Effects of high oleic full-fat soybean meal on broiler live performance, carcass and parts yield, and fatty acid composition of breast fillets.

The effects of high oleic oil full-fat (HO-FF) soybean meal (SBM) on broiler meat quality could lead to value-added food products. This experiment evaluated the effects of dietary normal oleic extruded expelled (NO-EE), normal oleic full-fat (NO-FF), or HO-FF SBM on live performance, carcass and parts yield, and breast fatty acid composition. Diets were formulated to be isoenergetic and isonitrogenous. A total of 540 Ross-708 male broilers were raised on floor pens with 18 broilers/pen and 10 replicates/treatment. Data were analyzed in a completely randomized design. Chickens were fed with a starter (0-14 d), grower (15-35 d), or a finisher diet (36-47 d) up to 47 d. Chickens were weighed at 7, 14, 35, and 47 d. At 48 d, 4 broilers per pen were processed. Breast samples were collected and evaluated for quality and fatty acid content. Broilers fed diets with NO-EE were heavier (P < 0.05) than chickens fed diets with full-fat SBM (NO-FF and HO-FF) at d 7, 14, 35 while feed conversion ratio (FCR) of NO-EE was best (P < 0.05) at 7 and 47 d. Carcass yield was also higher for broilers fed NO-EE than the other treatments. Diet did not affect parts yield, breast meat color, cooking, drip loss, white stripping, or SM quality parameters. More breast fillets without wooden breast (score 1) were observed (P < 0.05) for NO-FF than the other 2 treatments. The breast meat fatty acid profile (g fatty acid/100 g of all fatty acids) was significantly affected (P < 0.001) by diet. Broilers fed the HO-FF SBM diet had 54 to 86% more oleic acid, 72.5% to 2.2 times less linoleic acid, and reduced stearic and palmitic acid levels in the breast meat than NO-FF and NO-EE. In conclusion, feeding HO-FF to broilers enriched the oleic acid content of their breast meat while reducing the saturated fatty acid content relative to the NO-FF and NO-EE treatment groups.

Standardized ileal amino acid digestibility of high-oleic full-fat soybean meal in broilers.

High-oleic (HO) soybean may serve as a value-added feed ingredient to enrich poultry meat due to its fatty acid content. However, the amino acid (AA) nutrient digestibility of soybean meal (SBM) made from these soybeans has yet to be determined. The objective of this study was to determine apparent ileal AA digestibility (AID) and standardized ileal AA digestibility (SID) of high-oleic full-fat (HO-FF) SBM compared to normal oleic full-fat (NO-FF), normal oleic extruded expeller (NO-EE), and solvent-extracted SBM (SE-SBM) in broilers. A nitrogen-free basal diet (NFD) was fed to 1 treatment group with 10 chicks/cage to determine basal endogenous losses (BEL). Titanium dioxide was used as an inert marker. The test diets contained 57.5% of the basal NFD and 42.5% of 1 of the 4 soybean sources. A total of 272 Ross-708 male broilers were placed in 40 battery cages with 5 treatments and 8 replicates per treatment. A common starter diet was provided to all the chickens for 14 d. Experimental diets were provided as a mash for 9 d before sample collection. Chickens were euthanized with CO2 on d 23, and contents of the distal ileum were collected, frozen, and freeze-dried. The BEL were similar to the values found in the literature. At d 23, broilers fed the SE-SBM had the highest body weight gain and best FCR compared to chickens fed the HO-FF and NO-FF treatments (P < 0.001). Broilers fed the SE-SBM and NO-EE experimental diets had (P < 0.001) higher apparent ileal AA digestibility and AA SID than broilers fed the HO-FF and NO-FF treatments. In conclusion, the SID of AA from HO-FF is similar to the digestibilities of other full-fat soybeans found in the literature and is lower than that of NO-EE and SE-SBM.

Genotype imputation for soybean nested association mapping population to improve precision of QTL detection.

