Restriction Fragment Length Polymorphism Markers Research Articles

PCR-RFLP analysis of cpDNA in Gigantochloa scortechinii (Poaceae: Bambuseae) in Peninsular Malaysia and implications for the use of cpDNA markers in systematic studies

Abstract Gigantochloa is a paleotropical woody bamboo genus that has been widely cultivated in SE Asia because of its usefulness. Recent studies have shown that species of this genus enter into an introgression complex with other genera of the same subtribe Bambusinae. Within G. scortechinii, a common species indigenous to Malay Peninsula and common in Peninsular Malaysia, two distinct chloroplast DNA (cpDNA) lineages, the Gombak- and Langat-type, were recovered. We report the development of a PCR-based restriction fragment length polymorphism (RFLP) marker for depicting the genetic differentiation in G. scortechinii based on cpDNA. We determined a cpDNA region and its corresponding restriction enzyme which can produce different RFLP profiles for the two cpDNA lineages. Our design was verified with empirical studies. The Gombak-type was the dominant cpDNA genotype for G. scortechinii in Peninsular Malaysia. Implications for the continued use of cpDNA markers in systematic studies are discussed.

Intron loss in the chalcone-flavanone isomerase gene of rye

The Chi gene encodes the flavonoid synthesis enzyme chalcone-flavanone isomerase. The complete coding sequence of the Chi gene was isolated by PCR from four cultivars of cereal rye (Secale cereale L.). Unlike most monocot and dicot plant species, S. cereale has one, rather than three introns in the Chi gene. Screening of a panel of 63 Triticeae accessions, representing 31 species, showed two intron loss events in the Triticeae tribe. One intron loss occurred early in the evolution of the Triticeae tribe, while another intron loss was only detected in S. cerealeChi. A new rye-specific PCR marker was developed based on Chi intron loss polymorphism and was shown to be effective for analysis of a wide range of intergenera Triticeae hybrids for the presence of rye genome. In addition, precise genetic mapping of the rye Chi gene was carried out based on insertion/deletion polymorphism between parents of a rye mapping population. The Chi gene was mapped on the long arm of chromosome 5R 9.3 cM distal to the restriction fragment length polymorphism marker Xscb35 and 4.4 cM proximal to the locus 3Rt encoding another flavonoid synthesis enzyme, anthocyanidin-3-glucoside rhamnosyltransferase.

Prospective molecular markers for the identification of illegally traded angelsharks (Squatina) and dolphin (Sotalia guianensis).

Endangered angelsharks and a protected dolphin species are illegally traded in Brazil. In this study, we determined prospective molecular markers for detecting these species in the trade of angelshark carcasses and 'dolphin' eyeball amulets. We compiled publicly available as well as new and unpublished cytochrome b (cyt b) DNA sequences for species involved in these trades. These sequences were digested in silico using restriction enzymes. We then described prospective polymerase chain reaction (PCR)-restriction fragment length polymorphism markers for distinguishing between protected species and the species whose trade was legally allowed in these two trade groups. The prospective marker for identifying angelshark carcasses consists of cyt b PCR and digestion by BstXI, BsgI, BspMI, BsrDI, and HaeII restriction enzymes. The prospective marker for identifying eyeball amulets consists of cyt b PCR and digestion by ApoI, BtsI, HindII, BsaAI, BplI, and SspI restriction enzymes. This is the first study to deposit in GenBank cyt b sequences for the angelshark species Squatina argentina, Squatina guggenheim, and Squatina occulta. Moreover, the S. argentina haplotype is the first DNA sequence for this species deposited in GenBank.

