Research Project: Genetic Improvement of Sugarcane for Adaptation to Temperate Climates

Location: Sugarcane Research

2021 Annual Report

Objectives
Objective 1: Develop and release sugarcane cultivars and germplasm with improved agronomic traits, adaptability, stress tolerance and genetic diversity. Sub-objective 1.A. Develop improved sugarcane cultivars utilizing parental germplasm derived from the SRU’s germplasm enhancement program (sub-objective 1.B.) that possess highly desirable traits. Sub-objective 1.B. Characterize and broaden the genetic base of Saccharum to support both sugarcane breeding for commercial cultivars, with specific emphasis on adaptation to temperate environments, disease resistance, and sugar content. Objective 2: Develop and deploy clone-and trait-specific genetic markers for sugarcane, and work with breeders to accelerate breeding and release improved sugarcane cultivars and germplasm.

Approach
The program’s breeding strategy is to increase the genetic diversity of parental clones through: (1) acquisition and maintenance of germplasm from wild species of Saccharum and related genera; (2) characterization of parents and progeny for traits (cold tolerance, stubbling ability, disease resistance, and sugarcane borer resistance) that will increase the adaptation of sugarcane to Louisiana’s temperate climate; (3) utilization of crossing and molecular marker techniques to produce interspecific and intergeneric hybrids containing new sources of disease and insect resistance and cold tolerance; and (4) recombination of progeny through backcrossing to develop parental material containing a concentration of desirable genes for the commercial breeding program. Screening procedures will be developed to determine relative cold tolerance among clonal material in the basic breeding program. Cultivar development will emphasize increased sugar yield, along with other import traits such as yield components (stalk number, height, and diameter), fiber concentration, rate of maturation, ratooning ability (stand longevity), harvestability (resistance to lodging, stalk erectness, and stalk brittleness), hardiness (winter survival, early spring vigor, and stalk and ratoon freeze tolerance), abiotic stress tolerance (droughts, floods, and heavy clay soils), and resistance to stalk boring insects (sugarcane borer and Mexican rice borer) and diseases (smut, rust, leaf scald, mosaic, yellow leaf virus, and ratoon stunting). Recurrent selection techniques will be utilized to accelerate the rate of genetic improvement for these important traits. In addition, trait-specific markers closely associated with traits such as sucrose accumulation, cold tolerance, and resistance to the sugarcane borer will be developed to assist breeders in eliminating undesirable plants early in the selection process.

