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.

Identification of new sources of reniform nematode (Rotylenchulus reniformis Linford and Oliveira) resistance in cotton is critical to expand-ing host plant resistance to manage this important pathogen. Phenotyping plants in early breeding generations without destructive sampling would be useful for introgression of nematode resistance from exotic germplasm resources; therefore, a rapid, nondestructive method was developed to assess host plant resistance to the reniform nematode based on the number of females infecting the roots. In one set of experiments, the root system was cut off at 0, 1, 2.5, or 5 cm below the soil line and used to assess the number of females infecting this portion of the root system. Resistance could be accurately determined while leaving up to 5 cm of roots with the shoot. In a second set of experiments, the rate of plant recovery and reproductive development was evaluated using a combination of root retention (0, 1, 2.5 cm, or all root) and shoot retention (leaves at top two nodes, leaves at bottom two nodes, no leaves, all leaves) treatments. Plants more rapidly recovered using a treatment combination in which the top leaves and 2.5 cm roots were kept. This combination per-formed similarly to plants with neither shoots nor roots modified and was harvested 20 days sooner compared to some other treatment combinations.

The cotton species Gossypium arboreum L. is self-pollinated, and most accessions in the United States Department Agriculture, National Plant Germplasm System, produce flowers with red pigmented petal spots, which represents the primitive phenotype and more attractive to pollinators. Few accessions have the ghost spot phenotype, and genetic analysis of this trait was conducted for PI 615742. Two F-2 populations were developed by crossing PI 615742 with G. arboreum accessions PI 452097, having red petal spots, and with PI 529708, showing the spotless phenotype. The PI 615742 x PI 529708 population data indicate a single completely dominant gene which confers the ghost spot phenotype. Petal spot phenotypes observed for the PI 615742 x PI 452097 population indicate red pigmented spots are also conferred by a single completely dominant gene. These results support a two-gene model in which the presence of petal spots and pigment development are controlled by independent genes that co-segregate with stem pigmentation. The accessions and population data can aid in characterizing the mechanisms controlling floral traits and pigment expression.

Flower corolla colour is an important trait for the attraction of pollinators and for the horticultural industry. Gossypium arboreum (L.) accessions from the United States Department of Agriculture germplasm collection frequently show flowers with a yellow coloured corolla. Accession PI 529731 is unique in that the flowers have a red coloured corolla. Genetic characterization of corolla pigmentation was conducted by crossing PI 529731 with two white flower accessions. Flowers with a red corolla were observed in the F-1 generations suggesting a dominant trait. Variation in corolla colour was observed for plants in the F-2 populations including dark red, red, light red, white, yellow and white with petals having red coloured margins. These data support a single dominant gene conferring the four red corolla phenotypes. The yellow corolla phenotype also supported a single dominant gene model. Dominant alleles at both loci are required for expression of the PI 529731 phenotype and data support a two gene model with a 9:3:3:1 segregation ratio. These data are useful for the characterization of genetic mechanisms controlling tissue-specific pigmentation.

Cotton (Gossypium spp.) is a major source of fibre for the textile industry and naturally coloured cotton fibre does not require the use of synthetic dyes making it an eco-friendly option for textile production. Coloured cotton cultivars are however associated with lower fibre yields and reduced lint quality. A greater understanding of the genetic mechanisms underlying fibre colouration will aid in the development of improved cultivars. The brown-fibred Gossypium arboreum L. accession PI 615733 was identified from the United States Department of Agriculture germplasm collection and crossed with three white-fibred accessions PI 529712, PI 529729 and PI 615740 to develop F-2 populations for genetic evaluation. Population data indicated a single dominant gene conferred the brown fibre phenotype. Variation in the intensity of fibre colouration was observed within populations. These data failed to support an incomplete dominance model suggesting genetic modifiers may also play a role in fibre colouration. Additionally, chimeras were frequently observed in the PI 615733 x PI 615740 population. These data will be useful to further characterize the brown fibre trait.

Cotton fibre can show a range in brown colouration, and these coloured lint fibres can be used for textile production reducing the environmental and human health hazards associated with synthetic dyes; however, coloured cotton cultivars have lower yield potential and reduced lint quality. A greater understanding of the genetics of lint colouration would be useful for the development of improved cultivars. Gossypium arboreum L. accession PI 408765 (cv. 'Sanguineum-1') having the brown lint phenotype was identified from the United States Department Agriculture, National Plant Germplasm System cotton collection and crossed with G. arboreum accessions PI 529729 (cv. 'Garo Hill') having white lint and with PI 615733 (cv. 'Zi-Hua Guang Zi') having brown lint. Lint colour data from the F-2 populations support a single incomplete dominant gene model for the brown lint phenotype. Additionally, the brown lint phenotypes for PI 408765 and PI 615733 are controlled by two independent genes. Plants in these populations showed a range of brown lint colouration, and these data would be useful to further characterize the mechanisms controlling brown lint development.

