Restriction Fragment Length Polymorphism Loci Research Articles

Comparative mapping of the oat Dw6/dw6 dwarfing locus using NILs and association with vacuolar proton ATPase subunit H

Seven pairs of oat near-isogenic lines (NILs) (Kibite in Crop Sci 41:277-278, 2001) contrasting for the Dw6 dwarfing gene were used to test for correlation between tall/dwarf phenotype and polymorphic genotype using restriction fragment length polymorphism (RFLP) and other molecular markers selected from the Kanota×Ogle (K×O) (Wight et al. in Genome 46:28-47, 2003) and Terra×Marion (De Koeyer et al. in Theor Appl Genet 108:1285-1298, 2004) recombination maps. This strategy located the Dw6/dw6 locus to a small chromosomal region on K×O linkage group (LG) KO33, near or at a putative RFLP locus aco245z. Aco245z and other tightly linked flanking markers have potential for use in marker-assisted selection (MAS), and PCR-based markers were developed from several of these. RFLP genotyping of the Dw6 NILs indicated that 13 of the 14 individual lines were homogeneously maternal or paternal for a large genomic region near Dw6/dw6, an unexpected result for NILs. The cDNA clone aco245 codes for a vacuolar proton ATPase subunit H, a potential candidate gene for Dw6. Vacuolar proton ATPase enzymes have a central role in plant growth and development and a mutation in subunit C is responsible for the det3 dwarfing mutation in Arabidopsis thaliana (Schumacher et al. in Genes Dev 13:3259-3270, 1999). Aco245 affords the potential of designing highly precise diagnostic markers for MAS for Dw6. The Dw6 NILs have potential utility to investigate the role of vacuolar proton ATPases in growth and development in plants.

Thermal adaptation in the fungal pathogen Mycosphaerella graminicola

Genetic differentiation in thermal adaptation can result from a trade-off between the performance of organisms across different temperatures or from the accumulation of deleterious mutations. In this experiment, we assayed thermal sensitivity of 138 genetically distinct Mycosphaerella graminicola isolates sampled from five host populations in four locations under two temperature regimes (22 and 15 °C) and found significant differences in growth rate and response to temperature among populations. On average, genetic differentiation accounted for more than 50% of phenotypic variation in thermal adaptation while plasticity contributed less than a quarter of phenotypic variation. Populations originating from warm places performed better under the high-temperature regime and had a larger positive response to increasing temperature. Pairwise population differentiation (Q(ST) ) in temperature sensitivity, measured by taking the ratio of growth rates at 22 to 15 °C, was positively and significantly correlated to the pairwise difference in annual mean temperature at the collection sites. Because overall Q(ST) in temperature sensitivity was significantly higher than overall G(ST) in neutral restriction fragment length polymorphism loci, we believe that the primary mechanism underlying this thermal adaptation is antagonistic pleiotropy. Our results indicate that temperature sensitivity is a better indicator of thermal adaptation than growth rate at individual temperatures.

Genotypic and phenotypic correlates with proliferative kidney disease-induced mortality in Atlantic salmon

Heritable variation in resistance to pathogens has been reported in many fish species, but little is known about its genetic architecture. To extend understanding, an investigation was made of the association of resistance to proliferative kidney disease (PKD) in 4 second filial generation (F2) families of Atlantic salmon with molecular markers from different genetic linkage groups in the species' genome, following a natural disease outbreak. PKD causes serious mortality in cultured salmonids. In addition to mortality, associations with growth-related traits were also examined, as immune responses are energetically costly and have been observed to reduce growth. Associations were investigated for 34 microsatellite markers and 5 restriction fragment length polymorphism (RFLP) loci from 3 regions of the growth hormone 1 gene (GH1). The phenotypic and genotypic character of survivors was compared with unexposed fish derived from the same families. Mortality was not size-selective, but growth in the survivors was reduced, and fish had a lower condition factor than unexposed fish, suggesting an energetic cost to resistance. Five markers showed significant allele frequency differences between survivors and unexposed fish, albeit in single families. Prior to correction for multiple tests, 2 of these markers were also linked to variation in growth-related traits among survivors, along with a further 7 markers. Though sample sizes constrained the power of the analysis, the study points to regions of the salmon genome that may contain quantitative trait loci related to PKD resistance, on which further work on the genetic architecture of PKD resistance in this species could focus.

