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ARS Home » Southeast Area » Tifton, Georgia » Crop Genetics and Breeding Research » Research » Research Project #436116

Research Project: Genetic Improvement and Cropping Systems of Warm-season Grasses for Forage, Feedstocks, Syrup, and Turf

Location: Crop Genetics and Breeding Research

2022 Annual Report

1. Characterize and improve internode length and stem maggot resistance in bermudagrass. 1A. Using RNA Sequencing, identify candidate genes that regulate internode length in bermudagrass. 1B. Develop integrated pest management strategies for mitigation of the Bermudagrass Stem Maggot (BSM). 2. Develop genetic markers and biocontrol agents to reduce root-knot nematode and aphid damage in sweet sorghum. 2A. Determine if the root-knot nematode resistance gene can be moved from Honey Drip to susceptible or moderately resistant sorghum cultivars by marker-assisted selection and thus confer or improve resistance. 2B. Identify new genetic loci for root-knot nematode resistance and develop markers associated with resistance. 2C. Investigate the use of entomopathogenic fungi to control sugarcane aphid in sorghum. 3. Assess lupin and carinata as renewable bio-based products and soil enhancement cover crops. 3A. Assess the economic and environmental impact of lupin as a winter crop cover within a summer row crop rotation. 3B. Determine the effects of Brassica carinata grown as a winter crop on soil quality and subsequent summer row crop production. 4. Develop genomic technologies for centipede grass and use those technologies to understand and improve desirable ecological and aesthetic traits for this species. Work may include, but is not limited to, water and nutrient efficiency, resilience to foot traffic, color, and pollinator support.

Objective 1: For characterization of internode length in turf bermudagrass, total ribonucleic acid (RNA) will be extracted from the leaf and stem tissue of bermudagrasses. RNA samples will be sent for library preparation and sequencing. The transcriptome will be reconstructed and differentially expressed genes will be identified and then confirmed for internode length via real-time Polymerase chain reaction (PCR). For stem maggot resistance, forage bermudagrass germplasm will be selected from the bermudagrass core collection for further evaluation for yield, quality and tolerance to Bermudagrass Stem Maggot (BSM) and tested in the field in two side by side plots (one sprayed and one not sprayed) and replicated four times in a randomized complete block design. Most tolerant lines for further analysis for yield and quality traits will be determined and used for release and use for crosses. Objective 2: The root-knot nematode resistance gene will be moved from ‘Honey Drip’ to susceptible or moderately resistant sorghum cultivars by marker-assisted selection. Furthermore, new genetic loci for root-knot nematode resistance will be identified by creating a mapping population using a source of resistance different than ‘Honey Drip’. In collaboration with ARS fungal curator, naturally occurring entomopathogenic fungal isolates will be obtained from sugarcane aphids. Entomopathogenic fungi will be applied to susceptible sorghum to determine if these strains can control sugarcane aphids. Objective 3: The economic and environmental impact of lupin with and without rye as a winter crop cover within a summer row crop rotation will be determined using rotating main crops of peanut and cotton over years with different cover crops during the winter (narrow leaf lupin, white lupin, white lupin + cereal rye, narrow leaf lupin + cereal rye, cereal rye, and fallow. Half the covers will be harvested and the other half rolled. Changes in soil fertility and yields will be determined. The effects of Brassica carinata grown as a winter crop on soil quality and subsequent summer row crop production an experiment will be determined by rotating carinata and rye planted as a winter cover with sorghum and soybean as rotating summer crops. Objective 4: For the genetic mapping of desirable turf traits in centipedegrass, a genome-wide association study will be conducted using a population of approximately 300 vegetatively propagated lines replicated in the field. Morphological traits will be measured for two years after establishment. Single nucleotide polymorphisms (SNPs) will be created from each line using genotyping by sequencing and the genome of a centipedegrass line will be sequenced. SNPs will be aligned to the reference sequence and SNPs will be identified that are associated with the traits. For the identification of pollinators of centipedegrass inflorescences, a collection of centipedegrass lines will be grown in large field plots. In collaboration with an entomologist, pollinators will be documented that transit into each plot and those directly pollinating the inflorescences.