Progress Report
The yearly cycle of crossing, field evaluations, and selections were made as part of the USDA-ARS commercial variety development program in Houma, Louisiana. Crosses were made by ARS scientists at USDA-ARS facilities in both Canal Point, Florida, and Houma, Louisiana, with the production of approximately 852,250 viable commercial seed. In fiscal year 2021, approximately 67,673 commercial seedlings were planted by ARS scientists at Houma, Louisiana. Of the approximately 58,520 seedlings planted by ARS scientists at Houma, Louisiana, in fiscal year 2020, a total of 7,641 were selected and advanced to the first-line trials. Selections planted in the fiscal year 2019 second-line trial were evaluated by ARS scientists at Houma, Louisiana, in the first ratoon. Of the 467 potential varieties in this trial, 69 received a permanent numerical assignment and were advanced for further testing (Milestones 1 and 2). In advanced stages of the program, 34 experimental varieties were advanced to off-station nurseries from the 2014 crossing series, and 14 were advanced to infield testing in fiscal year 2019 from crosses made in 2013. Stages in the commercial breeding program fall under the subordinate agreement, “Three-way (LSUAC, ASCL, ARS) Sugarcane Breeding and Variety Development” (Non-Funded Cooperative Agreement #58-6052-7-003N). Remote sensing images were taken by ARS scientists at Houma, Louisiana, of the first-ratoon seedling plot experiments (Milestone 1). Thirty-eight newly named and selected varieties entering large-plot, off-station testing were transferred by ARS scientists at Houma, Louisiana, under the subordinate agreement entitled “Evaluating Sugarcane Varieties for the Rio Grande Valley Sugarcane Industry” (Trust Fund Cooperative Agreement #58-6052-9-0001) to Rio Farms, Inc. for testing in the Lower Rio Grande Valley of Texas. This program has proven to be valuable to the growers in Texas and has resulted in a sustained effort for variety improvement in the region. Germplasm Enhancement Program: All stages of breeding and selection were carried out by ARS scientists at Houma, Louisiana, in the Houma, Louisiana-based, germplasm enhancement program. Approximately 320 crosses were made by ARS scientists at Houma, Louisiana, for this program at the crossing facilities in Houma, Louisiana, producing roughly 65,500 seeds. Approximately 14,700 seedlings enhanced with traits of interest from wild relatives of sugarcane were planted by ARS scientists at Houma, Louisiana, in May 2020. Newly planted first-line trials contain 1,448 selections, and basic second-line trials contained 197 potential new varieties. Fifty-six newly selected parents enhanced with wild germplasm traits were planted by ARS scientists at Houma, Louisiana, as material for the 2020 crossing season (Milestone 1). Approximately 550 samples were DNA fingerprinted by ARS scientists at Houma, Louisiana, from the sugarcane crossing cart parents, pathology greenhouse, historical nursery, and seed-increase plots. In addition, samples were collected by ARS scientists from Canal Point, Florida, for variety validation. High-throughput DNA fingerprinting was conducted by ARS scientists at Houma, Louisiana, in collaboration with the USDA-ARS at Stoneville, Mississippi. Approximately 11,000 fingerprint files were produced and analyzed by ARS scientists at Houma, Louisiana. Sixty-nine molecular identities were assigned by ARS scientists at Houma, Louisiana to newly named 2019-series USDA commercial clones and were deposited into the USDA’s sugarcane molecular identity database. Fingerprints of the remaining 481 samples were compared either among themselves or with the fingerprints already present in the database. Analysis showed that 40 pairs of 2018-series commercial clones from the HWT Primary Increase and Outfield Increase Plots shared identical molecular fingerprints. However, the DNA fingerprints of a commercial variety planted in 2019 and 2020 did not match, indicating a possible seed-cutting error in 2019 due to lodged field plots. In addition, the fingerprints of HoCP 05-918 from Canal Point, Florida, did not match with those of HoCP 05-918 from Houma, Louisiana. DNA fingerprinting is routinely used in the program to validate varieties and avoid errors in reporting final results (Milestone 1). Artificial inoculations give a good indication of varietal response to both sorghum and sugarcane mosaic virus in sugarcane. Efforts to screen material early in the crossing program were increased and inoculated tests have been conducted on all parents for the past four years. All varieties (basic and commercial) used in the crossing program were screened for the virus prior to the beginning of the 2020 crossing season as well as all newly named varieties that will be used as parents in 2021 (Milestones 1 & 2). In addition, all 2019 varieties remaining active in the program were re-screened in 2020 to validate the previous results. To increase confidence in the test, a sub-set of samples were collected and screened using PCR markers to validate the visual assessments. Breeders made targeted crosses to avoid an increase in susceptibility in the current breeding population based on results from these greenhouse tests. Crosses were not generated between two susceptible varieties. The development of new and resistant varieties for the Louisiana program will be hastened in the future based on this decision. In addition to inoculated testing, USDA continues to work on a modified genotyping by sequencing project with collaborators at Louisiana State University to identify genetic markers associated with mosaic. A susceptible and resistant sugarcane parent were crossed to generate a family of offspring that are segregating for mosaic resistance. The seedlings were planted in the field in April of 2020. In August, stalks were cut from each stool of cane and propagated in the greenhouse and field. The greenhouse-propagated stalks were inoculated and evaluated for mosaic resistance. Leaf tissue was collected from the inoculated plants for genotyping in 2021. This population will be used to validate DNA markers for mosaic resistance that are identified from the genotyping by sequencing project (Milestone 3). One hundred twenty newly named 2019 parents (used in the 2020 crossing season) were screened for a molecular marker, BRU1, indicating resistance to brown rust as part of a collaborative project with colleagues at the LSU AgCenter. Of these varieties, 32 tested positive for the BRU1 marker for rust resistance. Two of these varieties were from the germplasm enhancement program, and the remaining 30 were commercial. Four genotypes from the germplasm enhancement program and 30 from the commercial breeding program tested positive. An additional 132 varieties from 2020 were screened, and information on their BRU1 status was added to the database for the 2021 crossing season (Milestone 2). Targeted crosses were made during the 2020 crossing season to maximize the frequency of BRU1 in the progeny. Older parents continue to be used in the breeding program, many of which contain the BRU1 marker, and there has been an increase in frequency of the marker after targeting selection for the past several years. While it used to be unusual to find a parent in the breeding program possessing BRU1, it is now much more common (Milestone 2). There has been a long-standing effort by USDA to breed for cold tolerant sugarcane varieties. Twenty-eight sugarcane lines previously selected for having excellent yields after exposure to damaging freezes were planted and evaluated in replicated trials by the USDA in Houma and by Mississippi State University in Starkville, Mississippi. Cold tolerance has been validated in Mississippi with high yields despite a harsh winter following planting. The three most broadly adapted varieties were selected for breeding to enhance cold tolerance in future populations. The cane remains in the fields for testing of ratoon-harvest yields. This work was conducted as part of a subordinate agreement entitled “Center for Advanced Bioenergy and Bioproducts Innovation” (Interagency Reimbursable Agreement #60-6052-8-002). Fiber components were analyzed from the second clonal trial of the germplasm enhancement program according to the National Renewable Energy Laboratory’s Structural Carbohydrate Analysis protocol. Fiber quality was assessed using acid hydrolysis followed by quantification of soluble and insoluble lignin as well as structural carbohydrates (cellulobiose, arabinose, xylose, glucose, mannose, galactose, and acetic acid). Fiber work was conducted as part of a subordinate agreement entitled “Center for Advanced Bioenergy and Bioproducts Innovation” (Interagency Reimbursable Agreement #60-6052-8-002). Breeders conducted a study to compare maternal DNA markers (cytoplasmic) with cold tolerance in sugarcane. A collection of wild sugarcane was previously assessed for tolerance to freezing conditions and data was recorded as the percentage of surviving plants. This information was compared to DNA that is passed on by the mother (cytoplasmic DNA) to determine if the cold tolerance is based on maternal inheritance. Few differences were found among the cytoplasm types in the wild relatives, and they did not appear to be related to cold tolerance based on this study (Milestone 3). The fiber content of sugarcane stalks is important for both sugar and bioenergy production. Five unique DNA markers were identified using an enriched linkage map of sugarcane and fiber content data from two crops (plant cane and first ratoon) of a sugarcane population. Three QTL (quantitative trait loci) markers, namely, qFC020, qFC044 and qFC053, explained a total of 22.46%, 23.93% and 25.14% of fiber content variations in plant-cane, first ratoon and combined data, respectively. As fiber content increased, the combined effect of these QTL markers also increased.

