Genetic variation of the genus Kengyilia by ISSR markers

We investigated the genetic variation within 32 accessions distributed to 14 species and one variety by using ISSR (inter-simple sequence repeat) markers.The results showed that genetic variation was relatively higher among the accessions.A total of 593 bands were amplified by 12 ISSR primers,of which 535 bands (90.2%) were polymorphic.Eleven to 80 polymorphie bands were amplified from each prime,with an average of 44.6 bands.The interspecies GS (genetic similarity)value ranged from 0.430 to 0.866,and the average was 0.620.Cluster analysis showed that all accessions could be classified into 4 groups by ISSR markers.The different accessions in a species were clustered together,but they had genetic variation in molecular levels.There was obvious interspecies genetic variation.Species with similar morphological characteristics and from the same areas or neighboring geographical regions were clustered together and had close relationships.ISSR markers are useful in analyzing interspecies variation in Kengyilia.     

Isolation and characterization of dinucleotide repeat microsatellites in Drosophila ananassae

Drosophila ananassae is a cosmopolitan species with a geographic range throughout most of the tropical and subtropical regions of the world. Previous studies of DNA sequence polymorphism in three genes has shown evidence of selection affecting broad expanses of the genome in regions with low rates of recombination in geographically local populations in and around India. The studies suggest that extensive physical and genetic maps based on molecular markers, and detailed studies of population structure may provide insight into the degree to which natural selection affects DNA sequence polymorphism across broad regions of chromosomes. We have isolated 85 dinucleotide repeat microsatellite sequences and developed assay conditions for genotyping using PCR. The dinucleotide repeats we isolated are shorter, on average, than those isolated in many other Drosophila species. Levels of genetic variation are high, comparable to Drosophila melanogaster. The levels of variation indicate the effective population size of an Indonesian population of D. ananassae is 58,692 (infinite allele model) and 217,284 (stepwise mutation model), similar to estimates of effective population size for D. melanogaster calculated using dinucleotide repeat microsatellites. The data also show that the Indonesian population is in a rapid expansion phase. Cross-species amplification of the microsatellites in 11 species from the Ananassae, Elegans, Eugracilis and Ficusphila subgroups indicates that the loci may be useful for studies of the sister species, D. pallidosa, but will have limited use for more distantly related species.     

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.     

Genetic Variation Within and Among Populations of Rhodiola alsia (Crassulaceae) Native to the Tibetan Plateau as Detected by ISSR Markers

Genetic variation of 10 Rhodiola alsia (Crassulaceae) populations from the Qinghai–Tibet Plateau of China was investigated using intersimple sequence repeat (ISSR) markers. R. alsia is an endemic species of the Qinghai–Tibet Plateau. Of the 100 primers screened, 13 were highly polymorphic. Using these primers, 140 discernible DNA fragments were generated with 112 (80%) being polymorphic, indicating pronounced genetic variation at the species level. Also there were high levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 63.4 to 88.6%. Analysis of molecular variance (AMOVA) showed that the genetic variation was mainly found among populations (70.3%) and variance within populations was 29.7%. The main factors responsible for the high level of differentiation among populations are probably the isolation from other populations and clonal propagation of this species. Occasional sexual reproduction might occur in order to maintain high levels of variation within populations. Environmental conditions could also influence population genetic structure as they occur in severe habitats. The strong genetic differentiation among populations in our study indicates that the conservation of genetic variability in R. alsia requires maintenance of as many populations as possible.     

Genetic variation of the genus Kengyilia by ISSR markers

We investigated the genetic variation within 32 accessions distributed to 14 species and one variety by using ISSR (inter-simple sequence repeat) markers. The results showed that genetic variation was relatively higher among the accessions. A total of 593 bands were amplified by 12 ISSR primers, of which 535 bands (90.2%) were polymorphic. Eleven to 80 polymorphic bands were amplified from each prime, with an average of 44.6 bands. The interspecies GS (genetic similarity) value ranged from 0.430 to 0.866, and the average was 0.620. Cluster analysis showed that all accessions could be classified into 4 groups by ISSR markers. The different accessions in a species were clustered together, but they had genetic variation in molecular levels. There was obvious interspecies genetic variation. Species with similar morphological characteristics and from the same areas or neighboring geographical regions were clustered together and had close relationships. ISSR markers are useful in analyzing interspecies variation in Kengyilia. __________ Translated from Guihaia, 2006, 26 (4): 375–380 [译自: 广西植物]     