Addition of Restriction Fragment Length Polymorphism Markers to the Genetic Linkage Map of Brassica rapa L. (syn. campestris)

Genetic linkage analysis of 151 restriction fragment length polymorphism (RFLP) loci, that included eight new loci, detected by the six probes in the present study, and four trait loci including seed colour, leaf pubescence, resistance to white rust caused by Albugo candida race-2 (AC-2) and race-7 (AC-7) employing the MAPMAKER/EXP 3.0 programme led to the development of 10 linkage groups (LGs) spanning over 44.4 centiMorgan (cM) to 130.4 cM containing 9 to 22 loci and two short LGs with two or three marker loci in Brassica rapa. The enriched map covers 993.1 cM of B. rapa genome with an average marker interval of 6.41. Eight new RFLP loci occupied new map positions on five linkage groups, LG 2, 3, 6, 8 and 9. Addition of these RFLP loci led to appreciable changes in the corresponding linkage groups and resulted in an increase of the total map length by 102.8 cM and of the marker interval by 0.35 cM. Interval mapping by using the computer programme MAPMAKER/ QTL 1.1 for scanning the genetic map led to the detection of one major quantitative trait locus (QTL) in LG 4 and one minor QTL in LG 8 governing resistance to AC-7. Both QTLs contributed 7.89 to the interaction phenotype (IP) score with 96.3% genetic variation. The multi-locus model suggested additive gene action with 96.8% genetic variation.

Susceptibility of Populus balsamifera to Septoria musiva: A Field Study and Greenhouse Experiment.

A greenhouse inoculation experiment and field study were conducted to determine the cause of an outbreak of Septoria musiva cankers on Populus balsamifera in a northern Alberta plantation. Four clones of P. balsamifera, five clones of putatively resistant P. deltoides, and one susceptible hybrid poplar clone, Northwest, were inoculated with seven isolates identified as S. musiva. Four of the isolates were from P. balsamifera in Alberta and the others were from P. deltoides in Quebec. Results indicated that disease severity was similar for Alberta and Quebec isolates (P = 0.243) and that P. balsamifera had the greatest mean disease severity (x-bar = 4.20), P. deltoides had the lowest (x-bar = 2.76), and Northwest was intermediate (x-bar = 3.45). A genetic analysis comparing six polymorphic polymerase chain reaction restriction fragment length polymorphism loci and the mitochondrial small subunit ribosomal DNA sequences of the seven isolates indicated that the Alberta population was made up of at least three distinct genotypes. Canker incidence and age on Northwest and 56 different clones of P. balsamifera in a plantation were recorded. Canker incidence (P = 0.726) and the canker age distributions (P = 0.994) were similar for the two species. In conclusion, contrary to what has been reported in the literature, P. balsamifera appears to be quite susceptible to Septoria canker.

Phylogeographical analyses reveal global migration patterns of the barley scald pathogen Rhynchosporium secalis

A phylogeographical analysis of the scald pathogen Rhynchosporium secalis was conducted using nuclear DNA sequences from two neutral restriction fragment length polymorphism loci and the mating-type idiomorphs. Approximately 500 isolates sampled from more than 60 field populations from five continents were analysed to infer migration patterns and the demographic history of the fungus. Migration rates among continents were generally low, consistent with earlier reports of significant population subdivision among continents. Northern Europe was mainly a source population for global migration. We hypothesize that the pathogen only recently moved out of its centre of origin, resulting in founder populations that are reproductively isolated due to the contemporary absence of long-distance gene flow.

Construction of a consensus linkage map for red clover (Trifolium pratense L.)

BackgroundRed clover (Trifolium pratense L.) is a major forage legume that has a strong self-incompatibility system and exhibits high genetic diversity within populations. For several crop species, integrated consensus linkage maps that combine information from multiple mapping populations have been developed. For red clover, three genetic linkage maps have been published, but the information in these existing maps has not been integrated.ResultsA consensus linkage map was constructed using six mapping populations originating from eight parental accessions. Three of the six mapping populations were established for this study. The integrated red clover map was composed of 1804 loci, including 1414 microsatellite loci, 181 amplified fragment length polymorphism (AFLP) loci and 204 restriction fragment length polymorphism (RFLP) loci, in seven linkage groups. The average distance between loci and the total length of the consensus map were 0.46 cM and 836.6 cM, respectively. The locus order on the consensus map correlated highly with that of accession-specific maps. Segregation distortion was observed across linkage groups. We investigated genome-wide allele frequency in 1144 red clover individuals using 462 microsatellite loci randomly chosen from the consensus map. The average number of alleles and polymorphism information content (PIC) were 9.17 and 0.69, respectively.ConclusionA consensus genetic linkage map for red clover was constructed for the first time based on six mapping populations. The locus order on the consensus map was highly conserved among linkage maps and was sufficiently reliable for use as a reference for genetic analysis of random red clover germplasms.