Accomplishments
1. Release of commercial sugarcane variety ‘HoCP 14-885’. ARS scientists at Houma, Louisiana, suggests there is no private breeding program for sugarcane in the United States. Thus,the industry is founded 100% on publicly developed varieties. ARS scientists from Houma, Louisiana, in collaboration with the American Sugar Cane League of the U.S.A., Inc. and the Louisiana State University Agricultural Center, developed and released a new sugarcane variety in 2021. The new variety, ‘HoCP 14-885’ is resistant to sugarcane smut caused by Sporisorium scitamineum, mosaic caused by Sorghum Mosaic Virus (SrMV), and brown rust caused by Puccinia melanocephala. It is susceptible to ratoon stunt caused by Leifsonia xyil subsp xylil, and moderately susceptible to leaf scald caused by Xanthamonas albilineans. HoCP 14-885 is susceptible to the sugarcane borer (Diatraea saccharalis) and Mexican rice borer (Eoreuma loftini), both of which can be controlled through pesticide application. It has moderate cold tolerance. The variety’s most significant attributes are disease resistance, early maturity, and high sugar yields through multiple harvests. Early maturity is important in Louisiana because the harvest season is cut short due to late season freezing temperatures. The release of this variety offers growers a well-adapted variety that exceeds the sugar yields of the currently leading variety across the harvest cycle. Due to its yield stability and resistance to major diseases, the variety can increase industry profits in addition to expanding the genetic variability contained within the growing region.