The occurrence of floral petal spots is common among flowering plants and plays a major role in attracting pollinators. Cotton genotypes having large red petal spots are frequent in the United States Department of Agriculture, National Plant Germplasm System Gossypium arboreum (L.) collection. One accession, PI 408798, showed a unique faint red petal spot phenotype. To genetically characterise this trait, a F-2 population of 226 plants was developed by crossing PI 408798 with G. arboreum accession PI 529714 that lacked pigmented petal spots. From the population, 161 plants showed the presence of faint red petal spots; whereas, 65 plants lacked pigmented petal spots. These data supported the single dominant gene model for the presence of floral petal spots. Accession PI 408798 will provide an important genetic resource to characterise the genes involved in the pathway controlling floral pigmentation.

Lint and fuzz are the two types of fiber that develop on cotton seeds. Lint is an economically important fiber used in the textile industry. Fuzz fibers remain attached to seeds, and reducing the amount of fuzz could improve ginning efficiency. The Gossypium arboreum accession PI 615733 from the United States Department of Agriculture cotton germplasm collection showed the fuzzless trait, where no fuzz fibers were observed on seeds. Accession PI 615733 was crossed with the fuzzy seeded G. arboreum accession PI 529729 to develop an F2 population. Seeds from F1 plants showed no fuzz fiber. For the F2 population of 409 plants, 239 plants were classified as fuzzless and 170 were classified as fuzzy. These data support a two-gene model with incomplete dominance. A greater understanding of the genetic mechanisms controlling fiber development on cotton seeds could contribute to breeding efforts to improve lint fiber yields and quality.

Flower corolla colour is an important trait for the attraction of pollinators and for the horticultural industry. (L.) accessions from the United States Department of Agriculture germplasm collection frequently show flowers with a yellow coloured corolla. Accession PI 529731 is unique in that the flowers have a red coloured corolla. Genetic characterization of corolla pigmentation was conducted by crossing PI 529731 with two white flower accessions. Flowers with a red corolla were observed in the F generations suggesting a dominant trait. Variation in corolla colour was observed for plants in the F populations including dark red, red, light red, white, yellow and white with petals having red coloured margins. These data support a single dominant gene conferring the four red corolla phenotypes. The yellow corolla phenotype also supported a single dominant gene model. Dominant alleles at both loci are required for expression of the PI 529731 phenotype and data support a two gene model with a 9:3:3:1 segregation ratio. These data are useful for the characterization of genetic mechanisms controlling tissue-specific pigmentation.

Breeding for reniform nematode (Rotylenchulus reniformis) resistance is hindered by the lack of resistance in upland cotton (Gossypium hirsutum) cultivars. Resistance has been frequently identified in accessions from the Gossypium arboreum germplasm collection with accession PI 529740 rated as highly resistant. Accession PI 529740 was crossed with the susceptible G. arboreum accession PI 529729 to develop an F-2 population for genetic characterization. The population showed quantitative variation suggesting multiple genes conferred the resistant phenotype. Thirteen of the 216 F-2 plants showed resistance similar to the resistant parent and these data supported a two recessive gene model. Sixty plants were classified as resistant or moderately resistant, indicating a single recessive gene conferred the moderately resistant phenotype. The classification of 24 F-2:3 families for nematode resistance generally supported the classification of the corresponding F-2 plants; however, most families were highly variable for infection with no families rated as resistant. This information will aid in the introgression of resistance into upland cotton as larger populations will be required to successfully recover resistance conferred by multiple recessive genes.

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.

Rotylenchulus reniformis is an important root pathogen of cotton in the south-eastern United States, and management is hindered by the lack of host-plant resistance in upland cotton (Gossypium hirsutum). The G.arboreum accession PI 417895 is highly resistant to R.reniformis, and a segregating population of 300F(2) plants was developed for phenotypic characterization of resistance. The population showed quantitative variation for nematode infection. Twenty plants showed no infection and were classified as escapes. Fifty-four plants were classified as resistant or moderately resistant, whereas, 226 were classified as moderately susceptible or susceptible based on the nematode response of the susceptible parent, indicating resistance is a recessive trait, but these data did not support the single recessive gene model. Alternatively, this model would be supported if the 77 plants with a similar nematode response as observed for PI 417895 were classified as resistant. Twelve plants showed high levels of resistance and these data would support a two recessive gene model. Accession PI 417895 represents a new source of R.reniformis resistance with two major genes conferring resistance. Introgression of multiple resistance genes into G.hirsutum will require the development of larger populations to recover the resistant phenotype.

Cotton breeders have focused mainly on selecting for high yield and early maturity under the impact of the boll weevil (Anthonomus grandis Boh.) in the last century. Selection for high fiber quality was once a less important objective in cotton breeding. With the transition of the U.S. cotton industry from a domestic consumer to a major exporter of raw fibers into the global market and the technology advancements in the textile industry since the 1990s, the need for high fiber quality in cotton cultivars has increased. In recent years, genetic improvement in cultivars for insect resistance, disease resistance, and abiotic stress tolerance has become important for maintaining cotton yield. Under strong competition from other major crops, increasing profit in cotton production has become an urgent task for cotton breeders and increasing economic potential in cottonseed and other economic traits can help promote profits for cotton growers. In this paper, the major research projects related to cotton breeding at the USDA-ARS at Stoneville, MS since the 1960s are reviewed. These research projects reflect the changing needs in cotton production during the period and focus on broadening the genetic base of Upland cotton for improving agronomic traits and fiber quality in cotton cultivars by a group of scientists with different scientific disciplines. A comprehensive review of this research can help develop strategies and identify research fields to strengthen to meet challenges in future.