Progress Report
Objective 1A1. Bermudagrass was grown in the greenhouse in a RCBD, measured for internode length, and RNA was extracted. RNA sequencing was performed and differentially expressed genes were identified. Several genes with homology to gibberellin receptors showed large differences in gene expression between dwarf and parental (taller) lines. The same experimental design was regrown in the greenhouse, RNA was extracted (with DNase), and qPCR was performed for three replicates. As compared to Tifgreen, TifEagle is 65x more repressed and MiniVerde is 144x more repressed for a gene with homology to gibberellin receptor GID1. Melt curve analysis indicates temperature differences between TifEagle and MiniVerde (the dwarf lines) with Tifgreen and Tifway (the taller lines) for this gene. We suspect these dwarf lines are unable to perceive gibberellin and are working with a lab in Nebraska to help us determine endogenous GA levels (GA1, GA3, and GA4 are biologically active) in these lines. SSR markers were also created from the RNA sequencing data and are frequently used for bermudagrass genotyping. Objective 1B1. After multiple years of evaluation for bermudagrass stem maggot (BSM) tolerance, six plant introduction lines have been identified by an ARS researcher in Tifton, Georgia, for further testing and increase. These six lines have been grown and tested in north Georgia (Rome, Georgia) for cold tolerance and establishment. Two of the lines have been tested for prussic acid content compared to the standard Tifton 85 under high nitrogen fertilization. One more year of evaluations will be made before large scale increases are performed for potential release. The best lines have also been planted in crossing blocks with each other and other cultivars to produce hybrid seed. Objective 1B2. An ARS researcher in Tifton, Georgia, has determined that pyrethroid sprayed twice during the first 3 weeks after bermudagrass forage has been harvested for hay has the best control of BSM on susceptible cultivars. Trials have begun to determine whether a single spray timed after harvest can be as effective as two sprays. Objective 2A. In an experiment to determine if the root-knot nematode resistance gene can be moved from ‘Honey Drip’ to susceptible sorghum cultivars by marker-assisted selection (MAS) and thus confer or improve resistance, progeny from the BC1F6 progeny were created by originally crossing Honey Drip to susceptible forage, sweet and grain sorghum. The BC1F6 progeny were evaluated for root-knot nematode resistance in a greenhouse over two years. All BC1F6 progeny were all highly resistant and similar to the resistant standard ‘Honey Drip’. Thus, the resistance QTL can be introgressed using marker-assisted selection into many sorghum genotypes and confer a high level of resistance to root-knot nematodes. This project was written into this year’s accomplishments. Objective 2B. Objective completed. Objective 2C1. Sugarcane aphids were collected from multiple fields in GA from 2018-2020 primarily at the beginning of the aphid population crash and were shipped to an ARS entomopathogen curator. The curator isolated the entomopathogens, performed morphological measurements and extracted the DNA from each isolate. The curator amplified and sequenced the large subunit of nuclear ribosomal DNA from each sample and then mailed the DNA to the ARS researcher as the curator left ARS. The ARS researcher amplified and sequenced the translation elongation factor a and the nuclear small subunit of ribosomal DNA from each sample. Objective 2C2. Objective completed. Objective 3A. An ARS researcher in Tifton, Georgia, has completed four years of planting and harvesting winter cover lupin with or without rye at three locations. Peanut and cotton were rotated alternatively during the summers. Substantial biomass was produced by lupin each spring. No yield differences between cover crop treatments have been observed for peanuts each year. Soil and plant samples will be analyzed during the next two years. Objective 3B. ARS scientists have completed one cycle of sorghum/soybean summer crops in a test to determine the economic and environmental fit of planting either Brassica carinata (oilseed crop for renewable aviation fuel) or wheat. One more full cycle of the winter and summer crops will be completed prior to soil analysis. Objective 4. ARS researchers in Tifton, Georgia, are developing genomic technologies for centipedegrass and use those technologies to understand and improve desirable ecological and aesthetic traits for this species such as pollinator support. To determine genomic regions associated with morphological traits, a GWAS experiment was initiated. 295 centipedegrass lines were selected from a diverse clonal collection, were increased in the greenhouse, and planted in the field with four replicates in the fall of 2021. Plants are currently being maintained to increase the material into 6 x 6 field plots. DNA has been extracted, genotyping by sequencing has been performed, and the genome of a centipedegrass line has been sequenced but not yet assembled. ARS scientists are also examining how the ploidy of centipedegrass impacts turf traits and water usage. ARS scientists at Tifton, Georgia, created three tetraploid centipedegrass lines in 2018 using tissue culture (centipedegrass is a diploid) and two lines have been stable (one is now a mixoploid). The genome size of each line has been measured, the relatedness between each line has been evaluated using centipedegrass SSR markers, and chromosome counts have been performed. The lines are currently being evaluated in a greenhouse for morphological and physiological traits in collaboration with a University of Georgia plant physiologist.

