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BACKGROUND: Reniform nematode (Rotylenchulus reniformis) has emerged as one of the most destructive root pathogens of upland cotton (Gossypium hirsutum) in the United States. Management of R. reniformis has been hindered by the lack of resistant G. hirsutum cultivars; however, resistance has been frequently identified in germplasm accessions from the G. arboreum collection. To determine the genetic basis of reniform nematode resistance, a genome-wide association study (GWAS) was performed using 246 G. arboreum germplasm accessions that were genotyped with 7220 single nucleotide polymorphic (SNP) sequence markers generated from genotyping-by-sequencing. RESULTS: Fifteen SNPs representing 12 genomic loci distributed over eight chromosomes showed association with reniform nematode resistance. For 14 SNPs, major alleles were shown to be associated with resistance. From the 15 significantly associated SNPs, 146 genes containing or physically close to these loci were identified as putative reniform nematode resistance candidate genes. These genes are involved in a broad range of biological pathways, including plant innate immunity, transcriptional regulation, and redox reaction that may have a role in the expression of resistance. Eighteen of these genes corresponded to differentially expressed genes identified from G. hirsutum in response to reniform nematode infection. CONCLUSIONS: The identification of multiple genomic loci associated with reniform nematode resistance would indicate that the G. arboreum collection is a significant resource of novel resistance genes. The significantly associated markers identified from this GWAS can be used for the development of molecular tools for breeding improved reniform nematode resistant upland cotton with resistance introgressed from G. arboreum. Additionally, a greater understanding of the molecular mechanisms of reniform nematode resistance can be determined through genetic structure and functional analyses of candidate genes, which will aid in the pyramiding of multiple resistance genes.
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Resistencia a la Enfermedad/genética , Gossypium/genética , Infecciones por Nematodos , Enfermedades de las Plantas , Polimorfismo de Nucleótido Simple , Animales , Estudio de Asociación del Genoma Completo , Gossypium/fisiología , Análisis de Secuencia de ADN , TylenchoideaRESUMEN
The diploid cotton species Gossypium arboreum possesses many favorable agronomic traits such as drought tolerance and disease resistance, which can be utilized in the development of improved upland cotton cultivars. The USDA National Plant Germplasm System maintains more than 1600 G. arboreum accessions. Little information is available on the genetic diversity of the collection thereby limiting the utilization of this cotton species. The genetic diversity and population structure of the G. arboreum germplasm collection were assessed by genotyping-by-sequencing of 375 accessions. Using genome-wide single nucleotide polymorphism sequence data, two major clusters were inferred with 302 accessions in Cluster 1, 64 accessions in Cluster 2, and nine accessions unassigned due to their nearly equal membership to each cluster. These two clusters were further evaluated independently resulting in the identification of two sub-clusters for the 302 Cluster 1 accessions and three sub-clusters for the 64 Cluster 2 accessions. Low to moderate genetic diversity between clusters and sub-clusters were observed indicating a narrow genetic base. Cluster 2 accessions were more genetically diverse and the majority of the accessions in this cluster were landraces. In contrast, Cluster 1 is composed of varieties or breeding lines more recently added to the collection. The majority of the accessions had kinship values ranging from 0.6 to 0.8. Eight pairs of accessions were identified as potential redundancies due to their high kinship relatedness. The genetic diversity and genotype data from this study are essential to enhance germplasm utilization to identify genetically diverse accessions for the detection of quantitative trait loci associated with important traits that would benefit upland cotton improvement.