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.

Genome-wide association results for the 7220 SNPs included in the study. (XLSX 564 kb)

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.

The narrow genetic base in sweet sorghum [Sorghum bicolor (L.) Moench] breeding programs is limiting the development of new varieties for biofuel production. Therefore, the identification of genetically diverse sweet sorghum germplasm in the U.S. National Plant Germplasm System (NPGS) collection is imperative for biofuel breeding programs as biofuel production expands to new regions. Nine-hundred twenty-five sweet sorghum accessions from the NPGS collection were agronomically evaluated and a subset of 56 accessions selected for further evaluation. A 2 year replicated trial of this subset together with 17 U.S. sweet sorghum varieties were evaluated for agronomic and biofuel traits flowering time, plant height, fresh and dry weight, brix, juice volume, percent of moisture, and fermentable sugars [dinitrosalicylic (DNS) method] and disease response [anthracnose (Colletotricum sublineolum) and rust (Purcina purpurea)]. Nine accessions from the NPGS collection originally from South Africa, Ethiopia, Sudan, Zimbabwe, and the U.S. showed brix values ranging from 10 to 14, with five accessions having a higher amount of fermentable sugars than U.S. references accessions (DNS = 9.86-11.42). Likewise, the total dry matter content of three accessions originally from Ethiopia and U.S. were higher than the U.S. reference accessions (>156.87 g/plant). Multiple new sources of anthracnose and rust resistance were identified; being PI 156424 from Tanzania resistant to both diseases. The results demonstrated that accessions in the NPGS sorghum collection enclose valuable genes/alleles for biofuel traits that are not being used in U.S. biofuel breeding programs. Thus, the integration of these accessions into these programs will aid to increase genetic diversity and development of new biofuel varieties.

Ovule culture is one of the techniques currently used to introgress desirable traits from Gossypium arboreum germplasm into G. hirsutum cultivars.Twenty-six (26) G. hirsutum breeding lines were used as female parents in crosses with five G. arboreum accessions to determine if the G. hirsutum parent influenced the germination and recovery of plants from ovule culture.Variation in boll weight and the number of ovules per boll was observed for crosses with the G. hirsutum lines, but heavier bolls and a greater number of ovules per boll were not associated with a higher germination rate.Ovules derived from crosses with 16 G.hirsutum lines showed germination.Plants were recovered for seven of these lines (Acala GLS, DES 56, DES 119, Deltapine 50, Stoneville 132, Stoneville 506 and Stoneville 825) with vigorous growing plants derived from four crosses (DES 119 x PI 408763, Stoneville 506 x PI 408763, Acala GLS x PI 529779, and DES 119 x PI 615699).The breeding line DES 119 showed a better success rate and typically produced smaller bolls with fewer ovules.However, results would suggest the G. arboreum accessions had a greater influence on the success rate compared to the G. hirsutum lines.

Gossypium hirsutum flowers are easily emasculated by splitting the staminal column with the fingernail and removing the corolla and androecium. However, any damage to the ovary is considered detrimental to successful boll formation and damaged flowers are typically discarded. This study evaluated boll retention after different emasculation treatments. Removal of the membrane surrounding the ovary reduces boll retention compared to self-pollinated flowers, and showed a similar response across 18 genotypes. Damage to the ovary wall reduced boll retention compared to emasculate flowers without ovary damage. Damaged flowers could be cross-pollinated to produce additional bolls as compared to discarding the flowers.

The USDA-ARS National Plant Germplasm System maintains a Zimbabwe sorghum collection of 1235 accessions from different provinces. This germplasm has not been extensively employed in US breeding programmes due to the lack of phenotypic and genetic characterization. Therefore, 68 accessions from Zimbabwe were phenotyped, and evaluated for their anthracnose response for two consecutive years, and genetically characterized with 21 simple sequence repeat markers. Phenotypic analysis showed significant differences among accessions with plant height and panicle length being the most variable traits. Likewise, 25 accessions were anthracnose resistant, nine showed variable responses and 34 were susceptible. Genetic analysis identified 174 alleles with an average of 8.3 alleles and 11.8 genotypes per locus and a polymorphic information content of 0.60. These results reflect a moderate genetically diverse germplasm. Neighbour-joining clustering analysis revealed that the majority of anthracnose-resistant accessions showed high genetic relatedness; therefore, this germplasm might represent one to six new sources of resistances. Results presented herein show that the Zimbabwe collection contains valuable germplasm for breeding programmes and is an important source of anthracnose resistance.