Quantitative trait loci controlling aluminum tolerance in soybean: candidate gene and single nucleotide polymorphism marker discovery

Aluminum (Al) toxicity is an important abiotic stress that affects soybean production in acidic soils throughout the world. Development of Al-tolerant cultivars is an efficient and environmentally friendly solution to the problem. A previous report identified quantitative trait loci (QTL) for Al tolerance inherited from PI 416937, using restriction fragment length polymorphism markers, in a population of Young × PI 416937. The population was genotyped with 162 simple sequence repeats to enhance the power of QTL detection and enable the selection of candidate genes for functional marker development. Two QTL that explained 54 % of the phenotypic variation in root extension under Al stress conditions (HIAL) were refined on chromosomes (chr) Gm08 and Gm16. Three QTL located on chr Gm08, Gm16 and Gm19 explained 59 % of the phenotypic variation in root extension as a percent of control (PC). Two major QTL, designated qAL_HIAL_08 and qAL_PC_08, controlling HIAL and PC, respectively, were mapped to the same genomic region on chr Gm08 and inherited their favorable allele from PI 416937. These QTL explained 45 and 41 % of phenotypic variation in HIAL and PC, respectively. Six homologues for citrate synthase (CS) genes were found in the soybean genome sequence at chr Gm02, Gm08, Gm14, Gm15, and Gm18. Sixteen single nucleotide polymorphisms (SNPs) were identified in the CS homologue on chr Gm08. A SimpleProbe assay of Glyma08g42400-SNP was developed for the major QTL on chr Gm08. The SNPs identified from this region could be used for marker-assisted selection of Al tolerance.

English

Fifty-four Cymbidium kanran cultivars from China, Japan and South Korea were examined and analyzed by using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) markers to determine their molecular diversity and relationships. In chloroplast (cp) PCR-RFLP analyses, genetic differences were revealed in 4 of 6 primer sets (66.67%) and 19 of 72 primer-enzyme combinations (26.38%), 116 polymorphic bands were detected. For mitochondrion (mt) PCR-RFLP markers, genetic differences were revealed in 2 of 8 primer sets (25%) and 55 polymorphic bands (53.49%) were detected with 7 restriction primer-enzyme combinations. According to the PCR-RFLP marker profiling data, all the cultivars were classified into four groups. The group 1 has included three subclusters that two of them were generally consistent with geography classii¬cation. Subcluster 1 and Subcluster 2 were mainly composed of Chinese cultivars. Comparably, Subcluster 3 was composed of two cultivars which originated from Japan separately. Group 2 comprised Huangchengzhiyue and Xiongnu originated from Japan. Group 3 was included in seven Chinese cultivars and five Japanese cultivars. Group 4 was composed of eight Chinese cultivars and four Japanese cultivars. Therefore, we demonstrated that the PCR-RFLP technique could provide a powerful tool for cultivar identification and establishment of genetic relationships of cultivars in cymbidium kanran. Key words: Cymbidium kanran, chloroplast (cp) polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), mitochondrion (mt) PCR-RFLP.

Genetic variability within n-rDNA region of ectomycorrhizal isolates originating from temperate ecosystems

Identification of Ectomycorrhiza Fungi (ECM) based on morphological characters has been laborious and time consuming, especially samples collected from the environment. Additionally, due to its microscopic nature and limited morphological characters, intraspecies variation is difficult to detect. In view of this, this study aimed at confirming the earlier identification of these fungi, which was based on morphological characters, and also to find suitable molecular restriction fragment length polymorphism (RFLP) markers for the identification of ECM fungi up to the level of species or isolates as part of an expandable database of RFLP patterns of the internal transcribed spacers (ITS) region of ECM fungi. Mycelia of 14 species of ectomycorrhizal fungi representing five genera were isolated in pure culture and characterized by morphological and molecular methods. Molecular identification was performed by analysis of the internal transcribed spacers of the nuclear encoded ribosomal RNA (n-RNA) gene region using restriction fragment length polymorphism (RFLP). The region was first amplified by polymerase chain reaction with specific primers and then cleaved with different restriction enzymes. The degree of polymorphism, although extensive, proved inadequate for proper identification of most of the isolates. Depending on the restriction enzymes used, the genera or species could be grouped on the basis of common fragment patterns, thereby confirming the potential of the small subunit (SSU)-ITS region in PCR-RFLP in molecular characterization and identification of ectomycorrhizal fungi. Based on the results of this study, congruent of morphology and molecular RFLP analysis is recommended for characterization of species/strain of ectomycorrhizal fungi. Keywords: Classification, diversity, ectomycorrhizal fungi, ecosystem, ribosomal DNA African Journal of Biotechnology Vol. 12(22), pp. 3390-3398