DNA Markers in Eucalyptus with Emphasis on Species Identification

I review the current knowledge on molecular markers for Eucalyptus species, with particular emphasis on individual identification and the prevention of escape of transgenes from transgenic Eucalyptus. Species-specific bands have been found in inter simple sequence repeat (ISSR) markers. Microsatellites are highly polymorphic genetic regions that allow precise discrimination of closely related individuals. Random amplified polymorphic DNA (RAPD) markers are also very useful for individual identification and map construction, although they have limited transferability because they are dominant markers with only two alleles. The amplified fragment length polymorphism (AFLP) technique facilitates the production of high-coverage parental maps in interspecific crosses of eucalypts. Genetic diversity in the continuously distributed Eucalyptus species has been investigated using nuclear restriction fragment length polymorphism (RFLP) loci. Chloroplast DNA, 5S rDNA genes, and ITS regions have proven to be useful in phylogenetic, population, and evolutionary studies of Eucalyptus. Genetic association studies using SNPs are in progress with several Eucalyptus species. Furthermore, microarray technology holds the promise of parallel analysis of thousands of markers in Eucalyptus. Early screening of hybrids with transgenic Eucalyptus using DNA markers will be important to prevent escaping transgenes.

Frequency of Sexual Recombination by Mycosphaerella graminicola in Mild and Severe Epidemics

The importance of sexual recombination in determining fungal population structure cannot be inferred solely from the relative abundance of sexual and asexual spores and reproductive structures. To complement a previously reported study of proportions of Mycosphaerella graminicola ascocarps and pycnidia, we investigated the share of sexual recombinants among isolates randomly derived from the same field at the same time. Early in three successive growing seasons (those ending in 1998, 1999, and 2000), field plots of the susceptible winter wheat cultivar Stephens were inoculated with suspensions of two M. graminicola isolates that each had rare alleles at restriction fragment length polymorphism (RFLP) loci. Near harvest time, leaves were randomly sampled from the same plots, and a population of over 100 monopycnidial isolates was created for each year of the experiment. Natural populations were also sampled from noninoculated plots in the 1999 and 2000 seasons, in order to compare allele frequencies. Based on RFLP haplotypes and DNA fingerprints, isolates from the inoculated plots were categorized by both inspection and Bayesian methods as inoculant clones, recombinants, or immigrants. Inoculation in the 2000 season was delayed, and the recovery rate of inoculant types was just 1 to 2%. In 1998, a high-disease year, and 1999, a low-disease year, inoculants comprised 36 and 22 to 23% of end-of-season samples, respectively. In those 2 years, recombinants as a percentage of inoculant descendants (both sexual and asexual) were 35 and 32%, respectively. By comparison, the study of fruiting bodies had found 93 and 32% of M. graminicola fruiting bodies were ascocarps in 1998 and 1999, respectively. These findings support the hypothesis that sexual recombination makes a relatively consistent contribution to M. graminicola population structure, despite differences in epidemic severity and ascocarp proportions.

Evidence for utility of the same PCR‐based markers for selection of extreme resistance to Potato virus Y controlled by Rysto of Solanum stoloniferum derived from different sources

