Chloroplast DNA variation and postglacial recolonization of common ash (Fraxinus excelsior L.) in Europe

We used chloroplast polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and chloroplast microsatellites to assess the structure of genetic variation and postglacial history across the entire natural range of the common ash (Fraxinus excelsior L.), a broad-leaved wind-pollinated and wind-dispersed European forest tree. A low level of polymorphism was observed, with only 12 haplotypes at four polymorphic microsatellites in 201 populations, and two PCR-RFLP haplotypes in a subset of 62 populations. The clear geographical pattern displayed by the five most common haplotypes was in agreement with glacial refugia for ash being located in Iberia, Italy, the eastern Alps and the Balkan Peninsula, as had been suggested from fossil pollen data. A low chloroplast DNA mutation rate, a low effective population size in glacial refugia related to ash's life history traits, as well as features of postglacial expansion were put forward to explain the low level of polymorphism. Differentiation among populations was high (GST= 0.89), reflecting poor mixing among recolonizing lineages. Therefore, the responsible factor for the highly homogeneous genetic pattern previously identified at nuclear microsatellites throughout western and central Europe (Heuertz et al. 2004) must have been efficient postglacial pollen flow. Further comparison of variation patterns at both marker systems revealed that nuclear microsatellites identified complex differentiation patterns in south-eastern Europe which remained undetected with chloroplast microsatellites. The results suggest that data from different markers should be combined in order to capture the most important genetic patterns in a species.     

Hybridization and Rorippa austriaca (Brassicaceae) invasion in Germany

Introgressive hybridization between the invasive Rorippa austriaca and the native R. sylvestris in Germany has been studied using chloroplast DNA (trnL intron) and amplified fragment length polymorphism. Three hybrid zones between the invasive and native species were located in the Ruhr Valley (Mülheim) and at the River Main near Würzburg (Randersacker, Winterhausen). In each hybrid zone hybridization was indicated by additivity of region-specific amplified fragment length polymorphism markers proving independent hybridization events. The hybrids were either morphologically intermediate (R. x armoracioides) or were close to R. sylvestris. The trnL intron of R. austriaca is characterized by a species-specific deletion. This diagnostic chloroplast marker of R. austriaca was detected in three individuals of R. sylvestris providing evidence for introgression of the invasive chloroplast into the native species. Bidirectional introgression of R. austriaca markers into R. sylvestris and of R. sylvestris markers into R. austriaca was detected in the amplified fragment length polymorphism analysis. Some of the invasive R. austriaca populations showed high within-population variation. A possible association among introgression, within-population variation and invasion success is discussed. The morphologically intermediate hybrid R. x armoracioides is currently spreading in northern Germany. It forms large populations without its parent species R. austriaca and R. sylvestris. It is concluded that hybridization between invasive R. austriaca and native R. sylvestris may lead to the evolution of a new invasive species R. x armoracioides.     

The comparison of RFLP,RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.Abbreviations RFLP restriction fragment length plymorphism - RAPD random-amplified polymorphic DNA - AFLP amplified fragment length polymorphism - SSR simple sequence repeat - PCR polymerase chain reaction - TBE Tris-borate-EDTA buffer - MI marker index - SENA sum of effective numbers of alleles     

Comparative analyses of genetic diversities within tomato and pepper collections detected by retrotransposon-based SSAP, AFLP and SSR

The retrotransposon-based sequence-specific amplification polymorphism (SSAP) marker system was used to assess the genetic diversities of collections of tomato and pepper industrial lines. The utility of SSAP markers was compared to that of amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. On the basis of our results, SSAP is most informative of the three systems for studying genetic diversity in tomato and pepper, with a significant correlation of genetic relationships between different SSAP datasets and between SSAP, AFLP and SSR markers. SSAP showed about four- to ninefold more diversity than AFLP and had the highest number of polymorphic bands per assay ratio and the highest marker index. For tomato, SSAP is more suitable for inferring overall genetic variation and relationships, while SSR has the ability to detect specific genetic relationships. All three marker results for pepper showed general agreement with pepper types. Additionally, retrotransposon sequences isolated from one species can be used in related Solanaceae genera. These results suggest that different marker systems are suited for studying genetic diversity in different contexts depending on the group studied, where discordance between different marker systems can be very informative for understanding genetic relationships within the study group.     

