Alignment of the conserved C genomes of Brassica oleracea and Brassica napus

A population of 169 microspore-derived doubled-haploid lines was produced from a highly polymorphic Brassica oleracea cross. A dense genetic linkage map of B. oleracea was then developed based on the segregation of 303 RFLP-defined loci. It is hoped that these lines will be used by other geneticists to facilitate the construction of a unified genetic map of B. oleracea. When the B. oleracea map was compared to one ofB. napus (Parkin et al. 1995), based on the same RFLP probes (Sharpe et al. 1995), good collinearity between the C-genome linkage groups of the two species was observed.     

Molecular-marker analysis of seed-weight: genomic locations,gene action,and evidence for orthologous evolution among three legume species

The objectives of this study were to use molecular markers to: (1) identify quantitative trait loci (QTL) controlling seed-weight in soybean, (2) characterize the genetic basis of seed-weight expression, and (3) determine whether soybean shares orthologous seed-weight genes with cowpea and/or mung bean. An F₂ population was developed between a large-seeded Glycine max breeding line and a small-seeded G. soja plant introduction. DNA samples from 150 F₂ individuals were analyzed with 91 polymorphic genetic markers, including RFLPs, RAPDs and SSRs. Seed-weight was analyzed by randomly sampling 100 seeds from each of 150 greenhouse-grown F₂ individuals, and their 150 F₂₃ lines, from a replicated field trial. Markers associated with seed-weight were identified using the computer program MapMaker-QTL and a one-way analysis of variance. Three and five markers were significantly associated with seed-weight variation (P<0.01) in the F₂ and F₂₃ generations, respectively. Tests for digenic epistasis revealed three significant interactions in both generations. In a combined analysis, these markers and interactions explained 50 and 60% of the phenotypic variation for seed-weight in the F₂ and F₂₃ generations, respectively. Comparison of our results in soybean (Glycine) with those previously reported in cowpea and mung bean (Vigna) indicated that soybean and cowpea share an orthologous seed-weight gene. In both species, a genomic region significantly associated with seed-weight spanned the same RFLP markers in the same linkage order. A significant digenic interaction involving this genomic region was conserved in all three species. These results suggest that the exploitation of comparative QTL mapping is an invaluable tool for quantitative geneticists working with poorly characterized plant systems.     

灰背栎遗传多样性和遗传结构的AFLP指纹分析

用AFLP方法对灰背栎 (Quercussenescens) 8个居群进行了遗传多样性、居群遗传结构研究。TFPGA软件分析两组引物组合共产生 12 5个位点 ,其中 94个为多态位点 ,多态位点百分率为 75 2 % ,发现灰背栎居群的遗传变异水平有随着海拔升高而遗传多样性下降的趋势。Arliquin 2 0 0 0中的AMOVA分析表明灰背栎居群间分化大 ,分化指数达 φst=0 2 95 6。用PAUP软件对所有个体间的遗传关系进行了聚类分析     

Multi-locus genomic analysis reveals the genetic diversity and population structure of the rock carp (Procypris rabaudi) in the upper Yangtze River

Rock carp [Procypris rabaudi (Tchang)] is an endemic species widely distributed throughout the upper reaches of the Yangtze River and its tributaries. Recently, the wild genetic resources of this species have markedly declined and it has been listed as vulnerable in China. Thus, conservation policies for this species are required urgently. However, information supporting decision-making on the conservation of this species is insufficient, especially at the genetic level. In this work, eight populations covering the entire natural range of the species were investigated using amplified fragments length polymorphism markers to determine the genetic diversity and population genetic structure. The results indicated that this species is characterized by moderate levels of genetic polymorphism (46.4% polymorphic loci) and genetic diversity (He = 0.163), and by moderate to high levels of differentiation among the geographical populations. Principal coordinate and Bayesian assignment analysis were used to investigate the genetic structure of this species. No genetically distinct groups among the individuals were detected. The results provided new genetic information and have wide implications for genetic assessment, fishery management and conservation of the rock carp.     

Using molecular markers to map multiple quantitative trait loci: models for backcross,recombinant inbred,and doubled haploid progeny

Summary To maximize parameter estimation efficiency and statistical power and to estimate epistasis, the parameters of multiple quantitative trait loci (QTLs) must be simultaneously estimated. If multiple QTL affect a trait, then estimates of means of QTL genotypes from individual locus models are statistically biased. In this paper, I describe methods for estimating means of QTL genotypes and recombination frequencies between marker and quantitative trait loci using multilocus backcross, doubled haploid, recombinant inbred, and testcross progeny models. Expected values of marker genotype means were defined using no double or multiple crossover frequencies and flanking markers for linked and unlinked quantitative trait loci. The expected values for a particular model comprise a system of nonlinear equations that can be solved using an interative algorithm, e.g., the Gauss-Newton algorithm. The solutions are maximum likelihood estimates when the errors are normally distributed. A linear model for estimating the parameters of unlinked quantitative trait loci was found by transforming the nonlinear model. Recombination frequency estimators were defined using this linear model. Certain means of linked QTLs are less efficiently estimated than means of unlinked QTLs.     