AbstractThe tuber‐bearing wild potato species, Solanum stoloniferum, carries a dominant gene, Rysto, which confers extreme resistance (ER) to Potato virus Y (PVY). This gene was introgressed to cultivated potato germplasm (Solanum tuberosum) using accessions of S. stoloniferum maintained in European gene banks. It is mainly used in potato breeding programmes in Europe. Rysto was recently mapped to potato chromosome XII. However, in this study, a different accession of S. stoloniferum (PI275244; Haw1293) was used as a female parent in a cross to obtain a diploid (2n = 2x = 24) potato population of 112 F1 genotypes. From this accession, ER to PVY has been introgressed to the potato breeding programmes at the International Potato Center (Peru). As expected, ER to PVY was inherited in a dominant, monogenic fashion in the F1 population. Marker‐specific choices of DNA polymerase and adjustments of PCR conditions were made to optimise marker detection. The corresponding gene (Rysto) was mapped to the chromosome XII using the previously described and new cleaved amplified polymorphic sequence (CAPS) markers, which are based on the restriction fragment length polymorphism loci GP122 (six markers) and GP269 (one marker), and the simple sequence repeat marker STM0003. Four GP122‐based CAPS markers and STM0003 detected the same genotypes expressing ER to PVY. Because of a few recombinants, that is ER genotypes lacking the markers and the genotypes that react with necrosis but contain the markers, the marker distance from Rysto was estimated as 15.2 cM in this F1 population. However, the distance may be less if necrosis was considered an altered response also controlled by Rysto. The markers also specifically detected independent European potato cultivars that express ER to PVY derived from S. stoloniferum. Phylogenetic analysis of the sequences amplified from the GP122 locus of S. stoloniferum and potato cultivars further confirmed that the Rysto gene from independent accessions of S. stoloniferum can be selected using the same markers and the protocols described in this study.

Mapping and Comparative Analysis of QTL for Crown Rust Resistance in an Italian × Perennial Ryegrass Population

ABSTRACT Crown rust (Puccinia coronata f. sp. lolli) is a serious fungal foliar disease of perennial ryegrass (Lolium perenne L.) and Italian ryegrass (L. multiflorum Lam.), which are important forage and turf species. A number of quantitative trait loci (QTL) for crown rust resistance previously were identified in perennial ryegrass under growth chamber or greenhouse conditions. In this study, we conducted a QTL mapping for crown rust resistance in a three-generation Italian x perennial ryegrass interspecific population under natural field conditions at two locations over 2 years. Through a comparative mapping analysis, we also investigated the syntenic relationships of previously known crown rust resistance genes in other ryegrass germplasms and oat, and genetic linkage between crown rust resistance QTL and three lignin genes: LpOMT1, LpCAD2, and LpCCR1. The interspecific mapping population of 156 progeny was developed from a cross between two Italian x perennial ryegrass hybrids, MFA and MFB. Because highly susceptible reactions to crown rust were observed from all perennial ryegrass clones, including two grandparental clones and eight clones from different pedigrees tested in this study, two grandparent clones from Italian ryegrass cv. Floregon appeared to be a source of the resistance. Two QTL on linkage groups (LGs) 2 and 7 in the resistant parent MFA map were detected consistently regardless of year and location. The others, specific to year and location, were located on LGs 3 and 6 in the susceptible parent MFB map. The QTL on LG2 was likely to correspond to those previously reported in three unrelated perennial ryegrass mapping populations; however, the other QTL on LGs 3, 6, and 7 were not. The QTL on LG7 was closely located in the syntenic genomic region where genes Pca cluster, Pcq2, Pc38, and Prq1b resistant to crown rust (P. coronata f. sp. avenae) in oat (Avena sativa L.) were previously identified. Similarly, the QTL on LG3 was found in a syntenic region with oat genes resistant to crown rust isolates PC54 and PC59. This indicates that the ortholoci for resistance genes to different formae speciales of crown rust might be present between two distantly related grass species, ryegrass and oat. In addition, we mapped four restriction fragment length polymorphism loci for three key ryegrass lignin genes encoding caffeic acid-O-methyltransferase, cinnamyl alcohol dehydrogenase, and cinnamoyl CoA-reductase on LG7. These loci were within a range of 8 to 17 centimorgans from the QTL on LG7, suggesting no tight linkage between them. The putative ortholoci for those lignin biosynthesis genes were identified on segments of rice (Oryza sativa L.) chromosomes 6 and 8, which are the counterparts of ryegrass LG7. Results from the current study facilitate understanding of crown rust resistance and its relationship with lignin biosynthesis, and also will benefit ryegrass breeders for improving crown rust resistance through marker-assisted selection.