High density of long dinucleotide microsatellites in Drosophila subobscura

We isolated 96 dinucleotide repeats with five or more tandemly repeated units from a subgenomic Drosophila subobscura library. The mean repeat unit length of microsatellite clones in D. subobscura is 15, higher than that observed in other Drosophila species. Population variation was assayed in 32-40 chromosomes from Barcelona, Spain, using 18 randomly chosen microsatellite loci. Positive correlation between measures of variation and perfect repeat length measures (mean size, most common, and longest allele) is consistent with a higher mutation rate in loci with longer repeat units. Levels of microsatellite variation measured as variance in repeat number and heterozygosity in D. subobscura were similar to those of Drosophila pseudoobscura and higher than those of Drosophila melanogaster and Drosophila simulans. Our data suggest that higher levels of microsatellite variation, and possibly density, in D. subobscura compared with D. melanogaster are due to both a higher average effective population and a higher intrinsic slippage rate in the former species.     

Allelic variation of a Beauveria bassiana (Ascomycota: Hypocreales) minisatellite is independent of host range and geographic origin.

The minisatellite locus, BbMin1, was isolated from a partial Beauveria bassiana genomic library that consisted of poly(GA) flanked inserts. Polymerase chain reaction (PCR) of the BbMin1 repeat demonstrated allele size variation among 95 B. bassiana isolates. Amplification was also observed from single isolates of Beauveria amorpha, Beauveria brongniartii, and Beauveria caledonica. Eight alleles were identified at the haploid locus, where repeat number fluctuated between one and fourteen. AMOVA and theta (Fst) indicated that fixation of repeat number has not occurred within pathogenic ecotypes or geographically isolated samples of B. bassiana. Selective neutrality of allele size, the rate of BbMin1 mutation, and the age of the species may contribute to host and geographic independence of the marker. Presence of alleles with a large number of repeat units may be attributed to the rare occurrence of somatic recombination or DNA replication error. The molecular genetic marker was useful for the identification of genetic types of B. bassiana and related species.     

昆仑锦鸡儿(豆科)的遗传多样性研究

采用ISSR方法,对昆仑锦鸡儿及其近缘种吐鲁番锦鸡儿的遗传多样性进行检测。结果表明,两个种多态位点百分率分别为79.07%和41.86%,昆仑锦鸡儿遗传多样性水平明显高于吐鲁番锦鸡儿;两个种间的遗传距离为0.394 3;昆仑锦鸡儿居群间的遗传一致度较高(平均0.961 4),遗传分化很低(GST:0.083 6)。     

Microsatellite variation in populations of Drosophila pseudoobscura and Drosophila persimilis

We have isolated, characterized and mapped 33 dinucleotide, three trinucleotide and one tetranucleotide repeat loci from the four major chromosomes of Drosophila pseudoobscura. Average inferred repeat unit length of the dinucleotide repeats is 12 repeat units, similar to D. melanogaster. Assays of D. pseudoobscura and populations of its sibling species, D. persimilis, using 10 of these loci show extremely high levels of variation compared with similar studies of dinucleotide repeat variation in D. melanogaster populations. The high levels of variation are consistent with an average mutation rate of approximately 10(-6) per locus per generation and an effective population size of D. pseudoobscura approximately four times larger than that of D. melanogaster. Consistent with allozymes and nucleotide sequence polymorphism, the dinucleotide repeat loci reveal minimal structure across four populations of D. pseudoobscura. Finally, our preliminary recombinational mapping of 24 of these microsatellites suggests that the total recombinational genome size may be larger than previously inferred using morphological mutant markers.     