Molecular cloning and sequence analysis of the lysR gene from the extremely thermophilic eubacterium, Thermus thermophilus HB27

We have isolated a lysine-auxotrophic and kanamycin-resistant mutant from an extreme thermophile, Thermus thermophilus HB27. This mutant showed the lysA⁻ or lysR⁻ genotype since it could not grow on the minimal plate which contained diaminopimelic acid. Sequence analysis of the clones which could rescue the Lys⁻ mutant indicated the lysR gene. The lysR gene overlapped with the rimK gene for the modification enzyme of ribosomal protein S6. In the Lys⁻ mutant, the lysR gene was disrupted and the C-terminus region of the RimK protein was different from that of the wild-type, which contributed to the Lys⁻ and kanamycin-resistant phenotype. The deduced amino acid sequence of the lysR gene showed 20.9% identity with the LysR protein of Escherichia coli. The percentage of use of cytosine or guanine in the third letter of the codons in the lysR gene was only 67.4%. We also determined that the argC gene encoding N-acetyl-γ-glutamyl phosphate reductase and the argB gene encoding acetylglutamate kinase were located immediately upstream of the lysR gene.     

Simulation of genetic control. Homozygous-viable pericentric inversions in field-female killing systems

Summary The GENCON simulation program GC5 is designed to simulate genetic population control using field-female killing (FK) systems carying pericentric inversions in addition to Y-linked translocations and deleterious mutations. Homozygous-viable pericentric inversions are included on the same chromosomes as the deleterious mutations, in repulsion to the Y-linked translocation. Released males transmit the inversions and mutations to their daughters and the translocation to their sons. Daughters are semisterile regardless of the type of male they mate with, because products of crossing-over within the inversions carry inviable duplications and deficiencies. Compared to present FK systems, inversion-containing strains give higher levels of genetic death, with both faster initial suppression and greater persistence of genetic death from field-reared descendants if releases are interrupted. At low release rates, both types of FK system are more effective than sterile males.     

AFLP标记在小香羊遗传多态性检测中的应用

研究了AFLP标记在研究小香羊遗传多态性方面应用的可行性和该山羊个体基因组DNA的AFLP扩增结果。实验应用10条AFLP引物,用PstI酶切,对15只小香羊基因组DNA进行AFLP反应,共获得113个AFLP标记,单引物获得的标记数在2～19之间,小香羊群体相似系数AFLP研究结果为0．913(0．814～0．980)。该研究为评价小香羊的遗传稳定性提供了相关的参数,准确评价尚待和其它品种对比研究后确定。     

Analysis of the barley and rice genomes by comparative RFLP linkage mapping

Comparative genetic mapping of rice and barley, both major crop species with extensive genetic resources, offers the possibility of uniting two well-established and characterized genetic systems. In the present study, we screened 229 molecular markers and utilized 110 polymorphic orthologous loci to construct comparative maps of the rice and barley genomes. While extensive chromosomal rearrangements, including inversions and intrachromosomal translocations, differentiate the rice and barley genomes, several syntenous chromosomes are evident. Indeed, several chromosomes and chromosome arms appear to share nearly identical gene content and gene order. Seventeen regions of conserved organization were detected, spanning 287 cM (24%) and 321 cM (31%) of the rice and barley genomes, respectively. The results also indicate that most (72%) of the single-copy sequences in barley are also single copy in rice, suggesting that the large barley genome arose by unequal crossing over and amplification of repetitive DNA sequences and not by the duplication of single-copy sequences. Combining these results with those previously reported for comparative analyses of rice and wheat identified nine putatively syntenous chromosomes among barley, wheat and rice. The high degree of gene-order conservation as detected by comparative mapping has astonishing implications for interpreting genetic information among species and for elucidating chromosome evolution and speciation.     