QTL mapping with near-isogenic lines in maize

A set of 89 near-isogenic lines (NILs) of maize was created using marker-assisted selection. Nineteen genomic regions, identified by restriction fragment length polymorphism loci and chosen to represent portions of all ten maize chromosomes, were introgressed by backcrossing three generations from donor line Tx303 into the B73 genetic background. NILs were genotyped at an additional 128 simple sequence repeat loci to estimate the size of introgressions and the amount of background introgression. Tx303 introgressions ranged in size from 10 to 150 cM, with an average of 60 cM. Across all NILs, 89% of the Tx303 genome is represented in targeted and background introgressions. The average proportion of background introgression was 2.5% (range 0-15%), significantly lower than the expected value of 9.4% for third backcross generation lines developed without marker-assisted selection. The NILs were grown in replicated field evaluations in two years to map QTLs for flowering time traits. A parallel experiment of testcrosses of each NIL to the unrelated inbred, Mo17, was conducted in the same environments to map QTLs in NIL testcross hybrids. QTLs affecting days to anthesis, days to silking, and anthesis-silk interval were detected in both inbreds and hybrids in both environments. The testing environments differed dramatically for drought stress, and different sets of QTLs were detected across environments. Furthermore, QTLs detected in inbreds were typically different from QTLs detected in hybrids, demonstrating the genetic complexity of flowering time. NILs can serve as a valuable genetic mapping resource for maize breeders and geneticists.

Global Hierarchical Gene Diversity Analysis Suggests the Fertile Crescent Is Not the Center of Origin of the Barley Scald PathogenRhynchosporium secalis

A total of 1,366 Rhynchosporium secalis isolates causing scald on barley, rye, and wild barley (Hordeum spontaneum) were assayed for restriction fragment length polymorphism loci, DNA fingerprints, and mating type, to characterize global genetic structure. The isolates originated from 31 field populations on five continents. Hierarchical analysis revealed that more than 70% of the total genetic variation within regions was distributed within a barley field. At the global level, only 58% of the total genetic variation was distributed within fields, while 11% was distributed among fields within regions, and 31% was distributed among regions. A significant correlation was found between genetic and geographic distance. These findings suggest that gene flow is common at the local level while it is low between regions on the same continent, and rare between continents. Analyses of multilocus associations, genotype diversity, and mating type frequencies indicate that sexual recombination is occurring in most of the populations. We found the highest allele richness in Scandinavia followed by Switzerland. This suggests that R. secalis may not have originated at the center of origin of barley, the Fertile Crescent, nor in a secondary center of diversity of barley, Ethiopia.

Selection for increased cyproconazole tolerance in Mycosphaerella graminicola through local adaptation and in response to host resistance

SUMMARY Sterol demethylation inhibitors (DMIs) represent one of the largest groups of systemic fungicides that have been used to control agriculturally important fungal pathogens. Knowledge regarding the evolution of fungicide resistance in agricultural ecosystems is fragmentary and a better understanding of the processes driving the development of DMI resistance in populations of fungal pathogens is needed by plant pathologists and the agrochemical industry. We considered some of these processes using approaches based on molecular population and quantitative genetics. Five Mycosphaerella graminicola populations sampled from unsprayed wheat fields on four continents were assayed for eight restriction fragment length polymorphism (RFLP) markers and their level of tolerance to cyproconazole. DMI fungicides such as cyproconazole inhibit the enzyme eburicol 14-alpha-demethylase. The gene encoding this target, CYP51, was sequenced for all isolates. We found unimodal, continuous variations in cyproconazole tolerance among the M. graminicola isolates sampled from individual fields, consistent with a polygenic mode of inheritance. We also found that population differentiation for cyproconazole tolerance (Q(ST)) among the five M. graminicola populations was significantly higher than the corresponding population differentiation for neutral RFLP markers (G(ST)), suggesting that selection for cyproconazole tolerance in the Swiss population has already led to local adaptation that can be seen even in an unsprayed population. The Swiss population displayed the highest level of tolerance to cyproconazole, in addition to a lower than expected quantitative variation in fungicide tolerance and a skewed distribution, indicating that selection had increased the overall tolerance of this population. Further analysis with DNA sequencing showed that the population from Switzerland was dominated by isolates with several point mutations and a 6-bp deletion in CYP51. This deletion and one of the point mutations were previously related to increased resistance in field isolates. The fungal population from Oregon sampled from an unsprayed resistant host cultivar displayed the same gene diversity in RFLP loci but higher cyproconazole tolerance and quantitative variation in tolerance than the fungal population from the same field sampled from an unsprayed susceptible host cultivar.