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Variación Genética , Gossypium/clasificación , Gossypium/genética , Análisis por Conglomerados , ADN de Plantas , Marcadores Genéticos , Genética de Población , Técnicas de Genotipaje , Filogenia , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
In the southeastern United States, reniform nematode (Rotylenchulus reniformis) is a serious pest of upland cotton (Gossypium hirsutum), a species which has no naturally occurring resistance against this nematode. To identify sources of reniform nematode resistance in species closely related to upland cotton, 222 G. arboreum accessions from the U.S. germplasm collection were evaluated in repeated growth chamber experiments. In initial screenings, root infection was measured 4 wks after inoculation. The 15 accessions supporting the fewest infections (PI 529992, PI 615755, PI 615766, PI 615788, PI 615848, PI 615856, PI 615950, PI 615977, PI 615991, PI 616008, PI 616016, PI 616062, PI 616126, PI 616159, and A2 553) were evaluated again in confirmation tests lasting 8 wk. The combined totals of nematodes extracted from soil and eggs extracted from roots were analyzed. All 15 accessions tested supported significantly smaller reniform nematode populations than the susceptible controls (G. hirsutum cultivar Deltapine 16 and G. arboreum accession PI 529729). Nine accessions (PI 529992, PI 615755, PI 615766, PI 615788, PI 615856, PI 615950, PI 615991, PI 616008, and PI 616159) supported reniform nematode populations comparable to the resistant control (G. arboreum accession PI 615699), and accession PI 615848 had significantly fewer reniform nematodes than the resistant control. Cotton breeders would benefit from introgressing the newly identified resistance from these accessions into their upland cotton improvement programs.
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Reniform nematode (Rotylenchulus reniformis) is an important microparasite for Upland cotton (Gossypium hirsutum L.) production. Growing resistant cultivars is the most economical management method, but only a few G. barbadense genotypes and some diploid Gossypium species confer high levels of resistance. This study conducted a transcriptome analysis of resistant genotypes to identify genes involved in host plant defense. Seedlings of G. arboreum accessions PI 529728 (A2-100) and PI 615699 (A2-190), and G. barbadense genotypes PI 608139 (GB 713) and PI 163608 (TX 110), were inoculated with the reniform nematode population MSRR04 and root samples were collected on the fifth (D5) and ninth (D9) day after inoculation. Differentially expressed genes (DEGs) were identified by comparing root transcriptomes from inoculated plants with those from non-inoculated plants. Accessions A2-100 and A2-190 showed 52 and 29 DEGs on D5, respectively, with 14 DEGs in common, and 18 DEGs for A2-100 and 11 DEGs for A2-190 on chromosome 5. On D9, four DEGs were found in A2-100 and two DEGs in A2-190. For GB 713, 52 and 43 DEGs were found, and for TX 110, 29 and 117 DEGs were observed on D5 and D9, respectively. Six DEGs were common at the two sampling times for these genotypes. Some DEGs were identified as Meloidogyne-induced cotton (MIC) 3 and 4, resistance gene analogs, or receptor-like proteins. Other DEGs have potential roles in plant defense, such as peroxidases, programmed cell death, pathogenesis related proteins, and systemic acquired resistance. Further research on these DEGs will aid in understanding the mechanisms of resistance to explore new applications for the development of resistant cultivars.
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BACKGROUND: Previous loblolly pine (Pinus taeda L.) genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats), also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective of this study was to integrate a large set of SSR markers from a variety of sources and published cDNA markers into a composite P. taeda genetic map constructed from two reference mapping pedigrees. A dense genetic map that incorporates SSR loci will benefit complete pine genome sequencing, pine population genetics studies, and pine breeding programs. Careful marker annotation using a variety of references further enhances the utility of the integrated SSR map. RESULTS: The updated P. taeda genetic map, with an estimated genome coverage of 1,515 cM(Kosambi) across 12 linkage groups, incorporated 170 new SSR markers and 290 previously reported SSR, RFLP, and ESTP markers. The average marker interval was 3.1 cM. Of 233 mapped SSR loci, 84 were from cDNA-derived sequences (EST-SSRs) and 149 were from non-transcribed genomic sequences (genomic-SSRs). Of all 311 mapped cDNA-derived markers, 77% were associated with NCBI Pta UniGene clusters, 67% with RefSeq proteins, and 62% with functional Gene Ontology (GO) terms. Duplicate (i.e., redundant accessory) and paralogous markers were tentatively identified by evaluating marker sequences by their UniGene cluster IDs, clone IDs, and relative map positions. The average gene diversity, He, among polymorphic SSR loci, including those that were not mapped, was 0.43 for 94 EST-SSRs and 0.72 for 83 genomic-SSRs. The genetic map can be viewed and queried at http://www.conifergdb.org/pinemap. CONCLUSIONS: Many polymorphic and genetically mapped SSR markers are now available for use in P. taeda population genetics, studies of adaptive traits, and various germplasm management applications. Annotating mapped genes with UniGene clusters and GO terms allowed assessment of redundant and paralogous EST markers and further improved the quality and utility of the genetic map for P. taeda.