小麦蜡质基因座位上SSR的多态性及其与直链淀粉含量的关系

分析了蜡质基因(Wx)3′端的一个简单重复序列(SSR)(AT(AT)n AT)在32份小麦中的多态性及其与直链淀粉含量(AC)的关系。这些材料包括了山东省内的不同小麦品种,其AC含量差别较大。在扩增的小麦品种中均出现两条带,一条204bp(Wx-Dla)位于7DS染色体上,另一条225—346bp(Wx-Ala)位于7AS染色体上。也就是说在扩增的小麦品种中均存在Wx-Ala和Wx- Dla基因,并且较长的片段显示了长度多态性。分析表明AC与7D上这个SSR序列基因的多态性呈正相关,高AC小麦品种扩增的片段较长,电泳迁移率较小;相反,低AC小麦品种扩增的片段较短,迁移率较大,不同长度SSR品种间AC差异显著。虽然目前还不能确定这个SSR序列在直链淀粉合成中的直接功能,但SSR变异与AC之间的相关性可作为分子标记直接用于小麦品种改良。     

草莓DNA分子标记及其应用

综述了限制性长度多态性(RFLP)、随机扩增多态性DNA(RAPD)、扩增片段长度多态性(AFLP)、简单重复序列(SSR)等不同类型分子标记在草莓指纹图谱构建、品种鉴别、遗传多样性、进化、遗传作图以及相关性状的标记等方面的应用,分析了草莓分子标记研究中的关键问题,提出了今后研究方向。     

Correlation between molecular markers and adaptively significant genetic variation in Bromus tectorum (Poaceae), an inbreedingannual grass

Single sequence repeat (SSR) and amplified fragment length polymorphic (AFLP) molecular marker genotypes in cheatgrass (Bromus tectorum) were compared to published data on phenotypic variation in seed dormancy, vernalization requirement, and resistance to the pathogen Ustilago bullata. Several features of cheatgrass facilitated this study: it is a recent invader in the western United States, has considerable phenotypic polymorphism, and is an obligate self-pollinator. Forty self-pollinating lines from four populations common to the three phenotypic data sets were analyzed for molecular genetic variation using seven SSR loci and 31 AFLP loci. We examined correlations between distance matrices using the Mantel test for each pair of studies. The two molecular data sets were significantly correlated (r = 0.636). The AFLP markers often distinguished among several lines with identical SSR genotypes. The AFLP data were also significantly correlated with the phenotypic data (r values from 0.4640 to 0.5658), but the SSR data were much more highly correlated (r values from 0.677 to 0.844). The difference between molecular marker systems was especially notable when an outlier population from Potosi Pass, Nevada, was excluded from the analysis. These results suggest that SSR markers may be good surrogates for phenotypic traits in population genetic studies of strongly inbreeding species such as cheatgrass.     

Reproducibility testing of RAPD,AFLP and SSR markers in plants by a network of European laboratories

A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.     

Identification of a 118-kb DNA fragment containing the locus of blast resistance gene Pi-2(t) in rice

Rice blast disease, caused by the fungal pathogen Pyricularia grisea Sacc., is one of the most devastating crop diseases worldwide. Previous studies have shown that the dominant blast resistance gene Pi-2(t) confers resistance to a broad spectrum of pathogenic strains. Using a population of 292 recombinant inbred lines combined with bioinformatic analysis, we mapped Pi-2(t) between the SSR (simple-sequence repeat) marker SSR140 and the RFLP (restriction fragment length polymorphism) marker JSH12, 0.9 cM from both SSR140 and JSH12. A physical map consisting of six overlapping BAC (bacterial artificial chromosome) clones was anchored to the region containing the Pi-2(t) locus. By analyzing recombination events in this region, the Pi-2(t) locus was localized to a DNA fragment of 118 kb in length. The detailed genetic and physical maps of the Pi-2(t) locus will facilitate both molecular isolation of the gene and marker-assisted transfer of the gene in breeding programs.     

Genetic variation at AFLPs for the Dipterocarpaceae and its relation to molecular phylogenies and taxonomic subdivisions

Genetic differentiation was investigated among 54 Indonesian species of Dipterocarpaceae, a dominant tree family in Asian tropical rainforests, using amplified fragment length polymorphism markers. The tree developed from the resultant unweighted pair group method using arithmetic averages clearly separated all investigated dipterocarps into two major groups that corresponded to tribe Dipterocarpeae and tribe Shoreae, respectively. These results are in accordance with the topology of molecular phylogenetic trees derived from PCR–restriction fragment length polymorphism analysis of chloroplast DNA and generally support the traditional taxonomic assessments. The possibility of interspecific hybridization is also discussed.     