Molecular Markers for the Classification of Switchgrass (Panicum virgatum L.) Germplasm and to Assess Genetic Diversity in Three Synthetic Switchgrass Populations

Information regarding the amount of genetic diversity is necessary to enhance the effectiveness of breeding programs and germplasm conservation efforts. Genetic variation between 21 switchgrass genotypes randomly selected from two lowland (‘Alamo’ and ‘Kanlow’) and one upland (‘Summer’) synthetic cultivars were estimated using restriction fragment length polymorphism (RFLP) markers. Comparison of 85 RFLP loci revealed 92% polymorphism between at least two genotypes from the upland and lowland ecotypes. Within ecotypes, the upland genotypes showed higher polymorphism than lowland genotypes (64% vs. 56%). ‘Kanlow’ had a lower percent of polymorphic loci than ‘Alamo’ (52% vs. 60%). Jaccard distances revealed higher genetic diversity between upland and lowland ecotypes than between genotypes within each ecotype. Hierarchical cluster analysis using Ward's minimum variance grouped the genotypes into two major clusters, one representing the upland group and the other the lowland group. Phylogenetic analysis of chloroplast non-coding region trnL (UAA) intron sequences from 34 switchgrass accessions (6 upland cultivars, 2 lowland cultivars, and 26 accessions of unknown affiliation) produced a neighbor-joining dendrogram comprised of two major clusters with 99% bootstrap support. All accessions grouped in the same cluster with the lowland cultivars (‘Alamo’ and ‘Kanlow’) had a deletion of 49 nucleotides. Phenotypic identification of greenhouse-grown plants showed that all accessions with the deletion are of the lowland type. The deletion in trnL (UAA) sequences appears to be specific to lowland accessions and should be useful as a DNA marker for the classification of upland and lowland germplasm.

Genetic differentiation among morphological variants of Acacia saligna (Mimosaceae)

This study investigated the pattern of genetic variation in Acacia saligna (Labill.) H.L. Wendl. to facilitate its development as a crop species for dryland salinity management. A. saligna is a morphologically variable species and four main variants are recognized. The genetic structure within A. saligna was investigated in populations across the geographic range of the species using nuclear restriction fragment length polymorphism loci. The analysis identified considerable genetic variation within A. saligna that was genetically structured into three groups. Two of the three groups corresponded to variants recognized in the field study; the third group encompassed the other two variants which, though morphologically different, were not genetically differentiated. The level of genetic differentiation between the groups suggests they may represent different taxa and a taxonomic revision of the species may be required. Identification of different taxa within A. saligna will have implications for the utilization and domestication of the species, as the taxa will need to be evaluated separately to determine their suitability for agroforestry. The high genetic variation between and within groups suggests there is a large genetic base available for breeding improved cultivars.

New evidence fromSinapis albaL. for ancestral triplication in a crucifer genome

We present clear evidence of ancestral genome triplication in Sinapis alba, a close relative of the cultivated Brassica species. Exceptionally high levels of heterozygosity in the parents of an F1 intercross permitted the mapping of an estimated 87% of all detected restriction fragment length polymorphism (RFLP) loci, with each RFLP probe typically detecting 2 or 3 loci. These duplicated loci were arranged in 8 triplicated homologous linkage blocks and 2 small, duplicated, homologous linkage blocks covering the majority of the S. alba genome. Several large-scale inversions and translocations appear to have rearranged the order of loci within homologous blocks. The role of successive polyploidization events on the evolution of crucifer species is discussed.

Molecular and phenotypic description of the widespread root symbiont Acephala applanata gen. et sp. nov., formerly known as dark-septate endophyte Type 1