Converting restriction fragment length polymorphism to single‐strand conformation polymorphism markers and its application in the fine mapping of a trichome gene in cotton

AbstractA well‐characterized and systematically organized collection of genetic markers is crucial in the study of any crop species. It is the basis of map‐based gene cloning and crop improvements through marker‐assisted selections. Single‐strand conformation polymorphism (SSCP) has been a robust way of discovering new polymorphisms in marker development without the requirement of sequencing. Here, we report the first approach of applying SSCP marker discovery methods in the genetic map construction and gene mapping of cotton species. A total of 80 restriction fragment length polymorphism (RFLP) markers were selected from a region on published cotton genetic maps around the T1 gene related to cotton trichome. Among the 80 RFLPs, 28 showed polymorphisms through SSCP, showing a polymorphic rate of approximately 35%, which is much higher than that of simple sequence repeat (SSR) markers in the same region (7.8%). By integrating these newly generated SSCP markers, a detailed genetic map was reconstructed around this region using an F2 population derived from a cross between Gossypium arboreum and G. herboceum. The reconstructed region comprises 22 SSCP markers, eight SSR markers and the T1 gene, spanning 21.6 cM. The marker order of the new map agrees well with published reference RFLP maps. The above results suggest that SSCP method can be applied very efficiently and reliably to the marker development of cotton genomes. It will prove to be even more valuable and robust after the public release of cotton whole‐genome sequences.

Identification of QTLs Associated with Callogenesis and Embryogenesis in Oil Palm Using Genetic Linkage Maps Improved with SSR Markers

Clonal reproduction of oil palm by means of tissue culture is a very inefficient process. Tissue culturability is known to be genotype dependent with some genotypes being more amenable to tissue culture than others. In this study, genetic linkage maps enriched with simple sequence repeat (SSR) markers were developed for dura (ENL48) and pisifera (ML161), the two fruit forms of oil palm, Elaeis guineensis. The SSR markers were mapped onto earlier reported parental maps based on amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers. The new linkage map of ENL48 contains 148 markers (33 AFLPs, 38 RFLPs and 77 SSRs) in 23 linkage groups (LGs), covering a total map length of 798.0 cM. The ML161 map contains 240 markers (50 AFLPs, 71 RFLPs and 119 SSRs) in 24 LGs covering a total of 1,328.1 cM. Using the improved maps, two quantitative trait loci (QTLs) associated with tissue culturability were identified each for callusing rate and embryogenesis rate. A QTL for callogenesis was identified in LGD4b of ENL48 and explained 17.5% of the phenotypic variation. For embryogenesis rate, a QTL was detected on LGP16b in ML161 and explained 20.1% of the variation. This study is the first attempt to identify QTL associated with tissue culture amenity in oil palm which is an important step towards understanding the molecular processes underlying clonal regeneration of oil palm.

Genetic and molecular analyses of resistance to a variant of Puccinia striiformis in barley