KEY MESSAGE: Software for high imputation accuracy in soybean was identified. Imputed dataset could significantly reduce the interval of genomic regions controlling traits, thus greatly improve the efficiency of candidate gene identification. Genotype imputation is a strategy to increase marker density of existing datasets without additional genotyping. We compared imputation performance of software BEAGLE 5.0, IMPUTE 5 and AlphaPlantImpute and tested software parameters that may help to improve imputation accuracy in soybean populations. Several factors including marker density, extent of linkage disequilibrium (LD), minor allele frequency (MAF), etc., were examined for their effects on imputation accuracy across different software. Our results showed that AlphaPlantImpute had a higher imputation accuracy than BEAGLE 5.0 or IMPUTE 5 tested in each soybean family, especially if the study progeny were genotyped with an extremely low number of markers. LD extent, MAF and reference panel size were positively correlated with imputation accuracy, a minimum number of 50 markers per chromosome and MAF of SNPs > 0.2 in soybean line were required to avoid a significant loss of imputation accuracy. Using the software, we imputed 5176 soybean lines in the soybean nested mapping population (NAM) with high-density markers of the 40 parents. The dataset containing 423,419 markers for 5176 lines and 40 parents was deposited at the Soybase. The imputed NAM dataset was further examined for the improvement of mapping quantitative trait loci (QTL) controlling soybean seed protein content. Most of the QTL identified were at identical or at similar position based on initial and imputed datasets; however, QTL intervals were greatly narrowed. The resulting genotypic dataset of NAM population will facilitate QTL mapping of traits and downstream applications. The information will also help to improve genotyping imputation accuracy in self-pollinated crops.

Identification of SNP markers associated with soybean fatty acids contents by genome-wide association analyses.

Composition of fatty acids (FAs) in soybean seed is important for the quality and uses of soybean oil. Using gas chromatography, we have measured soybean FAs profiles of 621 soybean accessions (maturity groups I through IV) grown in five different environments; Columbus, OH (2015), Wooster, OH (2014 and 2015), Plymouth, NC (2015), and Urbana, IL (2015). Using publicly available SoySNP50K genotypic data and the FA profiles from this study, a genome-wide association analysis was completed with a compressed mixed linear model to identify 43 genomic regions significantly associated with a fatty acid at a genome wide significance threshold of 5%. Among these regions, one and three novel genomic regions associated with palmitic acid and stearic acid, respectively, were identified across all five environments. Additionally, nine novel environment-specific FA-related genomic regions were discovered providing new insights into the genetics of soybean FAs. Previously reported FA-related loci, such as FATB1a, SACPD-C, and KASIII, were also confirmed in this study. Our results will be useful for future functional studies and marker-assisted breeding for soybean FAs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01216-1.

RNA-Seq study reveals genetic responses of diverse wild soybean accessions to increased ozone levels.

BACKGROUND: Ozone is an air pollutant widely known to cause a decrease in productivity in many plant species, including soybean (Glycine max (L.) Merr). While the response of cultivated soybean to ozone has been studied, very little information is available regarding the ozone response of its wild relatives. RESULTS: Ozone-resistant wild soybean accessions were identified by measuring the response of a genetically diverse group of 66 wild soybean (Glycine soja Zucc. and Sieb.) accessions to elevated ozone levels. RNA-Seq analyses were performed on leaves of different ages from selected ozone-sensitive and ozone-resistant accessions that were subjected to treatment with an environmentally relevant level of ozone. Many more genes responded to elevated ozone in the two ozone-sensitive accessions than in the ozone-resistant accessions. Analyses of the ozone response genes indicated that leaves of different ages responded differently to ozone. Older leaves displayed a consistent reduction in expression of genes involved in photosynthesis in response to ozone, while changes in expression of defense genes dominated younger leaf tissue in response to ozone. As expected, there is a substantial difference between the response of ozone-sensitive and ozone-resistant accessions. Genes associated with photosystem 2 were substantially reduced in expression in response to ozone in the ozone-resistant accessions. A decrease in peptidase inhibitors was one of several responses specific to one of the ozone resistant accessions. CONCLUSION: The decrease in expression in genes associated with photosynthesis confirms that the photosynthetic apparatus may be an early casualty in response to moderate levels of ozone. A compromise of photosynthesis would substantially impact plant growth and seed production. However, the resistant accessions may preserve their photosynthetic apparatus in response to the ozone levels used in this study. Older leaf tissue of the ozone-resistant accessions showed a unique down-regulation of genes associated with endopeptidase inhibitor activity. This study demonstrates the existence of significant diversity in wild soybean for ozone response. Wild soybean accessions characterized in this study can be used by soybean breeders to enhance ozone tolerance of this important food crop.

Changes in gene expression between a soybean F1 hybrid and its parents are associated with agronomically valuable traits.

