Genetic diversity among wild and cultivated populations of Hevea brasiliensis assessed by nuclear RFLP analysis

Restriction fragment length polymorphism was assessed in wild and cultivated populations of Hevea brasiliensis using random probes from an Hevea nuclear library. One-hundred-and-sixty-four individuals were surveyed, and the results discussed in the light of previous work performed on isozyme variation. Both studies show that germplasm collections have led to an effective enrichment of the genetic resources available for Hevea breeding, and that cultivated clones have conserved a relatively high level of polymorphism, despite their narrow genetic base and their high level of inbreeding. An equivalent level of polymorphism is revealed by random nuclear probes and isozymes. However, the genetic structuring of the diversity appears more striking using RFLP markers. Wild accessions can be divided into three genetic groups according to their geographical origin. The present results are an essential guide to the incorporation of wild material in breeding schemes.     

Phylogeny of tall fescue and related species using RFLPs

The wild species of tall fescue (Festuca arundinacea var.genuina Schreb.) represent a wide range of genetic variation and constitute potential germplasm for tall fescue improvement. Our objective was to evaluate genome specificity of the previously-identified DNA probes and to examine the phylogenetic relationship of tall fescue with six related species by using RFLP data. A total of 29 DNA probes from aPstI-genomic library of tall fescue were hybridized toEcoRI-orHindIII-digested DNA of 32 plants from sixFestuca species and fromLolium perenne L. Fifteen probes hybridized to all seven species. The remaining 14 probes showed differential hybridization patterns (i.e., ±), especially at the diploid and tetraploid levels. This hybridization pattern reflected genome divergence in these species. The DNA probes will be useful markers in breeding programs involving interspecific and intergeneric hybridization. Cluster analyses were performed using the average genetic distances calculated with the RFLP data from 53 probe-enzyme combinations. Generally, genotypes from the same species were grouped in the same cluster. These data indicated that tall fescue has a close relationship withF. pratensis Huds. (diploid),F. arundinacea var.glaucescens Boiss. (tetraploid), andL. perenne L. (diploid) and thatFestuca pratensis andL. perenne had the closest degree of relationship.This paper is a contribution of the Missouri Agricultural Experimental Station, Journal Series no. 11,798     

DNA restriction fragment length polymorphisms correlate with isozyme diversity in Phaseolus vulgaris L.

Summary Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.     

Genetic diversity of Ustilago maydis strains

Thirty wild isolates belonging to five different locations in Mexico plus two laboratory strains of Ustilago maydis were characterized by restriction fragment length polymorphism (RFLP) analysis using 23 different clones as probes derived from a PstI library and two restriction enzymes. All loci analysed presented a high level of polymorphism, including one locus with thirty one different alleles. Geographical grouping of the populations was based on Nei's genetic distance and there was no correlation between genetic and geographic distances among these isolates. Our results suggest that DNA fingerprinting is a useful method for detecting genetic variation in populations of U. maydis. This work demonstrated that considerable genetic variation may be present within field populations of U. maydis.     

Molecular approaches to ectomycorrhizal diversity studies: variation in ITS at a local scale

In most ectomycorrhizal (EM) community studies involving molecular identification methods there is a poor correspondence between fungi that appear dominant as sporocarps and those that appear dominant on EM roots and the species richness belowground is higher than that above ground. As a consequence, many fungi from root tip samples remain unidentified. In most studies, genetic data from multiple samples of an EM morphotype collected from various sampling locations are compared to genetic data from one to a few sporocarps of each species for identification purposes. The mismatch between above- and belowground species richness may be influenced by these different sampling efforts. To address this, intra-specific variation in the ITS region first investigated in Kårén et al. (1997) is revisited here, but at a spatial scale in which variation is expected to be low. Sporocarps were collected across a 7 km region of the Oregon Dunes National Recreation Area in western North America. ITS–RFLP data are presented for 3-18 sporocarps from each of 44 EM species in 18 genera. A total of 311 sporocarps were analyzed. Fifty-three ITS–RFLP types were observed. Of the 44 species, 38 (86% of total) yielded a single, species specific, RFLP type. No 2 species had the same RFLP type. Polymorphic ITS–RFLP types were observed in six species (14%). The following three species had two ITS-RFLP types with one type dominating: Inocybe lacera, Laccaria proxima, and Rhizopogon subcaerulescens. The following three species had three RFLP types with no type dominating: Laccaria laccata, Lactarius deliciosus, and Tricholoma flavovirens. A phylogenetic analysis of ITS sequences in Tricholoma revealed that two of the RFLP types in T. flavovirenswere apparently the result of intra-specific variation, while the third RFLP type was likely a cryptic species. All the other RFLP types observed in Tricholoma represented unique phylogenetic species. These results suggest that ITS–RFLP data from single samples (sporocarp or EM) are robust for characterizing most of the species at this scale. However, restriction endonucleases detect a limited amount of existing nucleotide variation and thus have limited value to detect cryptic species. Therefore, additional analyses of sequence data should be added to the RFLP matching technique to identify unknown RFLP types. These data also suggest that polymorphic RFLP types within species do not adequately explain the mismatch between above- and belowground views of EM species richness.     