Seedlings of 62 Australian barley cultivars and two exotic barley genotypes were assessed for resistance to a variant of Puccinia striiformis, referred to as "Barley Grass Stripe Rust" (BGYR), first detected in Australia in 1998, which is capable of infecting wild Hordeum species and some genotypes of cultivated barley. Fifty-three out of 62 cultivated barley cultivars tested were resistant to the pathogen. Genetic analyses of seedling resistance to BGYR in six Australian barley cultivars and one Algerian barley landrace indicated that they carried either one or two major resistance genes to the pathogen. A single recessive seedling resistance gene, rpsSa3771, identified in Sahara 3771, was located on the long arm of chromosome 1 (7 H), flanked by the restriction fragment length polymorphism (RFLP) markers Xwg420 and Xcdo347 at genetic distances of 12.8 and 21.9 cM, respectively. Mapping resistance to BGYR at adult plant growth stages using the doubled haploid (DH) population Clipper × Sahara 3771 identified two major quantitative trait loci (QTL), one on the long arm of chromosome 3 (3 H) and the second on the long arm of chromosome 1 (7 H), accounting for 26 % and 18 % of the total phenotypic variation, respectively. The QTL located on chromosome 7HL corresponded to seedling resistance gene rpsSa3771 and the second QTL was concluded to correspond to a single APR gene, designated rpsCl, contributed by cultivar Clipper.

Stability of genome-wide QTL effects on malt α-amylase activity in a barley doubled-haploid population

Pre-harvest sprouting (PHS) can be a problem in malting barley (Hordeum vulgare L.) production as it results in lower yield and reduced grade. The presence of PHS is often associated with increased activity of α-amylase, a key enzyme for breakdown of starch into simple sugars. It is difficult to measure α-amylase activity (AA) associated with PHS however, and the same quantitative trait loci (QTL) may be responsible for both PHS AA and malt AA. The objective of this paper was to investigate co-occurrence and stability of QTL controlling malt AA in a barley doubled-haploid (DH) population that was evaluated in eight environments across North America. This population consisted of 150 DH lines developed from a cross between two barley varieties (Steptoe × Morex) for the North American Barley Genome Mapping Project. Malt AA was measured on a composite 400 g sample of each DH line and parent. There were a total of 223 restriction fragment length polymorphism markers covering a total genetic distance of 1221.9 cM across the entire genome. We used an empirical Bayesian method for detecting single QTLs and epistatic QTLs for individual environments. A common QTL was counted if it co-occurred in a pair of environments. Thus, the level of QTL × environment interaction was assessed based on the total frequency of co-occurred QTLs for all possible pairs of eight environments [(8 × 7)/2 = 28]. Heritability estimates (0.62–0.89) for individual environments were moderate to high. Genetic correlations between co-occurred additive and epistatic effects are essentially zero for most environment pairs. These results indicated strong QTL × environment interaction for malt AA. While the efficacy of genome-wide selection for PHS is still unknown especially in the presence of QTL × environment interaction, using the Bayesian approach may help us better understand appropriate methods for measuring and selecting for this trait.

Contemporary gene flow between “paired” silver (Ichthyomyzon unicuspis) and northern brook (I. fossor) lampreys: implications for conservation

In most lamprey genera, “paired” species exist in which the larvae are morphologically similar or indistinguishable but, following metamorphosis, one species becomes parasitic while the other bypasses the adult feeding phase and rapidly becomes sexually mature. Since DNA barcoding and similar studies using short segments of the mitochondrial genome do not provide sufficient resolution to distinguish between recent divergence and a lack thereof, the current study examined the relationship between the parasitic silver lamprey (Ichthyomyzon unicuspis) and nonparasitic northern brook lamprey (I. fossor) using up to 10,230 bp from the mitochondrial genome to make phylogenetic inferences and mitochondrial restriction fragment length polymorphism (RFLP) and microsatellite markers to test for significant differences in allele frequencies. We found that silver and northern brook lampreys are not reciprocally monophyletic; two lineages were observed but each occurred within both species. There also were no significant range-wide differences in RFLP haplotype or microsatellite allele frequencies between the species, nor were there significant differences where they occurred sympatrically within the Lake Huron basin. Significant genetic differentiation was found only within the Lake Michigan basin where the results were potentially confounded by geographic separation. Our results thus support suggestions that silver and northern brook lampreys represent ecotypes of a single species since, where they are sympatric, they appear to be experiencing ongoing gene flow. However, alternative life history strategies can be important for a species’ long-term persistence, and critical data are needed to decide whether these two feeding types should be managed as a single unit.