Soybean [Glycine max (L.) Merr.] genetic diversity is limited because domesticated soybean has undergone multiple genetic bottlenecks. Its progenitor, the wild soybean [Glycine soja Siebold & Zucc], has not undergone the same intense selection and is much more genetically diverse than domesticated soybean. However, the agronomic importance of diversity in wild soybean is unclear, and its weedy nature makes assessment difficult. To address this issue, we chose for study a highly selected, adapted F4-derived progeny of wild soybean, NMS4-44-329. This breeding line is derived from the hybridization between G. max cultivar N7103 and G. soja PI 366122. Agronomic comparisons were made among N7103, NMS4-44-329 and their F1 and F2 progeny in replicated yield trials at two North Carolina locations. Significant F1 mid-parent heterosis was observed at each location for seed yield (189 and 223 kgha-1, P<0.05 and P<0.10, respectively), seed protein content (1.1g/100g, P<0.01) and protein production per hectare (101 and 100 kgha-1, P<0.01 and P<0.06, respectively). Increased yield, seed protein content and protein production per hectare in the hybrids suggested that wild soybean has the potential to improve agronomic traits in applied breeding. Comparisons of differentially-expressed genes in the hybrid vs. parents identified genes associated with N metabolism. Non-additive changes in gene expression in the hybrids relative to the parents could reasonably explain the improved protein levels in the F1 hybrids. Changes in gene expression were influenced by environmental effects; however, allele specific bias in the hybrids were well correlated between environments. We propose that changes in gene expression, both additive and non-additive, and changes in allele specific expression bias may explain agronomic traits, and be valuable tools for plant breeders in the assessment of breeding populations.

Comparisons of the Effects of Elevated Vapor Pressure Deficit on Gene Expression in Leaves among Two Fast-Wilting and a Slow-Wilting Soybean.

Limiting the transpiration rate (TR) of a plant under high vapor pressure deficit (VPD) has the potential to improve crop yield under drought conditions. The effects of elevated VPD on the expression of genes in the leaves of three soybean accessions, Plant Introduction (PI) 416937, PI 471938 and Hutcheson (PI 518664) were investigated because these accessions have contrasting responses to VPD changes. Hutcheson, a fast-wilting soybean, and PI 471938, a slow-wilting soybean, respond to increased VPD with a linear increase in TR. TR of the slow-wilting PI 416937 is limited when VPD increases to greater than about 2 kPa. The objective of this study was to identify the response of the transcriptome of these accessions to elevated VPD under well-watered conditions and identify responses that are unique to the slow-wilting accessions. Gene expression analysis in leaves of genotypes PI 471938 and Hutcheson showed that 22 and 1 genes, respectively, were differentially expressed under high VPD. In contrast, there were 944 genes differentially expressed in PI 416937 with the same increase in VPD. The increased alteration of the transcriptome of PI 416937 in response to elevated VPD clearly distinguished it from the other slow-wilting PI 471938 and the fast-wilting Hutcheson. The inventory and analysis of differentially expressed genes in PI 416937 in response to VPD is a foundation for further investigation to extend the current understanding of plant hydraulic conductivity in drought environments.

Leaf expansion of soybean subjected to high and low atmospheric vapour pressure deficits.

Vapour pressure deficit (VPD) is considered an important environmental factor that might affect leaf expansion and transpiration rate (TR) in plants. Two slow-wilting soybean (Glycine max (L.) Merr.) genotypes PI 416937 and PI 471938 along with commercial cultivar Hutcheson were subjected to low (1.2-1.6 kPa) and high VPD (2.8-3 kPa) environments to study their leaf expansion and TR over five days. Among the three genotypes, PI 416937 had the lowest increase in its TR (34%) at high VPD compared with low VPD and the greatest decrease in leaf area (31%). In contrast, Hutcheson had the highest increase in TR (87%) under high VPD and the lowest decrease in leaf expansion rate (18%). Expansin and extensin genes were isolated in PI 416937 to determine if changes in leaf expansion were associated with changes at the molecular level. The four studied genes were all suppressed after five days in the high VPD environment.

Identification of epitopes of the ß subunit of soybean ß-conglycinin that are antigenic in pigs, dogs, rabbits and fish.