1. Southern root-knot nematode resistance can be transferred to forage, sweet, and grain sorghum and confer resistance. The southern root-knot nematode causes significant economic damage to many crops in the U.S. such as cotton and vegetables. ARS researchers in Tifton, Georgia, had previously identified a region on sorghum chromosome 3 that confers root-knot nematode resistance in the sweet sorghum cultivar ‘Honey Drip’. These researchers wanted to ascertain if the resistance gene could be transferred using crossing and markers to forage, sweet, and grain sorghum and confer resistance. The resistance gene from ‘Honey Drip’ sorghum was crossed into five different sorghum backgrounds and the presence of the resistance gene was verified by DNA markers. Repeated greenhouse experiments documented that prior to incorporating the resistance gene the sorghum lines were all highly susceptible to the nematode, but after incorporating the resistance gene the lines were all highly resistant. The newly created lines were as resistant as their resistant parent, ‘Honey Drip’. These results suggest that this resistance gene could be moved into new lines using marker assisted selection and confer a high level of resistance. Thus, this gene and its associated markers will be useful for sorghum breeding programs that want to incorporate southern root-knot nematode resistance into their sorghum lines. The use of resistant sorghum lines will reduce the amount of southern root-knot nematodes in the soil, will decrease nematode damage in the next susceptible crop grown, and may reduce the use of fumigants/nematicides.

2. Aphid super-clone persists in the U.S. and is now present in Brazil. Sugarcane aphids have been a perennial economically important pest to U.S. sorghum since 2013. Previous research has shown this recent invader has been spreading as a super-clone in the U.S. To continuously monitor the genotypes present in the U.S. and to determine the genotype present in Brazil on sorghum, sugarcane aphids were collected in 2019 and 2020. Genotyping of sugarcane aphid samples by an ARS researcher in Tifton, Georgia, with microsatellite markers revealed that the super-clone predominated in the U.S. in 2019 and 2020 and Brazil in 2020. Thus, the sugarcane aphid super-clone remains in the U.S. on sorghum, Johnsongrass, and giant miscanthus and has spread to Brazil on sorghum. This information is useful to growers especially in Brazil as the methods used to control sugarcane aphids in the U.S. will be effective in Brazil as both countries have the same super-clone.

3. Release of sugarcane aphid-resistant sweet sorghum lines. Few insecticides are available to control sugarcane aphid in sweet sorghum, which is used to produce edible syrup, and no existing sweet sorghum cultivars have a high level of resistance to this pest. Sugarcane aphid damage includes leaf discoloration and desiccation and delayed or aborted flowering. Sugarcane aphids also reduce the quality and sugar content of sweet sorghum juice. ARS researchers in Tifton, Georgia, developed GTS1903, GTS1904, and GTS1905 sweet sorghum lines from an initial cross of an aphid-resistant Ethiopian line (No. 5 Gambela) with a sweet sorghum seed parent (AN109), followed by multiple generations of selection for aphid resistance, good agronomic traits, and high sugar content. Across four environments in 2019 and 2020, these three lines had higher juice sugar content and showed less visible damage from sugarcane aphids than the popular susceptible cultivar Top 76-6. They also harbored fewer aphids than Top 76-6 in one environment. With GTS1903, GTS1904, or GTS1905, growers should be able to produce a sweet sorghum crop without the need to spray insecticide to control sugarcane aphids. This will be particularly beneficial to organic producers. These lines were released in 2022 and seeds have been requested by at least 20 sweet sorghum growers across six states.

Review Publications
Akter, H., Dwivedi, P., Alam, A., Anderson, W.F. 2022. Does intercropping carinata with loblolly pine for sustainable aviation fuel production save carbon? A case study from the southern United States. BioEnergy Research.
Anderson, W.F., Knoll, J.E., Olson, D.M., Scully, B.T., Strickland, T.C., Webster, T.M. 2022. Winter legume cover effects on yields of biomass-sorghum and cotton in Georgia. Agronomy Journal. 114(2):1298-1310.
Harris-Shultz, K.R., Armstrong, J.S., Carvalho, G., Pereira Segundo, J., Ni, X. 2022. Melanaphis sorghi (Hemiptera: Aphididae) clonal diversity in the United States and Brazil. Insects. 13(5):416.
Davis, R.F., Harris-Shultz, K.R., Knoll, J.E., Wang, H. 2021. Transfer of Meloidogyne incognita resistance using marker-assisted selection in sorghum. Journal of Nematology. 53:e-2021-087.
Dong, H., Philley, H., Harris-Shultz, K.R. 2022. Registration of ‘MSB-264’ and ‘MSB-285’ bermudagrasses. Journal of Plant Registrations. 16(2):185-197.
Knoll, J.E., Uchimiya, S., Harris-Shultz, K. 2021. Juice chemical properties of 24 sorghum cultivars under varying levels of sugarcane aphids (Melanaphis sacchari) infestation. Arthropod-Plant Interactions. 15(5):707-719.