Molecular mapping of rice chromosomes

Summary We report the construction of an RFLP genetic map of rice (Oryza sativa) chromosomes. The map is comprised of 135 loci corresponding to clones selected from a PstI genomic library. This molecular map covers 1,389 cM of the rice genome and exceeds the current classical maps by more than 20%. The map was generated from F₂ segregation data (50 individuals) from a cross between an indica and javanica rice cultivar. Primary trisomics were used to assign linkage groups to each of the 12 rice chromosomes. Seventy-eight percent of the clones assayed revealed RFLPs between the two parental cultivars, indicating that rice contains a significant amount of RFLP variation. Strong correlations between size of hybridizing restriction fragments and level of polymorphism indicate that a significant proportion of the RFLPs in rice are generated by insertions/delections. This conclusion is supported by the occurrence of null alleles for some clones (presumably created by insertion or deletion events). One clone, RG229, hybridized to sequences in both the indica and javanica genomes, which have apparently transposed since the divergence of the two cultivars from their last common ancestor, providing evidence for sequence movement in rice. As a by product of this mapping project, we have discovered that rice DNA is less C-methylated than tomato or maize DNA. Our results also suggest the notion that a large fraction of the rice genome (approximately 50%) is single copy.     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

RFLP mapping of resistance to the blackleg disease [causal agent,Leptosphaeria maculans (Desm.) Ces. et de Not.] in canola (Brassica napus L.)

We report the tagging of genes involved in blackleg resistance, present in the French cultivar Crésor of B. napus, with RFLP markers. A total of 218 cDNA probes were tested on the parental cultivars Crésor (resistant) and Westar (susceptible), and 141 polymorphic markers were used in a segregating population composed of 98 doubled-haploid lines (DH). A genetic map from this cross was constructed with 175 RFLP markers and allowed us to scan for specific chromosomal associations between response to blackleg infection and RFLP markers. Canola residues infested with virulent strains of Leptosphaeria maculans were used as inoculum and a suspension of pycnidiospores from cultures of L. maculans, including the highly virulent isolate Leroy, was sprayed to increase disease pressure. QTL mapping suggested that a single chromosomal region was responsible for resistance in each of the four environments tested. This QTL accounted for a high proportion of the variation of blackleg reaction in each of the assays. A second QTL, responsible for a small proportion of the variation of blackleg reaction, was present in one of four year-site assays. A Mendelian approach, using blackleg disease ratings for classifying DH lines as resistant or susceptible, also allowed us to map resistance in the region of the highly significant LOD scores observed in each environment by interval mapping. Results strongly support the presence of a single major gene, named LmFr ₁ controlling adult plant resistance to blackleg in spring oil-seed rape cultivar Crésor. Several RFLP markers were found associated with LmFr ₁.     

Qualitative Traits Variation in Sorghum (<Emphasis Type="Italic">Sorghum Bicolor</Emphasis> (L.) Moench) Germplasm from,Eastern Highlands of Ethiopia

Forty-five sorghum germplasm growing in the Eastern Highlands of Ethiopia were evaluated for 10 qualitative traits. Phenotypic frequencies between the accessions from each of the nine Aanaas and Alemaya University, grouped in 10 localities were tabulated. Phenotypic diversity index, H′, was analysed and the result indicated the between localities component of diversity to be relatively smaller than the variation in H′ among characters within localities. The value of H′ for all sample germplasm ranged from 0.36 to 0.95 with a mean of 0.71. The results showed that there is a wide morpho-agronomical diversity among the sample germplasm studied. This information can be used for the conservation of these germplasm resources and future improvement work of the sorghum crop.     

Restriction fragment length polymorphism detected by cDNA and genomic DNA clones in Stylosanthes

A DNA isolation method suitable for genomic library construction and RFLP analyses of the forage legume Stylosanthes was developed. Probes isolated using this method were used to investigate the feasibility of constructing RFLP-based genetic maps in this genus. Two hundred and seventy-one PstI genomic DNA and 134 cDNA clones were analysed against four Stylosanthes accessions, including two tetraploids and two diploids, with the use of two restriction enzymes, DraI and HindIII. The proportion of clones which detected single-copy sequences from the PstI genomic library was higher than that from the cDNA library, but the percentage of clones which detected low-copy sequences was doubled in the latter. There was no significant difference in the level of RFLPs detected by gDNA and cDNA probes, although the level of polymorphism was lower in the diploids. A large proportion of RFLPs seemed to have resulted from mutation/base substitution events, and this was especially the case in diploids.     