CAPS Marker Linked to Tomato Hypocotyl Pigmentation

Tomato hypocotyl can generally be one of two colors, purple or green. Genetically, this trait is controlled by a single dominant gene. Hypocotyl tissue specific color expression is one of many visible genetic marker sources used to select tomato progeny. However, the visible marker does not show a clear distinction between homozygous genotype and heterozygous genotype from the breeding lines. Therefore, to identify a hypocotyl pigmentation related marker, we screened DNA polymorphisms in thirteen tomato lines showing purple or green hypocotyls. The markers used for screening consisted of primer set information obtained from anthocyanin related genes, conserved ortholog set II (COS II) marker sets localized near anthocyanin related genes, and restriction fragment length polymorphism (RFLP) markers localized near COS II markers, which produce polymorphisms between purple and green tomatoes. One primer from a RFLP fragment resulted in a polymorphism on agarose gel electrophoresis. From the RFLP fragment, a cleaved amplified polymorphic sequence (CAPS) marker was developed to distinguish between purple and green hypocotyls. The genotypes of 135 <TEX>$F_2$</TEX> individuals were analyzed using the CAPS marker, and among them, 132 individuals corresponded to the phenotypes of hypocotyl pigmentation.

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.

Quantitative trait loci controlling steamed-rice shape in a recombinant inbred population

To identify quantitative trait loci (QTLs) for steamed-rice shape in rice, we used one recombinant inbred (RI) population provided by Prof. A. Yoshimura of Kyushu University, Japan, derived from the cross between japonica cultivar Asominori and indica cultivar IR24 (IRRI). Tsunematsu et al (1996) constructed a restriction fragment length polymorphism (RFLP) map covering 1,275 cM of 12 chromosomes with 375 markers using 71 RI lines, which was used previously for mapping QTLs for important agronomic traits (Yoshimura et al 1998; Yamazaki et al 1999, 2000; Sasahara et al 1999). In this study, we used a subset of 289 RFLP markers from the original map to map QTLs affecting steamed-rice shape (with an average interval distance between marker pairs of 4.4 cM)

Hybrid Weakness Found in the Backcross Progeny of an Inter-Specific Cross of &lt;em&gt;Oryza Sativa / O. Glumaepatula&lt;/em&gt;

Hybrid breakdown expressed as sterility or weakness or both in F 2 or later generations is considered to be a reproductive barrier between two distantly related taxa. Hybrid breakdown that showed poor growth habit with fertile seeds was found in the backcross progenies (BC 4 F 2 ) which were derived from a cross between a cultivated rice Oryza sativa L. cv. Taichung 65 and a wild rice Oryza glumaepatula Steud (Acc. IRGC 105668) where Taichung 65 was used as a recurrent parent. Through segregation analysis of BC 4 F 2 population, a novel gene for weakness was identified, and this gene was designated as hwf1 (hybrid weakness f-1). RFLP (Restriction Fragment Length Polymorphism) analysis was employed to determine the chromosomal location of hwf1 gene. The result revealed that hwf1 was located between G3006 and C933 RFLP markers, with map distances of 0.5 cM and 1.9 cM, respectively, and co-segregated with C708 , C820 , and R288 on the short arm of chromosome 4. Since both O. glumaepatula (Acc. IRGC 105668) and Taichung 65 that was used as parents showed normal growth habit, the weakness in this study was assumed to be controlled by a set of complementary recessive genes. One of the complementary genes is hwf1 from O. glumaepatula , and another gene should be from Taichung 65, because the genetic background of the BC 4 F 2 populations that were used for analysis is Taichung 65. The gene from Taichung 65 has not been identified yet, therefore, it is necessary to conduct the experiment for finding out the complement of hwf1 gene in near future. Received: 20 November 2008; Revised: 15 May 2009; Accepted: 16 May 2009

Molecular mapping of powdery mildew resistance gene Eg-3 in cultivated oat (Avena sativa L. cv. Rollo)