Review Publications
Wu, J., Wang, Q., Pan, Y., Zie, J., Zhou, F., Xu, H., Qiu, Y., Liu, Z. 2021. Multivariate analysis of 31 phenotypic traits among major parental lines of sugarcane breeding programs in China. The Journal of Animal and Plant Sciences. 31(3):719-732. https://doi.org/10.36899/JAPS.2021.3.0262.
Todd, J.R., Dufrene Jr, E.O., Waguespack, H., Kimbeng, C., Pontif, M., Boykin, D.L. 2021. Data mining sugarcane breeding yield data for ratoon yield prediction. Euphytica. 217:54. https://doi.org/10.1007/s10681-021-02786-z.
Wu, Z., Hu, X., Zan, F., Pan, Y.-B., Burner, D.M., Luo, Z., Liu, J., Zhao, L., Yao, L., Zhao, Y., Liu, X., Xia, H., Yang, K., Zhao, J., Zhao, P., Qin, W., Chen, X., Wu, C. 2020. Subcellular localization of the D27 protein in sugarcane (Saccharum hybrids spp.) using an optimized protoplast isolation, purification, and transient gene expression protocol. Sugar Tech. 23(2):316-325. https://doi.org/10.1007/s12355-020-00879-y.
Islam, M.S., Pan, Y.-B., Lomax, L.E., Grisham, M.P. 2021. Identification of quantitative trait loci (QTL) controlling fiber content of sugarcane (Saccharum hybrids spp.). Plant Breeding. 140(2):360-366. https://doi.org/10.1111/pbr.12912.
Knoll, J.E., Anderson, W.F., Missaoui, A., Hale, A.L., Hanna, W.W. 2021. Biomass production and stability of five energycane cultivars at two latitudes in Georgia, USA. Agrosystems, Geosciences & Environment. 4:e20146. https://doi.org/10.1002/agg2.20146.
Ficket, N., Ebrahimi, L., Parco, A., Hale, A.L., Gutierrez, A.V., Pontif, M.J., Todd, J.R., Kimbeng, C.A., Hoy, J.W., Ayala Silva, T., Gravois, K.A., Baisakh, N. 2020. An enriched sugarcane diversity panel for utilization in genetic improvement of sugarcane. Scientific Reports. 10:1-13. https://doi.org/10.1038/s41598-020-70292-8.
Hu, X., Wu, Z., Luo, Z., Burner, D., Pan, Y.-B., Wu, C. 2020. Genome-wide analysis of the trehalose-6-phosphate synthase (TPS) gene family and expression profiling of ScTPS genes in sugarcane. Agronomy Journal. 10(7):969. https://doi.org/10.3390/agronomy10070969.
Yang, X., Luo, Z., Todd, J.R., Sood, S.G., Wang, J. 2020. Genome-wide association study of multiple yield traits in a diversity panel of polyploid sugarcane (Saccharum spp.). Plant Methods. https://doi.org/10.1002/tpg2.20006.
Luzaran, R.T., Pan, Y.-B. 2020. Cultivar-specific SSR markers as revealed through fluorescence- labeling and capillary electrophoresis in sugarcane (Saccharum hybrids spp.). Philippine Journal of Crop Science. 45(2):47-57.
Sanjel, S., Hincapie, M., Wang, Y., Todd, J.R., Chaulagain, B., Sood, S.G., Comstock, J.C., Raid, R.N., Rott, P. 2021. Occurrence of two races of Puccinia kuehnii causing orange rust of sugarcane in Florida. Plant Pathology. 00:1-10. https://doi.org/10.1111/ppa.13405.
Todd, J.R., Johnson, R.M. 2021. Prediction of ratoon sugarcane family yield and selection using remote imagery. Agronomy. 11(7):Article 1273. https://doi.org/10.3390/agronomy11071273.