Jeffery D. Ray

Identification of a new soybean rust resistance gene in PI 567102B

Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. and P. Syd. is one of the most economically important diseases of soybean (Glycine max (L.) Merr.). Durable resistance to P. pachyrhizi is the most effective long-term strategy to control SBR. The objective of this study was to investigate the genetics of resistance to P. pachyrhizi in soybean accession PI 567102B. This accession was previously identified as resistant to SBR in Paraguay and to P. pachyrhizi isolates from seven states in the USA (Alabama, Florida, Georgia, Louisiana, Mississippi, South Carolina, and Texas). Analysis of two independent populations, one in which F2 phenotypes were inferred from F2-derived F3 (F2:3) families and the other in which F2 plants had phenotypes measured directly, showed that the resistance in PI 567102B was controlled by a single dominant gene. Two different isolates (MS06-1 and LA04-1) at different locations (Stoneville, MS and Ft. Detrick, MD) were used to independently assay the two populations. Linkage analysis of both populations indicated that the resistance locus was located on chromosome 18 (formerly linkage group G), but at a different location than either Rpp1 or Rpp4, which were previously mapped to this linkage group. Therefore, the SBR resistance in PI 567102B appeared to be conditioned by a previously unreported locus, with an underlying single dominant gene inferred. We propose this gene to be designated Rpp6. Incorporating Rpp6 into improved soybean cultivars may have wide benefits as PI 567102B has been shown to provide resistance to P. pachyrhizi isolates from Paraguay and the US.

Glyphosate Resistance in Tall Waterhemp (Amaranthus tuberculatus) from Mississippi is due to both Altered Target-Site and Nontarget-Site Mechanisms

Abstract A tall waterhemp population from Missisippi was suspected to be resistant to glyphosate. Glyphosate dose response experiments resulted in GR50 (dose required to reduce plant growth by 50%) values of 1.28 and 0.28 kg ae ha−1 glyphosate for the glyphosate-resistant (GR) and -susceptible (GS) populations, respectively, indicating a five-fold resistance. The absorption pattern of 14C-glyphosate between the GR and GS populations was similar up to 24 h after treatment (HAT). Thereafter, the susceptible population absorbed more glyphosate (55 and 49% of applied) compared to the resistant population (41 and 40% of applied) by 48 and 72 HAT, respectively. Treatment of a single leaf in individual plants with glyphosate at 0.84 kg ha−1, in the form of 10 1-µl droplets, provided greater control (85 vs. 29%) and shoot fresh weight reduction (73 vs. 34% of nontreated control) of the GS plants compared to the GR plants, possibly indicating a reduced movement of glyphosate in the GR plants. The amount of 14C-glyphosate that translocated out of the treated leaves of GR plants (20% of absorbed at 24 HAT and 23% of absorbed at 48 HAT) was significantly lower than the GS plants (31% of absorbed at 24 HAT and 32% of absorbed at 48 HAT). A potential difference in shikimate accumulation between GR and GS populations at different concentrations of glyphosate was also studied in vitro. The IC50 (glyphosate concentration required to cause shikimate accumulation at 50% of peak levels measured) values for the GR and GS populations were 480 and 140 µM of glyphosate, respectively, resulting in more shikimate accumulation in the GS than the GR population. Sequence analysis of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the target site of glyphosate, from GR and GS plants identified a consistent single nucleotide polymorphism (T/C, thymine/cytosine) between GR/GS plants, resulting in a proline to serine amino acid substitution at position 106 in the GR population. The GR and GS plants contained equal genomic copy number of EPSPS, which was positively correlated with EPSPS gene expression. Thus, glyphosate resistance in the tall waterhemp population from Mississippi is due to both altered target site and nontarget site mechanisms. This is the first report of an altered EPSPS-based resistance in a dicot weed species that has evolved resistance to glyphosate. Nomenclature: Glyphosate; tall waterhemp, Amaranthus tuberculatus (Moq.) Sauer.

EPSPS amplification in glyphosate-resistant spiny amaranth (Amaranthus spinosus): a case of gene transfer via interspecific hybridization from glyphosate-resistant Palmer amaranth (Amaranthus palmeri).