Development and characterization of highly polymorphic microsatellite loci for the Western Spadefoot toad, Pelobates cultripes

Nine highly polymorphic microsatellite markers were isolated and characterized for the Western Spadefoot, Pelobates cultripes. Remarkably, for this amphibian species high numbers of microsatellites were found as part of larger repeat containing regions, making primer design difficult. For nine loci, primers were designed successfully and genotyping of individuals was reliable and consistent. Number of alleles and heterozygosity for these loci ranged from 9 to 34 and from 0.72 to 0.94, respectively. The high levels of polymorphism revealed by our developed loci should provide insight into population genetic structure and levels of dispersal for this typical Mediterranean temporary pond-breeding amphibian.     

Genetic Variation and Clonal Diversity of Psammochloa villosa(Poaceae) Detected by ISSR Markers

Genetic variation and clonal diversity of seven Psammochloavillosa(Poaceae) populations from northwest China were investigatedusing inter simple sequence repeat (ISSR) markers. Of the 84primers screened, 12 produced highly reproducible ISSR bands.Using these primers, 173 discernible DNA fragments were generatedwith 122 (70.5%) being polymorphic, indicating considerablegenetic variation at the species level. In contrast, there wererelatively low levels of polymorphism at the population levelwith the percentage of polymorphic bands (PPB) ranging from6.1 to 26.8. Analysis of molecular variance (AMOVA) showed thata large proportion of genetic variation (87.46%) resided amongpopulations, while only 12.54% resided among individuals withinpopulations. Clonal diversity was also high with 98 genets beingdetected from among 157 individuals using 12 ISSR primers. Theevenness of distribution of genotypes in P. villosa populationsvaried greatly, with all of the genotypes being local ones.No significant differences in genetic or clonal diversity werefound between populations in mobile or fixed dunes. The mainfactor responsible for the high level of differentiation amongpopulations and the low level of diversity within populationsis probably the clonal nature of this species, although selfingmay also affect the population genetic structure to some extent.The efficiency of ISSRs in identifying genetic individuals wasmuch higher than that of allozymes. An approximately asymptoticcorrelation was found between the number of genets detectedand the number of polymorphic loci used, suggesting that useof a high number of polymorphic bands is critical in genet identification.Copyright 2001 Annals of Botany Company Psammochloa villosa, ISSRs, genetic variation, clonal diversity     

Genetic similarity among cultivars of Phyllostachys pubescens

Phyllostachys pubescens is the most important economic bamboo species in China, which grows widely in the South of China. There are more than ten cultivars in this species but their genetic relationship still remains unknown. We used both amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat (ISSR) techniques to determine genetic similarity among ten cultivars of P. pubescens and two related species. Eight hundred and twenty seven bands, in which 495 are polymorphic, were detected using 15 pairs of AFLP primers whereas total 231 bands, in which 154 bands are polymorphic, were scored using 16 ISSR primers. Statistic analysis showed that the genetic similarity matrices obtained from these two sets of molecular markers had a significant correlation (R = 0.959, P = 0.013). The dendrogram generated with AFLP and ISSR markers could clearly genetically identify ten cultivars of P. pubescens that had high similarity with genetic distances ranging from 0.023 to 0.108, and could be divided into three groups based on their genetic variation and similarity. Our results suggest that these molecular markers are useful to genetically classify cultivars or varieties of a species, particularly a bamboo species.     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

Microsatellite variation among divergent populations of stalk-eyed flies, genus Cyrtodiopsis

Microsatellite primers are often developed in one species and used to assess neutral variability in related species. Such analyses may be confounded by ascertainment bias (i.e. a decline in amplification success and allelic variability with increasing genetic distance from the source of the microsatellites). In addition, other factors, such as the size of the microsatellite, whether it consists of perfect or interrupted tandem repeats, and whether it is autosomal or X-linked, can affect variation. To test the relative importance of these factors on microsatellite variation, we examine patterns of amplification and allelic diversity in 52 microsatellite loci amplified from five individuals in each of six populations of Cyrtodiopsis stalk-eyed flies that range from 2.2 % to 11.2% mitochondrial DNA sequence divergence from the population used for microsatellite development. We find that amplification success and most measures of allelic diversity declined with genetic distance from the source population, in some cases an order of magnitude faster than in birds or mammals. The median and range of the repeat array length did not decline with genetic distance. In addition, for loci on the X chromosome, we find evidence of lower observed heterozygosity compared with loci on autosomes. The differences in variability between X-linked and autosomal loci are not adequately explained by differences in effective population sizes of the chromosomes. We suggest, instead, that periodic selection events associated with X-chromosome meiotic drive, which is present in many of these populations, reduces X-linked variation.     