Characterization of pathogenic and nonpathogenic strains of Xanthomonas axonopodis pv. manihotis by PCR-based DNA fingerprinting techniques

Strains of Xanthomonas axonopodis pv. manihotis (Xam) were characterized for pathogenicity and for DNA polymorphism using different PCR-based techniques. Using amplified restriction fragment length polymorphism (AFLP), strains were distinguished from each other and also from other Xanthomonas strains. Cluster analysis showed a high correlation between DNA polymorphism and pathogenicity. Four Xam strains were further analyzed using three PCR-based techniques, AFLP, AFLP-pthB and RAPD-pthB. Various primer combinations were used including primers specific to a Xam pathogenicity gene (pthB) along with RAPD or AFLP primers. The AFLP primer combinations EcoRI+T/MseI+A and EcoRI+T/MseI+T were the most efficient to discriminate among pathogenic and nonpathogenic Xam strains. Polymorphic bands were excised from the gel, amplified and cloned. Sequences analysis showed significant homology with bacterial pathogenicity island, genes involved in pathogenic fitness and regulators of virulence. Three cloned AFLP fragments were used as probes in DNA blot experiments and two of them showed significant polymorphism.     

Comparative genome analysis of mungbean (Vigna radiata L. Wilczek) and cowpea (V. unguiculata L. Walpers) using RFLP mapping data

Genome relationships between mungbean (Vigna tradiata) and cowpea (V. Unguiculata) based on the linkage arrangement of random genomic restriction fragment length polymorphism (RFLP) markers have been investigated. A common set of probes derived from cowpea, common bean (Phaseolus vulgaris), mungbean, and soybean (Glycine max) PstI genomic libraries were used to construct the genetic linkage maps. In both species, a single F₂ population from a cross between an improved cultivar and a putative wild progenitor species was used to follow the segregation of the RFLP markers. Approximately 90% of the probes hybridized to both mungbean and cowpea DNA, indicating a high degree of similarity in the nucleotide sequences among these species. A higher level of polymorphism was detected in the mungbean population (75.7%) than in the cowpea population (41.2%). Loci exhibiting duplications, null phenotypes, and distorted segregation ratios were detected in both populations. Random genomic DNA RFLP loci account for about 89% of the currently mapped markers with a few cDNA and RAPD markers added. The current mungbean map is comprised of 171 loci/loci clusters distributed in 14 linkage groups spanning a total of 1570cM. On the other hand, 97 markers covered 684 cM and defined 10 linkage groups in the current cowpea map. The mungbean and cowpea genomes were compared on the basis of the copy number and linkage arrangement of 53 markers mapped in common between the two species. Results indicate that nucleotide sequences are conserved, but variation in copy number were detected and several rearrangements in linkage orders appeared to have occurred since the divergence of the two species. Entire linkage groups were not conserved, but several large linkage blocks were maintained in both genomes.     

Is urbanisation of European blackbirds (Turdus merula) associated with genetic differentiation?

Because of the extreme ecological and environmental changes along an urban–rural gradient, it has been proposed that urbanised and non-urbanised populations of the same species may be distinctly isolated. There is evidence that urban populations have become significantly different from the original forest populations in several aspects. However, little is known about the extent to which urban and non-urban populations are genetically isolated from each other. We tested the hypothesis of genetic differentiation by comparing the genomic DNA of an urban and a nearby forest-living European blackbird (Turdus merula) population. The present results suggest that, based on amplified fragment length polymorphism analysis, the urban population studied is very similar to a forest population at neutral genetic markers. Thus, despite indications of obvious functional genetic adaptation, the hypothesis of an overall genetic differentiation between our urban and forest populations could not be supported.Eberhard Gwinner died on 7 September 2004     

Modified AFLP analysis method for species with small genomes

A modified method for detecting amplified fragment length polymorphism (AFLP) has been developed for species with small genomes. We successfully used a combination of primers with a reduced number of selective bases to differentiate and fingerprint isolates ofColletotrichum acutatum, the causative agent of strawberry black spot, an important strawberry disease.     

Restriction fragment variation in the nuclear and chloroplast genomes of cultivated and wild Sorghum bicolor

Summary Fifty-six accessions of cultivated and wild sorghum were surveyed for genetic diversity using 50 low-copy-number nuclear DNA sequence probes to detect restriction fragment length polymorphisms (RFLPs). These probes revealed greater genetic diversity in wild sorghum than in cultivated sorghum, including a larger number of alleles per locus and a greater portion of polymorphic loci in wild sorghum. In comparison to previously published isozyme analyses of the same accessions, RFLP analysis reveals a greater number of alleles per locus. Furthermore, many RFLP alleles have frequencies between 0.25–0.75, while the vast majority of isozyme alleles are either rare (< 0.25) or near fixation (> 0.75). Correlations between genetic and geographic distances among the accessions were stronger when calculated with RFLP than with isozyme data. Systematic relationships revealed by nuclear and chloroplast restriction site analysis indicate that cultivated sorghum is derived from the wild ssp. arundinaceum. The portion of the wild gene pool most genetically similar to the cultivars is from central-northeastern Africa. Previous published data also suggested that this is most likely the principal area of domestication of sorghum. Introgression between wild and cultivated sorghum was inferred from disconcordant relationships shown by nuclear and chloroplast DNA markers. Introgression apparently occurs infrequently enough that the crop and its wild relatives maintain distinct genetic constitutions.     