BACKGROUND: ß-Conglycinin (conglycinin) is one of the major seed storage proteins of soybean. Conglycinin is a 7S trimer composed of different combinations of ß, α and α' subunits. All subunits of conglycinin have been reported to be allergenic in humans. The goal of this research is to identify epitopes of the ß subunit of conglycinin that are antigenic in multiple animal species. RESULTS: Sera from pigs, dogs, rabbits and hybrid striped bass that had antibodies against soybean conglycinin were identified by ELISA. Most of these sera recognized peptides that represent the ß subunit of conglycinin. One antigenic region of the ß subunit of conglycinin had considerable overlap among all species tested. One region that was similar to a peanut allergenic epitope in humans overlapped with a region that binds IgE from dogs. One region was antigenic in multiple rabbits and pigs, suggesting it may play a role in the response of pigs to soybean in the diet. CONCLUSION: One region of the ß subunit of conglycinin is an important antigen across species and abuts a region similar to the peanut allergen ARA h 1. A second region is particularly antigenic in pigs and rabbits. Variants of these antigenic regions of the ß subunit of conglycinin may be useful in determining the role these regions play in the health of animals fed soybean. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Epitopes from two soybean glycinin subunits are antigenic in pigs.

BACKGROUND: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of basic and acidic portions joined by disulfide bridges. There are five glycinin subunit isoforms designated Gy1-Gy5. The purpose of this study is to identify epitopes from selected glycinin subunits that are antigenic in pigs. RESULTS: Twenty-seven out of 30 pigs had antibodies against glycinin in their sera. Ten of these sera had immunoglobulin G (IgG) against the Gy4 (A5A4B3) or Gy1 (A1aBx) subunit. Three sera recognised overlapping regions between the two subunits tested, though no serum stained both A5A4B3 and A1aBx. Two sera stained a highly conserved region between A5A4B3 and A1aBx, though again neither serum stained both peptides. The basic part of the A1aBx subunit was not recognised by any of the sera tested even though immunoblot data indicated that the basic and acidic subunits of glycinin are nearly equally antigenic. CONCLUSION: Two antigenic regions of A5A4B3 and A1aBx were identified that bound antibodies in half of the sera that reacted with these two proteins. Half of the sera reacted with unique regions of A5A4B3 and A1aBx. The failure of the basic portion of A1aBx to bind pig antibodies may indicate that it is less antigenic than the basic portion of A5A4B3 and other glycinin subunits.

Characterization of an ADP-glucose pyrophosphorylase small subunit gene expressed in developing cotton (Gossypium hirsutum) fibers.

ADP-glucose pyrophosphorylase (ADPGp, EC 2.7.7.27) is a tetrameric protein composed of two small and two large subunits that catalyzes the biosynthesis of ADP-glucose from glucose-phosphate which is used to provide the glucose subunits for starch biosynthesis. A second cotton gene encoding an ADPGp small subunit has been cloned and characterized. The gene contains eight introns similar to previously reported potato and cotton ADPGp small subunit genes. The deduced translation of the gene contained a poorly conserved transit peptide and well conserved catalytic and regulatory elements typical of other plant ADPGps. The 5' end of the mRNA was cloned and sequenced to identify the transcriptional start site (TSS). The promoter region upstream of the TSS did not contain the core promoter sequence in the typical positions indicating this gene may not use a standard core promoter. Other sequence motifs associated with tissue specific expression and phytohormone response were present. Reverse transcription (RT)-PCR with gene specific primers identified the sites of expression of this gene. Expression was most abundant in the meristem region, and immature stem and relatively lower in starch accumulating roots demonstrating that this gene has a different pattern of expression than the previously reported cotton ADPGp small subunit gene. Additionally this gene was differentially expressed in cotton fibers. The presence of starch was confirmed in developing cotton fibers suggesting that starch metabolism plays a role in cotton fiber development.

Characterization of two cotton (Gossypium hirsutum L) invertase genes.

Two cotton vacuolar-invertase genes were identified and sequenced. Both genes had seven exons, including an unusually small second exon typical of acid invertases. These genes encode peptides with many features shared by acid invertases from other species including, leader sequences that probably target the peptide to the vacuole, active site motifs and substrate binding motifs. Expression analyses indicated that one of the genes was expressed in roots during the starch filling stage of development. However, expression of the same gene fluctuated during the starch utilization stage of development. Therefore this gene was unlikely to play a role in determining sink strength of this tissue. Both genes were expressed in elongating fibers where they were likely to play a role in cell expansion. The invertase gene uniquely expressed in fiber had a simple sequence repeat (SSR) in the third intron that was polymorphic among various cotton species. An EST was identified with an expansion of the SSR that included the third intron indicating this SSR is associated with a splice variant. The polymorphic SSR may be useful in investigating the function of this gene in fiber development.