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Mapeo Cromosómico , ADN Complementario , Marcadores Genéticos , Repeticiones de Minisatélite , Pinus taeda/genética , Ligamiento Genético , Genotipo , Polimorfismo GenéticoRESUMEN
Head smut, caused by the fungal pathogen Sporisorium reilianum, has been reported with increasing frequency in the grain sorghum growing areas of Texas. To facilitate analysis of changes in pathogen virulence, four inoculation techniques were examined: soil and teliospore mixture, seed coating, media placement, and syringe injection. Of the four, syringe injection was determined to be the most effective. Inoculations of sorghum host differentials BTx643, BTx7078, BTx635, SC170-6-17 (TAM2571), SA281 (Early Hegari), and Tx414 showed 23 of 32 Texas isolates were race 4. Two isolates from College Station, TX, were classified as race 1, but no race 2 or 3 isolates were found. New, virulent races 5 and 6 were identified among isolates from south Texas. Using 16 amplified fragment length polymorphism (AFLP) primer combinations, genetic diversity was assessed in DNA samples from 49 S. reilianum isolates, including 44 sorghum isolates from Texas, two from Uganda, and one from Mali; and two maize isolates from Mexico. Single-base extensions with EcoRI and MseI primers in the selective amplification increased the number of informative polymorphic bands. High genetic dissimilarity (50%) was observed between isolates originating from maize and those originating from sorghum. The resultant dendrogram, made using cluster analysis, grouped the Texas S. reilianum isolates into four small clusters with ≥82% similarity. Other than for two race 6 isolates from Weslaco, TX, no evidence for geographical or other restrictions on gene flow was evident.
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Sweet sorghum has the potential to become a versatile feedstock for large-scale bioenergy production given its sugar from stem juice, cellulose/hemicellulose from stalks, and starch from grain. However, for researchers to maximize its feedstock potential a first step includes additional evaluations of the 2,180 accessions with varied origins in the US historic sweet sorghum collection. To assess genetic diversity of this collection for bioenergy breeding and population structure for association mapping, we selected 96 accessions and genotyped them with 95 simple sequence repeat markers. Subsequent genetic diversity and population structure analysis methods identified four subpopulations in this panel, which correlated well with the geographic locations where these accessions originated or were collected. Model comparisons for three quantitative traits revealed different levels of population structure effects on flowering time, plant height, and brix. Our results suggest that diverse germplasm accessions curated from different geographical regions should be considered for plant breeding programs to develop sweet sorghum cultivars or hybrids, and that this sweet sorghum panel can be further explored for association mapping.
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Variación Genética , Genética de Población , Sorghum/genética , Biocombustibles , Productos Agrícolas/genética , Cruzamientos Genéticos , ADN de Plantas/genética , Marcadores Genéticos , Genotipo , Humanos , Modelos Genéticos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Estados UnidosRESUMEN
A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is described here in which the root system is visually examined for nematode infection. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.
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Resistencia a la Enfermedad , Genotipo , Gossypium/genética , Gossypium/parasitología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/parasitología , Tylenchoidea/fisiología , Animales , Femenino , Gossypium/inmunologíaRESUMEN
A simple, inexpensive design is presented for the rapid conversion of the popular MD-4 Polaroid land camera to a high quality digital gel documentation system. Images of ethidium bromide stained DNA gels captured using the digital system were compared to images captured on Polaroid instant film. Resolution and sensitivity were enhanced using the digital system. In addition to the low cost and superior image quality of the digital system, there is also the added convenience of real-time image viewing through the swivel LCD of the digital camera, wide flexibility of gel sizes, accurate automatic focusing, variable image resolution, and consistent ease of use and quality. Images can be directly imported to a computer by using the USB port on the digital camera, further enhancing the potential of the digital system for documentation, analysis, and archiving. The system is appropriate for use as a start-up gel documentation system and for routine gel analysis.