BACKGROUND Amaranthus spinosus, a common weed of pastures, is a close relative of Amaranthus palmeri, a problematic agricultural weed with widespread glyphosate resistance. These two species have been known to hybridize, allowing for transfer of glyphosate resistance. Glyphosate-resistant A. spinosus was recently suspected in a cotton field in Mississippi. RESULTS Glyphosate-resistant A. spinosus biotypes exhibited a fivefold increase in resistance compared with a glyphosate-susceptible biotype. EPSPS was amplified 33-37 times and expressed 37 times more in glyphosate-resistant A. spinosus biotypes than in a susceptible biotype. The EPSPS sequence in resistant A. spinosus plants was identical to the EPSPS in glyphosate-resistant A. palmeri, but differed at 29 nucleotides from the EPSPS in susceptible A. spinosus plants. PCR analysis revealed similarities between the glyphosate-resistant A. palmeri amplicon and glyphosate-resistant A. spinosus. CONCLUSIONS Glyphosate resistance in A. spinosus is caused by amplification of the EPSPS gene. Evidence suggests that part of the EPSPS amplicon from resistant A. palmeri is present in glyphosate-resistant A. spinosus. This is likely due to a hybridization event between A. spinosus and glyphosate-resistant A. palmeri somewhere in the lineage of the glyphosate-resistant A. spinosus plants. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Growth of Subtropical Forage Grasses under Extended Photoperiod during Short-Daylength Months

onstrated under field conditions that short daylengths induce decreased growth, then the possibility exists for One constraint on cattle production in the southeastern USA is increasing grass productivity during the cool, shortthe low productivity of perennial forage grasses during the shortdaylength months. Evidence indicates that total growth during these daylength months by genetic selection and development months could be enhanced by exposing these grasses to extended of photoperiod-insensitive cultivars. photoperiod. A detailed analysis of their year-round productivity and Studies in controlled environments showed that grass nutritive value is needed to understand fully the consequences of growth is sensitive to photoperiod length (Hay, 1990; overcoming the photoperiod-induced decline in growth. A 2-yr experiMarousky et al., 1991, 1992). In addition, sensitivity to ment was established at Ona, FL, in which photoperiod was extended photoperiod can have a large influence on the relative to 15 h throughout the short-daylength months. Four grasses were growth of grasses under natural conditions. Four grasses studied: ‘Pensacola’ bahiagrass, Paspalum notatum Flugge var. Saurde Parodi; ‘Tifton 85’ bermudagrass, Cynodon spp. L. Pers.; ‘Florakirk’ (Pensacola bahiagrass, Tifton 85 bermudagrass, Flobermudagrass; and ‘Florona’ stargrass, Cynodon nlemfuensis Vanderrakirk bermudagrass, and Florona stargrass) grown unyst var. nlemfuensis. Growth increases were observed in all grasses der field conditions had overall increases in forage yield during the short-daylength months as a result of the extended-photoduring the short-daylength months when plots were period treatment, with increases in the January through March hartreated with 15-h photoperiods (Sinclair et al., 2001). vests of 3-fold or more for Pensacola bahiagrass and 1.5to 2.5-fold In the 2-yr study, overall winter forage yield was signififor Tifton 85 bermudagrass. Generally, there was no evidence of cantly increased by the extended photoperiod in all adverse consequences from sustained growth during the short-daycases except for one season for Florona stargrass. Pensalength months either in the subsequent spring and summer growth or in traits measured in below-ground tissue. With one exception, cola bahiagrass and Tifton 85 bermudagrass had yield there was no major influence of the sustained growth on forage nutriincreases as a result of the extended photoperiod of 1.8tive value during the short-daylength months for any of the yearfold and greater. round harvests. Pensacola bahiagrass had decreased crude protein An objective of this paper was to extend the analysis under the extended-photoperiod treatment relative to the naturalof the results of overall yield (Sinclair et al., 2001) to a daylength treatment. Overall, these results indicated that the selection detailed examination of the year-round seasonal variaand genetic incorporation of photoperiod insensitivity into these grasses tion in forage yield in response to an extended-photopecould enhance productivity without adverse consequences. riod treatment. In particular, attention was given to the hypothesis that stimulated growth of these grasses during the cool-season, short-daylength months might reC production in the southeastern USA is limsult in depressed forage production in the subsequent ited to a large extent by low forage production spring and summer. Therefore, data are presented here during short-daylength months. The decrease in forage on year-round forage yield from harvests at 4or 5-wk production can be quite dramatic in these months. For example, in Florida the growth of Paspalum and Cynointervals throughout a 2-yr experiment. In addition, bedon species during October through March was relow-ground plant samples were obtained to examine the ported to account for only 14 and 27% of the annual hypothesis that possible decreases in forage production yield, respectively (Mislevy and Everett, 1981). The dein the spring and summer following growth stimulated crease in growth occurs in spite of the fact that there by extended daylengths might result from decreased appears to be adequate soil moisture, soil fertility, and partitioning of the materials to the below-ground tissues. sufficiently high temperatures to allow substantially Therefore, data are reported on mass, nitrogen congreater yields (Sinclair et al., 1997). The cause of decentration, and carbohydrate concentration of belowcreased grass growth may be a response to the short ground tissue. daylengths during these months. If it can be clearly demA second objective of the study was to determine if there was a change in nutritive value of the forage T. Sinclair and L. Premazzi, USDA-ARS, University of Florida, PO produced by sustained grass growth as a result of exBox 110965, Gainesville, FL 32611-0965 USA; J. Ray, USDA-ARS, tended photoperiod during the short-daylength months. Crop Genetics and Production, P.O. Box 345, Stoneville, MS 38776 Several types of data were collected to examine nutritive USA; P. Mislevy, University of Florida, IFAS, Range Cattle Research value, including leafiness, crude protein (CP) concentraand Education Center, 3401 Experiment Station, Ona, FL 338659706 USA. Mention of a trademark or proprietary product does not tion, and in vitro digestible organic matter (IVDOM) constitute a guarantee or warranty of the product by the U.S. Departconcentration. ment of Agriculture and does not imply approval or the exclusion of other products that may also be suitable. Received 17 Apr. 2002. *Corresponding author (trsincl@mail.ifas.ufl.edu). Abbreviations: CP, crude protein; IVDOM, in vitro digestible organic matter; TNC, total nonstructural carbohydrates. Published in Crop Sci. 43:618–623 (2003).