Isolation and characterization of a cotton cdh-like gene.

Cotton fiber cells elongate without dividing to form economically valuable spinnable fiber. Reports of the ploidy level of fiber cells are variable. Early reports indicated an increase in nuclear DNA content in young fibers; however, subsequent reports failed to observe such a significant increase in ploidy level. Evaluation and analysis of genes involved in regulation of DNA synthesis and other aspects of cell cycle regulation identified relevant genes that were present in fiber cells though usually at low levels. We report the isolation and characterization of another gene likely to be involved in cell cycle/DNA synthesis control. This gene was similar to a gene from Medicago species that controls entry into anaphase by regulating the activity of the anaphase promoting complex ability to ubiquinate selected proteins. The cotton gene was composed of nine exons and the deduced translational sequences have motifs similar to a Medicago gene expressed in highly polyploid cells. Based on this similarity the cotton gene was designated Ghcdh. Ghcdh is highly expressed in meristems and leaves but is present at much lower levels in fiber cells. These data are consistent with the lower levels of polyploidy reported for cotton fiber. A simple sequence repeat was identified in the gene that may be exploited as a marker to map this gene and associate it with important traits in cotton.

Expression of genes associated with carbohydrate metabolism in cotton stems and roots.

BACKGROUND: Cotton (Gossypium hirsutum L) is an important crop worldwide that provides fiber for the textile industry. Cotton is a perennial plant that stores starch in stems and roots to provide carbohydrates for growth in subsequent seasons. Domesticated cotton makes these reserves available to developing seeds which impacts seed yield. The goals of these analyses were to identify genes and physiological pathways that establish cotton stems and roots as physiological sinks and investigate the role these pathways play in cotton development during seed set. RESULTS: Analysis of field-grown cotton plants indicated that starch levels peaked about the time of first anthesis and then declined similar to reports in greenhouse-grown cotton plants. Starch accumulated along the length of the stem and the shape and size of the starch grains from stems were easily distinguished from transient starch. Microarray analyses compared gene expression in tissues containing low levels of starch with tissues rapidly accumulating starch. Statistical analysis of differentially expressed genes indicated increased expression among genes associated with starch synthesis, starch degradation, hexose metabolism, raffinose synthesis and trehalose synthesis. The anticipated changes in these sugars were largely confirmed by measuring soluble sugars in selected tissues. CONCLUSION: In domesticated cotton starch stored prior to flowering was available to support seed production. Starch accumulation observed in young field-grown plants was not observed in greenhouse grown plants. A suite of genes associated with starch biosynthesis was identified. The pathway for starch utilization after flowering was associated with an increase in expression of a glucan water dikinase gene as has been implicated in utilization of transient starch. Changes in raffinose levels and levels of expression of genes controlling trehalose and raffinose biosynthesis were also observed in vegetative cotton tissues as plants age.

Cotton benzoquinone reductase: up-regulation during early fiber development and heterologous expression and characterization in Pichia pastoris.

Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but not in the fiberless line SL 1-7-1. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify both full length coding regions of 609bp and once cloned, the restriction enzyme HindIII was used to distinguish the clones encoding the BR1 (one site) and BR2 (two sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D-PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the alpha-factor secretion sequence in pPICZalphaA vector and expressed with Pichia pastoris. Both BR1 and BR2 were approximately 26.5kDa and did enzymatically reduce 2,6-dimethoxybenzoquinone similar to the fungal BR.

Analysis of gene expression in cotton fiber initials.