Genetic diversity among wild and cultivated barley as revealed by RFLP

Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp. spontaneum from Israel, Iran and Turkey. Seventeen out of twenty-one studied cDNA and gDNA probes distributed across all seven barley chromosomes revealed polymorphism when DNA was digested with one of four restriction enzymes. A tree based on genetic distances using frequencies of RFLP banding patterns was estimated and the barley lines clustered into five groups reflecting geographical origin. The geographical groups of land-race lines showed less intragroup variation than the geographical groups of spontaneum lines. The group of European varieties, representing large variation in agronomic traits, showed an intermediate level. The proportion of gene diversity residing among geographical groups (F_ST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups.     

RFLP inheritance and linkage in walnut

Thirty-two low-copy-number genomic DNA clones from a walnut (Juglans sp.) Pst I genomic library were used to establish a molecular-marker linkage map for walnut. The clones were hybridized to restriction-endonuclease-digested DNA from parent walnut trees involved in an interspecific backcross of (J. hindsii x J. regia) x J. regia in order to identify parental polymorphism. Sixty-three backcross progeny were analyzed to determine the inheritance and linkage of 48 RFLP loci. Sixty-six percent of the walnut cloned sequences detected duplicated, but unlinked, loci. Twelve linkage groups were identified by 42 of the RFLP loci. A Poisson probability method for estimating genome size was utilized to calculate the approximate walnut genome length as 1660 cM and to estimate that 138 markers would be needed to cover 95% of the walnut genome within 20 cM of each marker.     

Evaluation of restriction fragment length polymorphism in Cucumis melo

Summary The objectives of this study were to assess the degree of restriction fragment length polymorphism (RFLP) in Cucumis melo and to determine interrelationships among cultivated varieties. Initial screening of a genomic PstI library revealed that approximately 40% of the clones were repetitive. A total of 162 unique and low-copy sequence clones were hybridized to seven diverse accesions of C. melo and a C. sativus cultivar Pacer to evaluate RFLP variation. Of these, 130 probes (80%) detected a polymorphism between C. melo accessions and C. sativus, and the majority were polymorphic with more than one enzyme digest. In contrast, only 53 probes (33%) were useful in differentiating at least one of the seven accessions. Of those, only 9% were informative with more than one enzyme digest. This indicates that within C. melo, the differences among accessions are due to infrequent base substitutions, whereas between the two species, differences are mainly due to genome rearrangements such as insertions and deletions or numerous base substitutions. Of the informative probes, 34 were used in analyzing 44 C. melo lines to establish a data base of RFLP hybridization patterns. Percent similarity based on RFLP profiles was computed among lines and analyzed by principal component analysis, to visualize relationships among lines. There were clear demarcations among, but not within, muskmelon and honeydew groups.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

基于SSR标记的紫花风铃木群体遗传多样性分析

为揭示紫花风铃木(Handroanthus impetiginosus)的群体遗传变异特征,对广东省6市12个群体72份种质材料进行遗传多样性和群体遗传结构分析。结果表明,9对引物共扩增出123个等位基因位点,引物的平均多态信息量为0.754,具有较高的多态性。12个群体均具有较高的遗传多样性,群体间平均有效等位基因数为3.272个,平均Shannon指数为1.159。AMOVA分析表明群体间遗传分化程度相对较低,群体内遗传分化程度较高。群体的总体遗传分化系数为0.077,处于中等程度。基于Structure分析、主坐标分析和NJ聚类分析均可将12个群体分为2大类群,分组结果具有一定相似性,表明供试紫花风铃木群体遗传结构较为简单。这为紫花风铃木优良种质资源的利用、遗传变异和科学育种提供了理论参考。     

Comparison and Characterization of Microbial Communities in Sulfide-rich Wastewater with and without Propidium Monoazide Treatment

A 16S rRNA gene-based culture-independent approach was used to study the bacterial and archaeal communities in a sulfide-rich wastewater. Propidium Monoazide (PMA) treatment was applied to limit the analysis to the fraction of viable cells in environment. A total of 104 and 68 clones respective from bacterial clone library and archaeal library were picked and analyzed by restriction fragment length polymorphism (RFLP). 35 RFLP patterns from bacterial clone library and 10 RFLP patterns from archaeal clone library were unique and the respective clones were selected for sequencing. BLAST analysis and RFLP analysis showed that the bacterial clone library mainly consisted of Gammaproteobacteria (73%), Anaerolineae (6%), Bacilli (5%), Deltaproteobacteria (7%), Clostridia (4%), Bacteroidetes (1%), and Chlorobia (1%); Methanomicrobia (99%) and Thermococci (1%) were the only two lineages of the archaeal domains. This study gave a first insight into the overall microbial structure in a cloth printing and dyeing wastewater treatment plant with high concentration of sulfide and increased knowledge on the applicability of the PMA treatment in combination with PCR-based molecular techniques to analyze only viable cells in microbial ecology.     