High-density Linkage Map of Cultivated Allotetraploid Cotton Based on SSR, TRAP, SRAP and AFLP Markers

A high-density linkage map was constructed for an F2 population derived from an Interspecific cross of cultivated allotetraploid species between Gossypium hirsutum L. and G. barbadense L. A total of 186 F2 individuals from the Interspecific cross of ＂CRI 36 × Hal 7124＂ were genotyped at I 252 polymorphic loci Including a novel marker system, target region amplification polymorphism （TRAP）. The map consists of 1 097 markers, including 697 simple se- quence repeats （SSRs）, 171 TRAPs, 129 sequence-related amplified polymorphisms, 98 amplified fragment length polymorphisms, and two morphological markers, and spanned 4 536.7 cM with an average genetic distance of 4.1 cM per marker. Using 45 duplicated SSR loci among chromosomes, 11 of the 13 pairs of homologous chromosomes were Identified In tetraploid cotton. This map will provide an essential resource for high resolution mapping of quantitative trait loci and molecular breeding in cotton.     

Advances in molecular marker techniques and their applications in plant sciences

Detection and analysis of genetic variation can help us to understand the molecular basis of various biological phenomena in plants. Since the entire plant kingdom cannot be covered under sequencing projects, molecular markers and their correlation to phenotypes provide us with requisite landmarks for elucidation of genetic variation. Genetic or DNA based marker techniques such as RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), SSR (simple sequence repeats) and AFLP (amplified fragment length polymorphism) are routinely being used in ecological, evolutionary, taxonomical, phylogenic and genetic studies of plant sciences. These techniques are well established and their advantages as well as limitations have been realized. In recent years, a new class of advanced techniques has emerged, primarily derived from combination of earlier basic techniques. Advanced marker techniques tend to amalgamate advantageous features of several basic techniques. The newer methods also incorporate modifications in the methodology of basic techniques to increase the sensitivity and resolution to detect genetic discontinuity and distinctiveness. The advanced marker techniques also utilize newer class of DNA elements such as retrotransposons, mitochondrial and chloroplast based microsatellites, thereby revealing genetic variation through increased genome coverage. Techniques such as RAPD and AFLP are also being applied to cDNA-based templates to study patterns of gene expression and uncover the genetic basis of biological responses. The review details account of techniques used in identification of markers and their applicability in plant sciences.     

A variable minisatellite sequence in the chloroplast genome of Sorbus L. (Rosaceae: Maloideae).

The chloroplast genome is now known to be more variable than was once thought. Reports of RFLP (restriction fragment length polymorphism) and sequence variation, as well as variation in chloroplast microsatellites, are common. Here, data are presented on the variability of a minisatellite sequence in the chloroplast genome of Sorbus species. RFLP analysis of a PCR product comprising the region between the trnM and rbcL genes of nine Sorbus species identified seven size variants. Sequencing revealed the observed size polymorphism to be due to differences in the number of copies of an imperfect 9-bp motif. A more intensive survey of the variability of the minisatellite was undertaken in populations of Sorbus aucuparia. The potential uses of such regions in chloroplast DNA are discussed and a possible mechanism for the evolution of the minisatellite is presented.     

Divergence in the chloroplast genome and nuclear rDNA of the rare western australian plant lambertia orbifolia gardner (Proteaceae)

The population genetic structure of the Australian plant Lambertia orbifolia was investigated for chloroplast DNA (cpDNA) and rDNA based on restriction fragment length polymorphism. Variation was assessed in 14-20 individuals from six populations with probes covering the majority of the chloroplast genome and the whole rRNA gene unit. For cpDNA, eight mutations were detected which were distributed over five haplotypes. Nucleotide diversity in the species was high and the majority of this diversity was distributed between populations with diversity within populations restricted to a single population. There was significant differentiation between the two regions in the species distribution with the Narrikup region being distinguished by a single haplotype that was characterized by six unique mutations. Variation in rDNA was detected with three gene length variants present in most individuals. However, the Narrikup region was characterized by homogenization of the gene unit to a single length variant in all individuals. The divergence of the Narrikup region suggests that the disjunction in the species distribution has been present for a long time and the two regions represent separate evolutionary lineages.     

Genetic comparison of Ug99 with selected South African races of Puccinia graminis f.sp. tritici

Using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) marker analyses, the genetic structure of selected South African wheat stem rust races was compared with Ug99. SSR analysis divided the population into two distinct groups with 24.5% similarity between them. A local race, UVPgt55 (North American race notation TTKSF), grouped with Ug99 (TTKSK) with a 100% similarity. When AFLP data were included, the same groups were found, but with an increased similarity of 66.7%. Although the SSR data were unable to distinguish between all individual isolates, the AFLP data alone and in combination with the SSR data discriminated between the isolates. The grouping of individual isolates resembled the pathogenicity profile of the different races. On the basis of its similarity with Ug99, it was concluded that UVPgt55 was most probably an exotic introduction into South Africa, whereas the other races specialized locally through mutational adaptation.     