杂交兰转录组SSR信息分析及EST-SSR标记开发应用

该研究主要开发筛选适用于杂交兰的EST-SSR引物,为杂交兰种质资源评价和遗传变异研究等提供可靠的分子标记。该研究对杂交兰进行转录组高通量测序,挖掘SSR位点和开发EST-SSR标记,并对不同种质的遗传多样性进行分析。结果表明,从31724条杂交兰Unigene中检测出18603个SSR位点,SSR出现频率为58.64%;SSR位点中的主导类型是单核苷酸重复,占总SSR的65.10%,其次是二核苷酸(23.56%)和三核苷酸(10.76%)重复;优势重复基元为A/T、AG/CT、AT/AT和AAG/CTT,分别占总位点的64.72%、13.74%、8.19%和2.51%。利用Primer Premier 5.0共设计了565对SSR引物,从筛选出的64对有效扩增引物中随机选择28对引物,对40份杂交兰种质进行多态性验证与遗传关系分析,其中16对(占57.14%)引物表现出可重复的高多态性,平均多态信息量(PIC)达0.789。基于扩增的多态性SSR信息,40份种质资源可聚为4类,聚类结果与其遗传背景基本一致。该研究印证了转录组测序获得的Unigene是SSR标记开发的有效来源,开发的EST-SSR引物可为杂交兰及近缘种的良种鉴别、遗传图谱构建、分子标记辅助育种及功能基因挖掘等提供有价值的候选标记。     

Sequence characterization of microsatellites in diploid and polyploid Ipomoea

 The objectives of the present study were to evaluate the inheritance and nucleotide sequence profiles of microsatellite genetic markers in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.] and its putative tetraploid and diploid ancestors, and to test possible microsatellite mutation mechanisms in polyploids by direct sequencing of alleles. Sixty three microsatellite loci were isolated from genomic libraries of I. batatas and sequenced. PCR primers were designed and used to characterize microsatellite loci in two hexaploid I. batatas populations, a tetraploid Ipomoea trifida population, and a diploid I. trifida population. Nine out of the sixty three primer pairs tested yielded a clearly discernible, heritable banding pattern; five showed Mendelian segregation. All other primer pairs produced either smeared banding patterns, which could not be scored, or no bands at all in I. batatas. All of the primers which produced discernible banding patterns from I. batatas also amplified products of similar size in tetraploid and diploid I. trifida accessions. The sequence analysis of several alleles in the three species showed differences due to mutations in the repeat regions consistent with small differences in the repeat number. However, in some cases insertions/deletions and base substitutions in the microsatellite flanking regions were responsible for polymorphisms in both polyploid and diploid species. These results provide strong empirical evidence that complex genetic mechanisms are responsible for SSR allelic variation in Ipomoea. Four I. batatas microsatellite loci showed polysomic segregation fitting tetraploid segregation ratios. To our knowledge this is the first report of segregation ratios for microsatellites markers in polyploids. Received: 4 January 1999 / Accepted: 4 January 1999     

多叶重楼遗传多样性的ISSR分析

采用ISSR分子标记对多叶重楼种下3个变种的8个居群共208份样品的遗传多样性进行了分析。l4条引物共检测到251个清晰的扩增位点,其中多态位点235个。在物种水平上,多态位点百分率(PPB)达93.63%,Nei′s基因多样性指数(HE)为0.2204、Shannon′s信息指数(HO)为0.3532。在居群水平上,多态位点百分率(PPB)为50.45%,Nei′s基因多样性指数(HE)为0.1405,Shannon′s信息指数(HO)为0.2194,这些均表明多叶重楼的遗传多样性水平较高。此外,还用NTSYS软件对样品进行了UPGMA聚类分析,结果显示滇重楼的6个居群聚为一支,滇重楼与狭叶重楼有较近的亲缘关系,而与长药隔重楼之间的遗传分化较大。本研究提出了合理保护重楼植物资源的若干措施,为进一步开展滇重楼优质种质引种驯化,实现种质资源多样性的合理保护和可持续利用提供参考。     