Genome-Wide Association Study of Ureide Concentration in Diverse Maturity Group IV Soybean [Glycine max (L.) Merr.] Accessions.

Ureides are the N-rich products of N-fixation that are transported from soybean nodules to the shoot. Ureides are known to accumulate in leaves in response to water-deficit stress, and this has been used to identify genotypes with reduced N-fixation sensitivity to drought. Our objectives in this research were to determine shoot ureide concentrations in 374 Maturity Group IV soybean accessions and to identify genomic regions associated with shoot ureide concentration. The accessions were grown at two locations (Columbia, MO, and Stuttgart, AR) in 2 yr (2009 and 2010) and characterized for ureide concentration at beginning flowering to full bloom. Average shoot ureide concentrations across all four environments (two locations and two years) and 374 accessions ranged from 12.4 to 33.1 µmol g−1 and were comparable to previously reported values. SNP–ureide associations within and across the four environments were assessed using 33,957 SNPs with a MAF ≥0.03. In total, 53 putative loci on 18 chromosomes were identified as associated with ureide concentration. Two of the putative loci were located near previously reported QTL associated with ureide concentration and 30 loci were located near genes associated with ureide metabolism. The remaining putative loci were not near chromosomal regions previously associated with shoot ureide concentration and may mark new genes involved in ureide metabolism. Ultimately, confirmation of these putative loci will provide new sources of variation for use in soybean breeding programs.

Genetic Resistance to Soybean Rust in PI567099A is at or Near the Rpp3 Locus

Our objective was to map the soybean rust (SBR) resistance genes(s) in PI 567099A. A population segregating for SBR resistance was evaluated in 2008 and 2009 in Paraguay. In both seasons, F2:3 families were grown in a field naturally infested with SBR. F2:3 families were rated as resistant, segregating, or susceptible on the basis of the lesion type present, and this classification was used to infer the F2-phenotype. Molecular markers flanking five SBR-resistance genes were applied to the F2 population, and markers flanking Rpp3 were significantly associated with the observed resistance (P < 0.0001) in both years. The phenotype of 24-F1 plants evaluated in the 2008 season indicated that the resistance was recessive. This is the first report of recessive resistance at or near the Rpp3 locus. Knowledge of the location and nature of resistance in PI 567099A will allow its more efficient utilization as an SBR-resistance source in breeding programs.

Identification of transcripts translated on free or membrane-bound polyribosomes by differential display

Differential display has been widely and successfully used to identify differentially expressed genes based on physiological treatments or genetic variation. We used differential display to identify genes based on their site of translation. Identification was made based on the fractionation of RNA from soybean leaves into total RNA, free polyribosomal RNA, and membrane-bound (MB) polyribosomal RNA. Sequences were identified representing RNAs uniquely translated on free or MB polyribosomes. The compartmentalization was confirmed on RNA blots. Differential display of free and MB polyribosomal RNA from genetic mutants or physiological studies has 2 potential advantages. First, the sensitivity of the method is increased. Second, localization of mRNAs to the free or MB compartments may identify genes that are controlled at the level of translation or that switch compartments in response to a treatment.