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Documentación , Electroforesis en Gel de Agar/instrumentación , Fotograbar/instrumentación , ADN/química , Electroforesis en Gel de Agar/métodos , Fotograbar/métodosRESUMEN
STUDY OBJECTIVE: To determine if 15 min of open-chest cardiac massage (OC-CPR) versus closed-chest compressions (CC-CPR) improves 72-h survival and neurologic outcome (behavioral and histologic) after 5 min of untreated cardiac arrest. METHODS: Mongrel dogs were anesthetized and instrumented. Cardiac arrest was induced by KCl injection and after a 5-min period of non-intervention, dogs were randomized to receive either CC-CPR (N = 7) or OC-CPR (N = 5) performed for 15 min. The dogs were then resuscitated and physiologic data was recorded. Surviving dogs were scored at 72 h using canine neurodeficit score of Safar et al. (NDS; 0 = behaviorally normal, 500 = brain death). Dogs that could not be resuscitated or died before 72 h were assigned a score of 500. Brain histology was performed on all survivors. RESULTS: All OC-CPR dogs were successfully resuscitated and were behaviorally normal at 72 h (NDS = 0). Histology in OC-CPR dogs showed little to no injury. Only three out of the seven CC-CPR dogs survived to 72 h. Of the survivors, one dog exhibited minor ataxia (NDS = 15), and two had incapacitating deficits (both NDS = 180). Two dogs died within 24 h after extubation, and one could not be resuscitated and the other could not be weaned from the ventilator (each NDS = 500). Histology of the CC-CPR survivors revealed moderate to severe lesions. NDS between groups was statistically significant (p < 0.0079). CONCLUSION: In our canine model of cardiac arrest, OC-CPR significantly improved 72-h survival and neurologic outcome when compared to CC-CPR.
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Reanimación Cardiopulmonar/métodos , Paro Cardíaco/complicaciones , Paro Cardíaco/terapia , Masaje Cardíaco/métodos , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/prevención & control , Animales , Temperatura Corporal , Encéfalo/patología , Modelos Animales de Enfermedad , Perros , Paro Cardíaco/fisiopatología , Hemodinámica , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/fisiopatología , Recuperación de la Función , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND: Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays. RESULTS: We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants. The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines. CONCLUSIONS: Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype.
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ADN Complementario/genética , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/genética , Regulación de la Expresión Génica de las Plantas/genética , Glycine max/genética , Glycine max/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis por Conglomerados , Cotiledón/genética , ADN de Plantas/genética , Perfilación de la Expresión Génica/estadística & datos numéricos , Biblioteca de Genes , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Especificidad de Órganos/genética , FenotipoRESUMEN
Whole-genome sequencing is fundamental to understanding the genetic composition of an organism. Given the size and complexity of the soybean genome, an alternative approach is targeted random-gene sequencing, which provides an immediate and productive method of gene discovery. In this study, more than 120000 soybean expressed sequence tags (ESTs) generated from more than 50 cDNA libraries were evaluated. These ESTs coalesced into 16928 contigs and 17336 singletons. On average, each contig was composed of 6 ESTs and spanned 788 bases. The average sequence length submitted to dbEST was 414 bases. Using only those libraries generating more than 800 ESTs each and only those contigs with 10 or more ESTs each, correlated patterns of gene expression among libraries and genes were discerned. Two-dimensional qualitative representations of contig and library similarities were generated based on expression profiles. Genes with similar expression patterns and, potentially, similar functions were identified. These studies provide a rich source of publicly available gene sequences as well as valuable insight into the structure, function, and evolution of a model crop legume genome.