Powdery mildew is a prevalent fungal disease affecting oat (Avena sativa L.) production in Europe. Common oat cultivar Rollo was previously shown to carry the powdery mildew resistance gene Eg-3 in common with cultivar Mostyn. The resistance gene was mapped with restriction fragment length polymorphism (RFLP) markers from Triticeae group-1 chromosomes using a population of F(3) lines from a cross between A. byzantina cv. Kanota and A. sativa cv. Rollo. This comparative mapping approach positioned Eg-3 between cDNA-RFLP marker loci cmwg706 and cmwg733. Since both marker loci were derived from the long arm of barley chromosome 1H, the subchromosomal location of Eg-3 was assumed to be on the long arm of oat chromosome 17. Amplified fragment length polymorphism (AFLP) marker technology featured as an efficient means for obtaining markers closely linked to Eg-3.

Comparative karyotypic analysis of A and C genomes in the genus Oryza with C 0 t-1 DNA and RFLP

Fluorescence in situ hybridization (FISH) was applied to somatic chromosomes preparations of Oryza sativa L. (AA), O. glaberrima (AA), and O. officinalis Wall. (CC) with a labeled probe of C 0 t-1 DNA. Genomic in situ hybridization to its own chromosomes (self-GISH) was conducted in a control experiment. The homologous chromosomes showed similar signal bands probed by C 0 t-1 DNA, while karyotypic analysis of chromosomes between A genome in the two cultivated species and C genome in O. officinalis were conducted based on the band patterns. The ideograms with C 0 t-1 DNA signal bands were also built. The nonuniform distribution of hybridization signals of C 0 t-1 DNA from O. sativa and that on its own chromosome of O. officinalis were observed. However, the similarity and correspondence between C 0 t-1 DNA signal patterns and genomic DNA signal patterns indicated that the self-GISH signals actually resulted from the hybridization of genomic repetitive sequences to the chromosomes. The restriction fragment length polymorphism (RFLP) marker, R2676, from the chromosome 8 of O. sativa and O. officinalis, was used as a probe to somatic hybrid on chromosomes for comparative karyotypic analysis between O. glaberrima and O. officinalis. The results showed that R2676 was located on the short arm of chromosome 7 in O. officinalis and chromosome 4 in O. glaberrima. The percentage distances from the centromere to hybridization sites were 91.56±5.62 and 86.20±3.17. Our results revealed that the relative length of O. officinalis chromosome 8 does not follow conventional chromosome length in descending order of number. C 0 t-1 DNA of A genome signals were detected in the end of the short arm of O. officinalis chromosome 8, indicating that the highly and moderately repetitive DNA sequences in this region were considerably similar between C and A genomes. However, the fluorescence intensity on the chromosomes of C 0 t-1 DNA of A genome was less than that of its own C genome from O. officinalis, which would be one of the causes for the fact that highly and moderately repetitive DNA sequences were amplified in O. officinalis. No homology signal of C 0 t-1 DNA from O. sativa was detected in the end of the long arm of O. glaberrima, indicating that repetitive DNA sequences of A genome in two cultivated rice were lost in the evolutional history. In this paper, using comparative karyotypic analysis of RFLP combined C 0 t-1 DNA signal bands, the evolutionary mechanism of genome in genus Oryza was also discussed.

Nivalenol-Type Populations of Fusarium graminearum and F. asiaticum Are Prevalent on Wheat in Southern Louisiana