Maturity Effects on Colony-Forming Units of Macrophomina phaseolina Infection as Measured using Near-Isogenic Lines of Soybeans

Charcoal rot (Macrophomina phaseolina) causes significant yield losses in soybean [Glycine max (L.) Merr.]. The actual effect of maturity on disease severity can be confounded by genotypic background. We evaluated disease severity using two sets of near-isogenic lines (NIL) differing in maturity genes. Field experiments were established on two soil types (sandy loam and clay) and evaluated over two years (2008 and 2009). Disease severity, expressed as colony-forming units (CFU), was evaluated for each line at physiological maturity. Within a year, similar levels of disease severity were observed on both soil types. Regression analysis indicated no significant (P < 0.05) relationship between maturity and CFU for either set of NIL on either soil type in either year. Adding selected environmental variables (rainfall, temperature, etc.) to the equation allowed the detection of a significant relationship in only one environment (P = 0.0306, F = 6.00). Results demonstrated little evidence of a relationship between maturity and disease severity.

A soybean mapping population specific to the early soybean production system

The objective of this research was to create a soybean [Glycine max (L.) Merr] genetic resource in the form of a publicly available, well-characterized mapping population specific to maturity groups (MG) used in the early soybean production system. A total of 568 simple sequence repeat (SSR) markers were tested for polymorphism between soybean breeding line DS97-84-1 (MG IV) and germplasm line DT97-4290 (MG IV). A 90-genotype subset of an F2 population from a cross between these lines was evaluated for genetic linkage using 162 polymorphic SSRs, plant height, pod color (L2/l2), flower color (W1/w1) and stem termination (Dt1/dt1). A 1514 cM (Kosambi) genetic map covering 65% of the soybean genome based on 157 linked SSR markers was created. Comparison with the composite soybean genetic map was used to verify map order. Loci for pod color, flower color and stem termination fell in the expected position on the map indicating this is a normally segregating mapping population. Loci for height were identified on linkage groups C2, D1a, D1b, H, L, M and O. MG IV and V soybean genotypes are critical for the early soybean production system widely used in the midsouthern US. However, only two mapping populations have been reported in Soybase for MG IV and V genotypes. Additionally, the parents used in this cross are known to differ in their response to soybean cyst nematode and charcoal rot, which constitute two major pathology threats to Midsouth soybean production. The population and map reported herein represent an important genetic resource for the early soybean production system.

Association Mapping of Total Carotenoids in Diverse Soybean Genotypes Based on Leaf Extracts and High-Throughput Canopy Spectral Reflectance Measurements

Carotenoids are organic pigments that are produced predominantly by photosynthetic organisms and provide antioxidant activity to a wide variety of plants, animals, bacteria, and fungi. The carotenoid biosynthetic pathway is highly conserved in plants and occurs mostly in chromoplasts and chloroplasts. Leaf carotenoids play important photoprotective roles and targeted selection for leaf carotenoids may offer avenues to improve abiotic stress tolerance. A collection of 332 soybean [Glycine max (L.) Merr.] genotypes was grown in two years and total leaf carotenoid content was determined using three different methods. The first method was based on extraction and spectrophotometric determination of carotenoid content (eCaro) in leaf tissue, whereas the other two methods were derived from high-throughput canopy spectral reflectance measurements using wavelet transformed reflectance spectra (tCaro) and a spectral reflectance index (iCaro). An association mapping approach was employed using 31,253 single nucleotide polymorphisms (SNPs) to identify SNPs associated with total carotenoid content using a mixed linear model based on data from two growing seasons. A total of 28 SNPs showed a significant association with total carotenoid content in at least one of the three approaches. These 28 SNPs likely tagged 14 putative loci for carotenoid content. Six putative loci were identified using eCaro, five loci with tCaro, and nine loci with iCaro. Three of these putative loci were detected by all three carotenoid determination methods. All but four putative loci were located near a known carotenoid-related gene. These results showed that carotenoid markers can be identified in soybean using extract-based as well as by high-throughput canopy spectral reflectance-based approaches, demonstrating the utility of field-based canopy spectral reflectance phenotypes for association mapping.