Genetic diversity and structure of the noxious alien grass Praxelis clematidea in southern China

Praxelis clematidea (Asteraceae), a plant species native to South America, is a noxious weed in southern China. We examined the genetic variation and population structure of 12 populations (76 individuals) of P. clematidea from Fujian, Guangdong, and Hainan Provinces in China using inter-simple sequence repeat (ISSR) analysis. From an initial set of 69 ISSR primers, 10 were selected which yielded 80 reproducible bands. Polymorphic bands (P) were 100%, Shannon's information index (I) was 0.4226, and Nei's gene diversity (H) was 0.2791. We infer that the high levels of genetic diversity exhibited by P. clematidea may have contributed to its invasiveness. Gene flow among populations was 2.4930, which has led to homogenization. The coefficient of population differentiation (Gst = 0.1671) indicated low levels of genetic variation among populations and high levels of genetic polymorphism within populations. There was a negative correlation between population elevation and genetic diversity, while there was a significant positive correlation between genetic distance and geographic distance based on a Mantel test (r = 0.5820, P < 0.01). Some populations from different provinces clustered together in principal coordinate and UPGMA analyses indicating that human-mediated events may have contributed to the dispersal of the species.     

Planchonella, first record of gynomonoecy for the family Sapotaceae

Inter-simple sequence repeat (ISSR) markers were used to investigate the levels and pattern of genetic variation within and among populations of Pteroceltis tatarinowii Maxim., an endangered plant endemic to China. Of the 76 primers screened, 11 produced highly reproducible ISSR bands. A total of 118 bands were presented from the 11 selected primers across all individuals of five natural populations, corresponding to an average of 10.73 bands per primer. The size of the ISSR bands ranged from 200 to 2,000 bp. The percentage of polymorphic loci at the population level ranged from 77.97 to 86.44%, with an average value of 82.54%. Genetic differentiation among populations was revealed based on Nei’s genetic diversity analysis (19.41%) and the nonparametric analysis of molecular variance (20.62%). The Mantel test showed a significant positive correlation between geographic distance and genetic distance (r = 0.7758, P < 0.05), indicating a role of geographic isolation in shaping the present population genetic structure of P. tatarinowii. The size of the natural populations of P. tatarinowii was noted in field observations to be very small, chiefly owing to habitat destruction and overexploitation in the past decades. Therefore, effective measures for preserving genetic diversity of this species at the population level are needed and should include protecting its natural habitats and increasing the numbers of individuals. To meet the commercial demand for this species, P. tatarinowii plantations and cultivation facilities should be established as an alternative source of raw materials.     

Genetic variation within and among populations of a dominant desert tree Haloxylon ammodendron (Amaranthaceae) in China

BACKGROUND AND AIMS: China is one of the countries most severely affected by desertification. Haloxylon ammodendron (Amaranthaceae) is an ecologically important component of the desert ecosystem and is one of the main tree species used for restoration, yet we know little about its genetic structure. METHODS: Genetic variation within and between nine populations of H. ammodendron from two regions of China was investigated using ISSR (inter-simple sequence repeat) markers. KEY RESULTS: Eight primers used in this study amplified 219 reproducible bands of which 184 (84 %) were polymorphic. Analysis of molecular variance (AMOVA) revealed high genetic variation within populations (97.63 %) and low genetic differentiation between regions (0.62 %) and among populations (1.75 %). CONCLUSIONS: It is suggested that the present genetic structure could have arisen by high levels of gene flow. The gene flow among populations observed here is probably mainly attributable to pollen movement. The genetic structure also has important implications in ecological restoration practice.