined using microsatellite and AFLP markers. By using five microsatellite loci a total of 21 alleles were detected. The average number of alleles per locus per population ranged from 1.667 to 3.000. The observed heterozygosity varied from 0.180 to 0.263, with an average of 0.210 indicating relatively low level of genetic variation comparing to the previous studies on A. marina in the worldwide range. The expected heterozygosity was larger than the observed heterozygosity leading to positive inbreeding coefficients in all the six populations. Highly significant departures from Hardy-Weinberg Equilibrium were detected in four populations. AFLP analysis revealed a total of 386 loci, of which 232 (60.1%) were polymorphic. In congruent with microsatellite markers relatively low levels of genetic variation were detected at both gene and nucleotide levels (H = 0.086; pi = 0.0054). Reduced level of genetic variation was found in the central population, and in the southern populations. Both microsatellite and AFLP markers revealed large genetic differentiation (F(ST) = 0.262 and 0.338, respectively) indicating strong genetic structure among regional populations. Pairwise genetic distance by AFLP showed two populations in the north and the other two in the south are closely related each other.     

Cryptic species in an endangered pondweed community (Potamogeton, Potamogetonaceae) revealed by AFLP markers

Cryptic species are morphologically indistinguishable, yet reproductively isolated. Morphological boundaries between species can also be obscured by hybridization and clonality. Determining the roles of reproductive isolation, hybridization, and clonality in morphologically indistinguishable taxa is essential to determining appropriate species-level taxonomic rankings for conservation purposes. The taxonomic status of the endangered Little Aguja pondweed of west Texas, Potamogeton clystocarpus, is uncertain due to a lack of fixed morphological differences between it and two sympatric congeners. Morphology, amplified fragment length polymorphisms (AFLPs), and sequences of the internal transcribed spacer (ITS) region and trnL-F intron and spacer were used to determine the degree of genetic distinctiveness, hybridization and clonality for this rare species. AFLPs indicate that P. clystocarpus is a genetically distinct lineage compared to P. pusillus and P. foliosus. No hybrids involving P. clystocarpus were detected, but two putative hybrids involving P. pusillus and P. foliosus were identified. Clonal growth was only detected in P. pusillus. A combination of morphological and molecular markers was successful in determining the genetic distinctiveness of an endangered cryptic species, Potamogeton clystocarpus. Further sampling in this and adjacent drainages is necessary to assess the degree of endemism of P. clystocarpus and confidently rule out hybridization and clonality in this taxon.     

Genetic relationships among Mediterranean Pistacia species evaluated by RAPD and AFLP markers

Polymorphisms among Mediterranean basin Pistacia species and accessions within species were assessed by random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. Twenty-eight Pistacia accessions representing six species from geographically diverse locations in the Mediterranean area were analyzed. With RAPD, a total of 259 DNA fragments were amplified by 27 pre-selected primers, 254 were polymorphic fragments. AFLP analysis with 15 primer sets, produced 954 (93%) polymorphic bands out of a total of 1026. A Mantel test revealed an extremely high correlation (r=0.99) between similarity matrices generated from RAPD and AFLP data sets, indicating that similar results were obtained by the two techniques. Dendrograms constructed from the similarity matrices showed that Pistacia species could be clustered into two groups, one group containing all the #E5/E5#. lentiscus and the second group containing all other accessions. The latter group was divided into two subgroups, one consisting of #E5/E5#. palaestina and #E5/E5#. terebinthus; the other consisting of #E5/E5#. atlantica, #E5/E5#. khinjuk and #E5/E5#. vera. P. vera and P. khinjuk were highly similar, as were P. palaestina and P. terebinthus.     

Genetic analyses of Oryza species by molecular markers for chloroplast genomes

Summary Relationships in a wide range of Oryza species (13 species) were analyzed using the large subunits (LS) of Fraction I protein (Rubisco) and the Bam HI restriction patterns of chloroplast DNA (ctDNA) as molecular markers. Four types of LS were detected by isoelectrofocusing with and without S-carboxymethylation. The close relation between AA and CCDD genome species was suggested by analyses of LS and ctDNA. Intraspecific variation in O. latifolia was detected at the levels of both LS and ctDNA. The LS of the BB, BBCC, and CC genomes and FF (O. brachyantha) were not distinguishable, although the native Rubisco of the latter was slightly different from those of the first three. It was also shown that O. australiensis, the only EE genome species, might have evolved differently than the other Oryza species.     