Acephala applanata gen. et sp. nov. is described. A. applanata is a dark-septate endophyte (DSE) of conifer roots and belongs to the Phialocephala fortinii species complex. Several genetic markers, including isozymes, inter-simple-sequence-repeat (ISSR) fingerprints, single-copy restriction fragment length polymorphisms (RFLP) and sequences of the internal transcribed spacers (ITS), let us unambiguously separate isolates of A. applanata from isolates of P. fortinii s.l. and other dark-septate endophytes. Alleles at four RFLP loci and two fixed nucleotides in the ITS region were diagnostic for A. applanata. One of the fixed nucleotides resulted in the addition of an Afa I restriction site. PCR amplification with primers prITS4 and the newly developed primer PF_ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) and restriction digestion with Afa I yielded three fragments (203 bp, 117 bp, 56 bp) in A. applanata but only two (260 bp and 117 bp) in P. fortinii s.l. Population differentiation (GST) between A. applanata and other cryptic species of P. fortinii was pronounced, and the index of association (IA) did not deviate significantly from zero, showing that recombination occurs or had occurred in A. applanata. Although isolates of A. applanata never were observed to sporulate, it can be distinguished morphologically from P. fortinii s.l. by the scarcity of aerial mycelium, significantly slower growth and denser mycelium on cellophane overlaid on water agar. These phenotypic characteristics, combined with diagnostic RFLP alleles and/or PCR-RFLP of the ITS fragment with the fixed Afa I restriction site, unequivocally allow identification of A. applanata.

QTL analysis and comparative genomics of herbage quality traits in perennial ryegrass (Lolium perenne L.)

Genetic control of herbage quality variation was assessed through the use of the molecular marker-based reference genetic map of perennial ryegrass (Lolium perenne L.). The restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) and genomic DNA-derived simple sequence repeat-based (SSR) framework marker set was enhanced, with RFLP loci corresponding to genes for key enzymes involved in lignin biosynthesis and fructan metabolism. Quality traits such as crude protein (CP) content, estimated in vivo dry matter digestibility (IVVDMD), neutral detergent fibre content (NDF), estimated metabolisable energy (EstME) and water soluble carbohydrate (WSC) content were measured by near infrared reflectance spectroscopy (NIRS) analysis of herbage harvests. Quantitative trait locus (QTL) analysis was performed using single-marker regression, simple interval mapping and composite interval mapping approaches, detecting a total of 42 QTLs from six different sampling experiments varying by developmental stage (anthesis or vegetative growth), location or year. Coincident QTLs were detected on linkage groups (LGs) 3, 5 and 7. The region on LG3 was associated with variation for all measured traits across various experimental datasets. The region on LG7 was associated with variation for all traits except CP, and is located in the vicinity of the lignin biosynthesis gene loci xlpomt1 (caffeic acid-O-methyltransferase), xlpccr1 (cinnamoyl CoA-reductase) and xlpssrcad 2.1 (cinnamyl alcohol dehydrogenase). Comparative genomics analysis of these gene classes with wheat (Triticum aestivum L.) provides evidence for conservation of gene order over evolutionary time and the basis for cross-specific genetic information transfer. The identification of co-location between QTLs and functionally associated genetic markers is critical for the implementation of marker-assisted selection programs and for linkage disequilibrium studies, which will enable future improvement strategies for perennial ryegrass.

Molecular genetic mapping of Gby, a new greenbug resistance gene in bread wheat.

The greenbug, Schizaphis graminum (Rhodani),is one of the major insect pests of wheat worldwide and it is important to develop a basic understanding of the chromosomal locations of known and new greenbug resistance genes. Gby is a new greenbug resistance gene in the wheat line 'Sando's selection 4040'. A mapping population used in this study was derived from a cross of Sando's 4040 and PI220127, a greenbug susceptible wheat land race from Afghanistan. A progeny test indicated that Gby is inherited as a single semi-dominant gene. A genetic linkage map consisting of Gby, Xgwm322 (a wheat microsatellite marker), XksuD2 (an STS marker) and 18 restriction fragment length polymorphism (RFLP) loci was constructed. We used DNA from Chinese Spring 7A deletion lines to show that the gwm332 and ksuD2 amplified fragments mapped in this study are located on along arm of chromosome 7A. This suggests that Gby is located on wheat chromosome 7A. Gby was mapped to the area in the middle of the 'island' of putative defense response genes that are represented by RFLP markers(Xpsr l9, XZnfp, Xbcd98 and Prl b) previously mapped to the distal part of the short arm of wheat chromosome group 7. This region of chromosome 7A is characterized by a high recombination rate and a high physical density of markers which makes Gby a very good candidate for map-based cloning. The selection accuracy when theRFLP markers Xbcd98, Xpsrll9 or XZnfp and Prlb flanking Gby are used together to tag Gby is 99.78%,suggesting that they can be successfully used in marker assisted selection.