BACKGROUND: Cotton (Gossypium hirsutum L.) fibers are trichomes that initiate from the ovule epidermis. Little is known about the developmental pathway causing fiber to differentiate from ovular epidermal cells even though limits on the number of cells that differentiate into fiber will limit yield. RESULTS: A method was developed to isolate RNA from fiber initials 1 day post anthesis (dpa). Complementary DNA libraries representing 1 dpa fibers and other cotton tissues were sequenced and analyzed. Assembly of G. hirsutum Expressed Sequenced Tags (ESTs) identified over 11,000 sequences not previously represented in GenBank. New genes identified among these ESTs were represented on microarrays. The microarrays were used to identify genes enriched in fiber initials (1 dpa fibers) and elongating fibers. Analyses of Gene Ontologies (GO) of differentially expressed genes determined that terms associated with the "membranes" were statistically over represented among genes increased in expression in fiber initials and 10 dpa fibers. Staining ovules with a fluorescent dye confirmed an increase in Endoplasmic Reticulum (ER) occurred in fiber initials on the day of anthesis, persisted through 3 dpa and was absent in a fiberless mutant. Two genes similar to the CAPRICE/TRIPTYCHON (CPC) gene that inhibits differentiation of leaf trichomes in Arabidopsis were also characterized. Genes associated with novel regulation of brassinosterols, GTP mediated signal transduction and cell cycle control and components of a Ca+2 mediated signaling pathway were identified. Staining of cellular Ca+2 indicated that fiber initials had more Ca+2 than other ovule cells supporting a role for Ca+2 in fiber development. CONCLUSION: Analysis of genes expressed in fiber initials identified a unique stage in fiber development characterized by an increase in ER and Ca+2 levels that occurred between 0 and 1 dpa. The gene similar to CPC has a MYB domain but appears to lack a transcription activating domain similar to the Arabisopsis gene. The method used to stain the ER also can be used to count fiber initials and showed fiber cells develop from adjacent cells unlike leaf trichomes.

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.

A global assembly of cotton ESTs.

Approximately 185,000 Gossypium EST sequences comprising >94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including drought stress and pathogen challenges. These libraries were derived from allopolyploid cotton (Gossypium hirsutum; A(T) and D(T) genomes) as well as its two diploid progenitors, Gossypium arboreum (A genome) and Gossypium raimondii (D genome). ESTs were assembled using the Program for Assembling and Viewing ESTs (PAVE), resulting in 22,030 contigs and 29,077 singletons (51,107 unigenes). Further comparisons among the singletons and contigs led to recognition of 33,665 exemplar sequences that represent a nonredundant set of putative Gossypium genes containing partial or full-length coding regions and usually one or two UTRs. The assembly, along with their UniProt BLASTX hits, GO annotation, and Pfam analysis results, are freely accessible as a public resource for cotton genomics. Because ESTs from diploid and allotetraploid Gossypium were combined in a single assembly, we were in many cases able to bioinformatically distinguish duplicated genes in allotetraploid cotton and assign them to either the A or D genome. The assembly and associated information provide a framework for future investigation of cotton functional and evolutionary genomics.

Characterization and expression of a putative retinoblastoma protein binding gene from Gossypium hirsutum.

A genomic clone representing a putative retinoblastoma binding (RBB) protein was isolated from a Gossypium hirsutum BAC library. Alignment of the gene sequence with the cDNA sequence indicated the gene consists of six exons that have standard eukaryotic splice junctions. The conceptual spliced transcript was 98% identical to TC37171 in the TIGR gene index, however it encoded an ORF 107 amino acids longer than best deduced protein from TC37171. The conceptual translation of the genomic clone was 56% identical to a tomato gene experimentally demonstrated to be a RBB protein and able to complement the yeast growth mutant IRA. The mRNA encoded by the genomic clone was abundantly expressed in meristems and expression levels increased as the cotton fiber matured. We propose that this gene may regulate growth and/or cell division in cotton based on homology of the clone with a protein of known function and sites of expression.

Incorporation of rhodamine B into male tobacco budworm moths Heliothis virescens to use as a marker for mating studies.

Rhodamine B, a dye commonly used in a variety of biological studies was incorporated into the bodies of male tobacco budworm moths, Heliothis virescens (Lepidoptera: Noctuidae), by allowing them to feed freely on 0.1% rhodamine dissolved in a 10% sucrose solution. After exposing males for one to three days to this pigment, rhodamine was clearly detectable in >82% of spermatophores extracted from untreated females. The intake of this dye did not affect the life span, the production of eggs or the capacity of moths to copulate when compared with moths fed only a sucrose solution or water. Rhodamine B was easily identifiable externally but was more apparent internally in males after only one day of exposure to the pigment. Even at this short feeding duration, rhodamine was detectable in >50% of males 5 days after feeding stopped. Longer exposure to the dye significantly increased the percentage stained. Detection of rhodamine was slightly enhanced by the use of ultraviolet light. The dye accumulation in internal abdominal organs was a better indicator of the presence of the pigment than external contamination of the moth. The use of the method described in this report can be a tool for the rapid incorporation of a low cost dye in the tobacco budworm for biological, behavioral and genetic studies.