Genetic relationships of Aglaonema species and cultivars inferred from AFLP markers

BACKGROUND AND AIMS: Aglaonema is an important ornamental foliage plant genus, but genetic relationships among its species and cultivars have not been reported. This study analysed genetic relatedness of 54 cultivars derived from nine species using amplified fragment length polymorphism (AFLP) markers. METHODS: Initially, 48 EcoRI + 2/MseI + 3 primer set combinations were screened, from which six primer sets that showed clear scoreable and highly polymorphic fragments were selected and used for AFLP reactions. AFLP fragments were scored and entered into a binary data matrix as discrete variables. Jaccard's coefficient of similarity was calculated for all pair-wise comparisons among the 54 cultivars, and a dendrogram was constructed by the unweighted pair-group method using the arithmetic average (UPGMA). KEY RESULTS: The number of AFLP fragments generated per primer set ranged from 59 to 112 with fragment sizes varying from 50 to 565 bp. A total of 449 AFLP fragments was detected, of which 314 were polymorphic (70 %). All cultivars were clearly differentiated by their AFLP fingerprints. The 54 cultivars were divided into seven clusters; cultivars within each cluster generally share similar morphological characteristics. Cluster I contains 35 cultivars, most of them are interspecific hybrids developed mainly from A. commutatum, A. crispum or A. nitidum. However, Jaccard's similarity coefficients among these hybrids are 0.84 or higher, suggesting that these popular hybrid cultivars are genetically much closer than previously thought. This genetic similarity may imply that A. nitidum and A. crispum are likely progenitors of A. commutatum. CONCLUSIONS: Results of this study demonstrate the efficiency and ease of using AFLP markers for investigating genetic relationships of ornamental foliage plants, a group usually propagated vegetatively. The AFLP markers developed will help future Aglaonema cultivar identification, germplasm conservation and new cultivar development.     

Taxonomic relationships among Arachis sect. Arachis species as revealed by AFLP markers.

Cultivated peanut, Arachis hypogaea L., is a tetraploid (2n = 4x = 40) species thought to be of allopolyploid origin. Its closest relatives are the diploid (2n = 2x = 20) annual and perennial species included with it in Arachis sect. Arachis. Species in section Arachis represent an important source of novel alleles for improvement of cultivated peanut. A better understanding of the level of speciation and taxonomic relationships between taxa within section Arachis is a prerequisite to the effective use of this secondary gene pool in peanut breeding programs. The AFLP technique was used to determine intra- and interspecific relationships among and within 108 accessions of 26 species of this section. A total of 1328 fragments were generated with 8 primer combinations. From those, 239 bands ranging in size from 65 to 760 bp were scored as binary data. Genetic distances among accessions ranged from 0 to 0.50. Average distances among diploid species (0.30) were much higher than that detected between tetraploid species (0.05). Cluster analysis using different methods and principal component analysis were performed. The resulting grouping of accessions and species supports previous taxonomic classifications and genome designations. Based on genetic distances and cluster analysis, A-genome accessions KG 30029 (Arachis helodes) and KSSc 36009 (Arachis simpsonii) and B-genome accession KGBSPSc 30076 (A. ipaensis) were the most closely related to both Arachis hypogaea and Arachis monticola. This finding suggests their involvement in the evolution of the tetraploid peanut species.     

Genetic structuring of important medicinal species of genus Warburgia as revealed by AFLP analysis

The genus Warburgia (Canellaceae) contains four tree species that are of valuable medicinal importance and are all found in Africa. Genetic diversity present in wild populations of these species is under great threat due to unsustainable harvesting for medicines and indiscriminate felling for timber and agricultural expansion. There is therefore an urgent need for conservation of these species. Some authors disagree about the taxonomy of the genus and list different species as synonyms. Amplified fragment length polymorphism (AFLP) technique was used to determine the genetic relationships between three species to resolve the taxonomic confusion. The amount of genetic variation within and among populations was assessed to guide strategies for their conservation and sustainable utilization. Four AFLP primer pairs (EcoRI/MseI) generated a total of 185 amplification products. Analysis of molecular variance revealed most variation among individuals within populations (63%, P < 0.0001), but variation among populations (37%, P < 0.0001) was highly significant as well. Constrained analysis of principal coordinates based on the Jaccard distance confirmed the separation among populations (38.2%, P < 0.0001). A phenetic tree and ordination graphs showed a clear distinction of W. ugandensis from W. salutaris and W. stuhlmannii. W. ugandensis populations from Uganda and western Kenya formed a subgroup that clustered away from the rest of the W. ugandensis populations. W. salutaris and W. stuhlmannii populations showed little genetic differentiation. An implication of the data to genetic management and taxonomic clarification is discussed.