Structure and evolution of cereal genomes

The cereal species, of central importance to our diet, began to diverge 50-70 million years ago. For the past few thousand years, these species have undergone largely parallel selection regimes associated with domestication and improvement. The rice genome sequence provides a platform for organizing information about diverse cereals, and together with genetic maps and sequence samples from other cereals is yielding new insights into both the shared and the independent dimensions of cereal evolution. New data and population-based approaches are identifying genes that have been involved in cereal improvement. Reduced-representation sequencing promises to accelerate gene discovery in many large-genome cereals, and to better link the under-explored genomes of 'orphan' cereals with state-of-the-art knowledge.     

Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean

Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F₂ families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.     

Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters

In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters.     

New 16Sr subgroups and distinct single nucleotide polymorphism lineages among grapevine Bois noir phytoplasma populations

Bois noir (BN) is an insect-transmitted grapevine yellows disease caused by phytoplasmas belonging to the stolbur subgroup 16SrXII-A. In Italy, increasing prevalence of stolbur phytoplasma strains in vineyards suggests progressive spread of the disease and potential for heavy impacts on the wine industry. In this study, we investigated the genetic diversity of stolbur phytoplasma strains in BN phytoplasma populations. Nucleotide sequences of 16S rRNA genes from stolbur phytoplasma strains affecting vineyards in the Lombardy region of Italy and stolbur phytoplasma 16S rDNA sequences retrieved from GenBank were subjected to virtual restriction fragment length polymorphism analysis. Calculation of virtual restriction similarity coefficients revealed the presence of new subgroups in group 16SrXII (stolbur phytoplasma group). Representative strains of confirmed new subgroups 16SrXII-F (XII-F) and XII-G and tentative new subgroups XII-A1 through XII-A19, XII-H, XII-I, and XII-J as well as known subgroup XII-A were from grapevines; strains representing three additional tentative new subgroups (XII-K, XII-L and XII-M) were from other plant hosts. Nucleotide sequence alignments identified no less than nine genetically distinct 16S rDNA single nucleotide polymorphism lineages from grapevine, indicating a high degree of genetic heterogeneity within BN phytoplasma populations. The findings open new opportunities for in-depth studies of the distribution of grapevine-associated 16SrXII phytoplasma strains in weeds, insect vector populations and grapevines from vineyards located in different geographic areas.     

Construction of integrated genetic linkage maps of the tiger shrimp (Penaeus monodon) using microsatellite and AFLP markers

The linkage maps of male and female tiger shrimp (P. monodon) were constructed based on 256 microsatellite and 85 amplified fragment length polymorphism (AFLP) markers. Microsatellite markers obtained from clone sequences of partial genomic libraries, tandem repeat sequences from databases and previous publications and fosmid end sequences were employed. Of 670 microsatellite and 158 AFLP markers tested for polymorphism, 341 (256 microsatellite and 85 AFLP markers) were used for genotyping with three F₁ mapping panels, each comprising two parents and more than 100 progeny. Chi‐square goodness‐of‐fit test (χ²) revealed that only 19 microsatellite and 28 AFLP markers showed a highly significant segregation distortion (P < 0.005). Linkage analysis with a LOD score of 4.5 revealed 43 and 46 linkage groups in male and female linkage maps respectively. The male map consisted of 176 microsatellite and 49 AFLP markers spaced every ～11.2 cM, with an observed genome length of 2033.4 cM. The female map consisted of 171 microsatellite and 36 AFLP markers spaced every ～13.8 cM, with an observed genome length of 2182 cM. Both maps shared 136 microsatellite markers, and the alignment between them indicated 38 homologous pairs of linkage groups including the linkage group representing the sex chromosome. The karyotype of P. monodon is also presented. The tentative assignment of the 44 pairs of P. monodon haploid chromosomes showed the composition of forty metacentric, one submetacentric and three acrocentric chromosomes. Our maps provided a solid foundation for gene and QTL mapping in the tiger shrimp.