U.S. populations of the Fusarium graminearum clade cause head blight on wheat and barley and usually contaminate grain with the trichothecene mycotoxin deoxynivalenol (DON). Recently, however, individual nivalenol (NIV)-type isolates from the United States were described that belonged to either the newly described species F. gerlachii or the genetically distinct Gulf Coast population of F. graminearum sensu stricto (s.s.). Here, we describe the discovery of NIV-type F. graminearum s.s. populations that were found in high proportion (79%) among isolates from small-grain-growing regions of Louisiana. We genotyped 237 isolates from Louisiana with newly developed polymerase chain reaction (PCR) restriction fragment length polymorphism markers and multiplex PCR primers that distinguish among the three trichothecene types: the two DON types (15ADON and 3ADON) and NIV. These isolates were compared with 297 isolates from 11 other U.S. states, predominantly from the Midwest. Using Bayesian-model-based clustering, we discovered a southern Louisiana population of F. graminearum s.s. that was genetically distinct from the previously recognized pathogen population in the Midwest (MW15ADON population). Population membership was correlated with trichothecene type. Most isolates from the southern Louisiana population were of the NIV type, while the majority of the isolates from the Midwest were of the 15ADON type. A smaller proportion of isolates from Louisiana belonged to the previously described Gulf Coast population that was mostly of the 3ADON type. The NIV type was also identified in collections from Arkansas (12%), North Carolina (40%), and Missouri (2%), with the collections from Arkansas and North Carolina being small and unrepresentative. F. asiaticum was detected from the two southern Louisiana parishes Acadia and Alexandria. All identified 41 F. asiaticum isolates were of the NIV type. Greenhouse tests indicated that U.S. NIV types accumulated four times less trichothecene toxin than DON types on inoculated wheat. This is the first report of NIV-type populations of F. graminearum s. s. and F. asiaticum in the United States.

Genetic Relationships among Populations of Florida Bass

AbstractFlorida bass Micropterus floridanus are endemic to peninsular Florida and co‐occur with largemouth bass M. salmoides in a natural intergrade zone in the northern portions of the state. In this study, we resolved the genetic population structure among populations of largemouth bass, Florida bass, and their interspecific hybrids from 48 lakes and streams across Florida, and we updated and refined the southernmost boundary of introgression. Nuclear (allozyme and microsatellite) and mitochondrial (restriction fragment length polymorphism) markers produced somewhat different results. Microsatellite genotypes alone and the combination of all nuclear genotypes resolved genetic structure among four regional groups within Florida: (1) northwest, (2) north‐central, (3) central‐west, and (4) south, central‐east, and northeast. Allozyme genotypes alone did not resolve well‐defined groups. The distribution of mitochondrial haplotypes suggested that populations from the St. Johns River drainage should be considered their own distinct group (rather than be included in the south—central‐east—northeast Florida group). Although mitochondrial markers failed to detect introgression throughout much of the intergrade zone, nuclear markers revealed an expected decline in introgression from west to east as well as from north to south. Largemouth bass alleles, however, were detected at low frequencies in 12 populations below the southern boundary of the accepted intergrade zone, some of which are the result of stocking. There was no evidence of nonrandom mating or selection against hybrids in the distribution of nuclear genotypes; however, the rarity of largemouth bass mitochondrial haplotypes suggests either assortative mating or reduced fitness in the progeny of female largemouth bass × male Florida bass crosses.

PCR markers for selection of adult plant leaf rust resistance in barley (Hordeum vulgare L.)

Adult plant resistance (APR) is considered potentially more durable for controlling barley leaf rust than seedling Rph (Resistance to Puccinia hordei) genes. A major gene for adult plant resistance to barley leaf rust has been mapped to the telomere region of chromosome 5HS. PCR-based molecular markers were developed for saturation of this region based on previously mapped simple sequence repeat, restriction fragment length polymorphism and Diversity Arrays Technology markers. In addition, defence gene homologue (DGH) and wheat expressed sequence tags mapped in specific bins were used to develop new PCR markers. Seventeen PCR-based markers were mapped to the short arm of chromosome 5H in 292 doubled haploid lines from a cross of Pompadour × Stirling, in which seven markers were mapped within 5 cM of the APR gene. The closest linked marker was about 0.7 cM from the APR gene. The wheat deletion bin map together with defence gene homologues was demonstrated to be an efficient tool for development of new molecular markers associated with the disease resistance gene. Four DGH markers were associated with the APR gene. The new molecular markers are a useful tool for marker-assisted selection of the APR gene and provided a better understanding of the molecular mechanism for leaf rust resistance.