Molecular marker analysis of Helianthus annuus L. 1. Restriction fragment length polymorphism between inbred lines of cultivated sunflower

cDNA and PstI genomic clones have been used to assess levels of restriction fragment length polymorphism (RFLP) in Helianthus annuus and to determine the inter-relationships between a diverse set of 24 inbred lines. Of the cDNA clones screened 45% were useful as RFLP probes, compared to less than 20% from the PstI library, which showed high levels of redundancy for high copy sequences. Fifty-seven low-copy DNA probes (23 PstI and 34 cDNA clones) were used to fingerprint 12 maintainer (B) lines and 12 restorer (R) lines. The average number of RFLP variants per probe was found to be 3.2, with a mean polymorphic index of 0.49, indicating that high levels of nuclear DNA polymorphism are to be found in cultivated sunflower. Cluster and principal coordinate analysis of the fingerprinting data clearly separated the maintainer and restorer lines, but there was a degree of association between 2 unbranched R-lines and the B-line germ plasm pool.     

First RFLP linkage map of red clover (<Emphasis Type="Italic">Trifolium pratense</Emphasis> L.) based on cDNA probes and its transferability to other red clover germplasm

We constructed a genetic linkage map of red clover (Trifolium pratense L., 2n=2x=14) using RFLP markers from cDNA probes of a backcrossed mapping population, and investigated the transferability of the markers to other red clover germplasm. The map contains 157 RFLP markers and one morphological marker on seven linkage groups. The total map distance was 535.7 cM and the average distance between two markers was 3.4 cM. All of the cDNA probes of the map were hybridized to the fragments of genomic DNA from 12 plants derived from three varieties, and 87% of the cDNA probes detected polymorphic bands that corresponded to those of mapping parents. This result indicated that RFLP markers on the present map were transferable to the genome analysis of other red clover germplasm. This is the first report to construct a linkage map of Trifolium species; it should provide fundamental and useful genetic information relevant to the breeding of red clover and genus Trifolium.Communicated by H.C. Becker     

Mapping loci controlling vernalization requirement in Brassica rapa

Brassica cultivars are classified as biennial or annual based on their requirement for a period of cold treatment (vernalization) to induce flowering. Genes controlling the vernalization requirement were identified in a Brassica rapa F₂ population derived from a cross between an annual and a biennial oilseed cultivar by using an RFLP linkage map and quantitative trait locus (QTL) analysis of flowering time in F₃ lines. Two genomic regions were strongly associated with variation for flowering time of unvernalized plants and alleles from the biennial parent in these regions delayed flowering. These QTLs had no significant effect on flowering time after plants were vernalized for 6 weeks, suggesting that they control flowering time through the requirement for vernalization. The two B. rapa linkage groups containing these QTLs had RFLP loci in common with two B. napus linkage groups that were shown previously to contain QTLs for flowering time. An RFLP locus detected by the cold-induced gene COR6.6 cloned from Arabidopsis thaliana mapped very near to one of the B. rapa QTLs for flowering time.     

Comparison of rapeseed cultivars and resynthesized lines based on allozyme and RFLP markers

It has frequently been suggested to use the resynthesis of rapeseed (Brassica napus) from B. campestris and B. oleracea to broaden its genetic base. The objective of the present study is twofold: (1) to compare the genetic variation within resynthesized rapeseed with a world-wide collection of oilseed rape cultivars, and (2) to compare genetic distances estimated from RFLP markers with distances estimated from a relatively small number of allozyme markers. We investigated 17 resynthesized lines and 24 rapeseed cultivars. Genetic distances were estimated either based on the electrophoresis of seven allozymes, with a total of 38 different bands, or based on RFLP data of 51 probe/enzyme combinations, with a total of 355 different bands. The results of allozyme and RFLP analyses agreed reasonably well. Genetic distances, estimated from two independent sets of RFLP data with 25 and 26 probe/enzyme combinations respectively, were highly correlated; hence about 50 RFLP markers are sufficient to characterize rapeseed material with a large genetic diversity. The cultivars were clustered into three groups: (1) spring rapeseed of European and Northern American origin, (2) winter rapeseed of European and Northern American origin, and (3) rapeseed of Asian origin. Several of the resynthesized rapeseed lines were similar to European winter rapeseed cultivars, whereas others had quite unique patterns. It is concluded, that resynthesized rapeseed is a valuable source for broadening the genetic variation in present breeding material of Brassica napus. However, different lines differ widely in their suitability for this purpose.