竹子叶绿体基因组SSR分子标记的开发及其应用

为探讨观赏竹叶片异质性的机理,根据麻竹(Dendrocalamus latiflorus)和绿竹(Bambusa oldhamii)叶绿体基因组序列开发SSR分子标记。结果表明,在麻竹和绿竹叶绿体基因组中分别存在87和86个SSR位点,其中三核苷酸重复类型最多,其次为单核苷酸重复类型。根据SSR位点设计21对引物,其中11对引物对6竹种能够扩增出稳定、清晰的条带,且具有多态性,引物有效率达到52.4%。聚类分析表明,6竹种可分为两大类群,与形态学分类结果基本一致。有4对引物在菲白竹(Pleioblastus fortunei)和白纹椎谷笹(Sasaella glabra f.albo-striata)的花叶中具有多态性,可作为区分观赏竹叶片异质性的分子标记。     

Development and use of anchored‐SSRs to study DNA polymorphism in bread wheat (Triticum aestivum L.)

In bread wheat, 21 anchored simple sequence repeat (SSR) primer pairs detecting SSR length polymorphism and 42 anchored SSR primers detecting microsatellite‐anchored fragment length polymorphisms (MFLPs) are reported. Eight bread wheat genotypes were used for detecting polymorphism. The number of alleles in SSR analysis ranged from two to six, with a mean of 2.9 alleles per SSR. The number of polymorphic bands in MFLP ranged from two to 40, with a mean of 12.74 polymorphic bands/primer combination, the SSRs with CT/GA motifs giving the highest level of polymorphism (a mean of 18.37 bands). The average value of polymorphic information content (PIC) was 0.473 for SSRs and 0.061 for MFLP.     

Cross-species microsatellite amplification in Vasconcellea and related genera and their use in germplasm classification.

To generate inexpensive and efficient DNA markers for addressing a number of population genetics problems and identification of wild hybrids in Vasconcellea, we have evaluated the use of simple sequence repeat (SSR) primers previously developed for other species. A set of 103 Vasconcellea accessions and some individuals of the related genera Carica and Jacaratia were analyzed with 10 primer pairs directing amplification of chloroplast microsatellites in Nicotiana tabacum and 9 nuclear SSR primer pairs recently identified in Vasconcellea x heilbornii. Heterologous amplification of chloroplast SSRs was successful for 8 of the 10 loci, of which 6 showed polymorphism. Seven of the 9 nuclear SSR primer pairs were useful in Vasconcellea and often also in Jacaratia and Carica, all revealing polymorphism. Exclusive haplotypes for each described taxon were identified based on chloroplast microsatellite data. Clustering based on separate nuclear and chloroplast data resulted in a clear grouping per taxon, but only low resolution was obtained above species level. The codominancy of nuclear SSRs and the general high polymorphism rate of SSR markers will make them more useful in future population genetics studies and diversity assessment in conservation programs.     

Genetic mapping of tomato cDNA clones encoding the chloroplastic and the cytosolic isozymes of superoxide dismutase

The isozyme pattern of superoxide dismutase (SOD) in tomato consists of two Cu,Zn isozymes located, respectively, in the chloroplast and in the cytosol, as well as additional isozymes of the Mn or Fe SOD type. We have shown that SOD-1 is the chloroplastic Cu,Zn SOD and is related to cDNA clone T10. Restriction fragment length polymorphism (RFLP) analysis was performed with two cDNA clones representing tomato Cu,Zn-superoxide dismutases. T10, coding for the chloroplast isozyme, was thus mapped to chromosome 11, between marker TG46 and TG108, while clone P31, coding for the cytosolic Cu,Zn SOD isozyme, was mapped to chromosome 1 between TG24 and TG81. SOD is associated with the response of plants to various environmental stresses; the mapping information presented here would permit the demonstration of this association by genetic analysis.     

Chloroplast and mitochondrial DNA diversity in Theobroma cacao

The variability of cocoa (Theobroma cacao) cytoplasmic genomes has been investigated. A total of 177 cocoa clones was surveyed for restriction fragment length polymorphism (RFLP) in chloroplast DNA and in mitochondrial DNA using two restriction endonucleases and various heterologous cytoplasmic probes. A high level of polymorphism was found for the mitochondrial genome. This study points up a structuring of the species that fits with the distinction between the Criollo and Forastero populations. In contrast to all previous analyses, a higher level of polymorphism is found among the Criollo clones while the Forastero clones form quite a homogeneous group.