During the year, the Noble Research Institute Small Grains Laboratory, led by Xuefeng Ma, Ph.D., in Ardmore, Oklahoma, has been collecting new germplasm; screening germplasm; conducting yield trials; and making new crosses of rye, triticale and oat for improving cover crop performance.
As of now, our major traits under selection include:
Below are the major activities for each crop.
During the 2018-19 season, 100 rye entries selected from last season were planted for further biomass yield evaluation; 75 rye lines were distributed to the five testing sites of the FFAR network for evaluating their cover crop potentials in each site; and five rye (and two triticale) lines were planted in 15 sites across several southern states for comparing biomass of continuous growth with accumulated regrowth under clipping treatment.
For the 2018-2019 season, 80 oat nurseries selected from the previous season were planted for further biomass and other agronomic trait evaluation; the SOAP accessions were also planted for collecting second year phenotyping data; all black oat lines will be planted in spring 2019 to ensure seed production due to limited amount of seeds for most of the black oat lines; and new germplasm lines and segregating nurseries from Quaker Oat and SunOat will also be planted (for evaluating agronomic and biomass traits) in spring 2019 due to the massive amount of rainfall in the fall of 2018.
In 2018-2019 season, a triticale panel consisting of 300 lines were planted for continuous screening of superior parents in addition to collecting agronomic traits that will be used for QTL mapping; 27 elite lines were in advanced yield trials. In addition, about 200 triticale crosses were made in 2018 and the crosses will be continued in 2019.
Wheat is not the focus of breeding for cover crops, but it has the most genomics resources that are valuable for other small grains. QTL and genes discovered in wheat can be used for triticale and rye improvement. During the last two years (2016-17 and 2017-18 seasons), a winter wheat association mapping panel has been used for QTL discoveries on several traits, such as seedling heat tolerance and drought tolerance that contribute to early vigor and quick establishment of small grain cover crops. In addition, three wheat RIL populations were evaluated in field for collecting segregating biomass traits.These research populations were also planted in 2018-2019 field trials for continuously evaluating agronomic traits that contribute to biomass. QTL analysis will be conducted after the 2018-2019 season.
The Legume Cover Crop Breeding (LCCB) team continued evaluation and selection of breeding material for hairy vetch, crimson clover and winter pea.
In 2018-19, 96 half-sib lines (selected from the initial 27 populations) were evaluated for emergence, vigor, cold tolerance, pubescence, disease, biomass production and flowering time at the breeding nurseries at North Carolina State University; Beltsville, Maryland; Noble Research Institute; and other collaborating sites in the LCCB network. The top ~5% of plants were advanced at each site and were planted in the fall of 2018 for the subsequent cycle of evaluation and selection.
Self-Pollination Study: The Noble Research Institute annual legume breeding program led by Suresh Bhamidamarri evaluated Different methods of self-pollination (bees, hand pollination and bagging) were evaluated to test self-incompatibility in hairy vetch. All the methods produced selfed seed in the variety AU merit. The selfed F1seed harvested from individual genotypes will be used to generate F2seed. Segregation patterns will be studied on F2seed if it is viable. Genotypes that were compatible to self-pollination will be further inbred to study the possibility of developing homozygous and homogeneous genotypes. The results from this study can indicate if a pureline and or a hybrid hairy vetch cultivar can be developed.
Stem Cutting Study: In this study, we are evaluating the possibility of propagating hairy vetch genotypes through stem cuttings. This will enable breeders to select parents for developing a synthetic population or variety based on progeny evaluations (genotypic recurrent selection). This breeding method is employed in various other crop/forage species for improving quantitative traits. Our initial studies on AU merit have shown that hairy vetch can be vegetatively propagated with a 90% success rate. Based on these preliminary results, we are studying the effect of variety and stem thickness (hollow, solid and semisolid) on the establishment of stem cuttings.
Hard Seed Trait Inheritance: In this study, we are testing the hypothesis that frequency of hard seed can be increased based on selection. Hard seeds from three experimental varieties, NFVV7B (30 hard seeds), NFVV21 (19 hard seeds) and NFVV22 (22 hard seed), were selected based on germination test and were intracrossed with bees in the greenhouse to generate synthetic 1 generation. Fifty syn1 seeds from each cross were tested for hard seed. Data shows that the frequency of hard seed varied from 21% in NFVV21 to 80% in NFVV7B. Based on these results, selections for hard seed and soft seed will be made from within high yielding varieties to generate two distinct populations of the same variety, i.e. soft and hard seeded varieties from the same population.
Transcript Profiling of Seed Hardiness in Bulk Segregant Analysis (BSA): Eighty-five plants have been generated from soft and hard seeds of AUVV-7 (AU Merit), NFVV-7AB and NFVV-22 hairy vetch. Tissue from leaf, flower, immature pods, seeds and seed coats were collected for RNA isolation. RNA are currently being isolated from these tissues. RNA will be sent to the Genomic Core Facility for sequencing in 2019.
Bi-Parental Mapping Population for Linkage Map and QTL Analysis: In order to construct a genetic linkage map, generate SNP markers and identify QTL governing the hard seeded trait, we are generating a bi-parental mapping population. Contrasting genotypes for seed hardiness are identified and plants are currently being grown in greenhouse to make a bi-parental cross and generate 200-300 F1. These will be phenotyped in multiple locations and genetic linkage maps will be created.
Evaluation of PI Accessions:In one study, 1,584 hairy vetch genotypes representing 66 different PI accessions (six genotypes per replications, four replications) obtained from the GRIN database were transplanted in spaced plant nursery in a randomized complete block design in October 2018. These accessions will be evaluated for vigor, winter hardiness, maturity, biomass production and forage composition in 2019. Superior genotypes from superior accessions will be selected for cultivar development.
In another selection study, 1,000 genotypes of AU Merit hairy vetch were transplanted in a spaced plant nursery near Ardmore, Oklahoma, for evaluation and selection. Superior genotypes will be selected based on performance for developing a potential synthetic cultivar.
In 2018-19, 1,200 crimson clover genotypes (selected from the initial 20 populations) were space planted as transplants and evaluated for emergence, vigor, uniformity, cold tolerance, biomass production and disease resistance at the breeding nurseries at North Carolina State University and in Beltsville, Maryland, for a total of 6,000 genotypes. The top ~5% of plants were advanced at each site, and seeds from these individuals were planted in the fall of 2018 for the subsequent cycle of evaluation and selection. Trials were also conducted of the best lines from each site (selected based on 2017 data) with 60 half-sib families direct-seeded, and the top 39 lines were planted from 2017 remnant seed in the fall of 2018 for the next year’s half-sib trial. In Ardmore, 1,380 genotypes of crimson clover representing 46 PI accessions (10/PI/Rep) were transplanted in a RCBD in three replications for evaluation. The accessions will be evaluated for winter hardiness, maturity, biomass and forage quality. Best performing individuals from the best accessions will be selected for developing a synthetic cross for evaluation.
In 2018-19, 50 lines were evaluated for emergence, vigor, cold tolerance, biomass production, disease resistance and flowering time at the breeding nurseries at North Carolina State University; Beltsville, Maryland; and another collaborating site in the LCCB network. The top-performing rows and individual plants were advanced at each site and 50 pea lines in Maryland and 80 in North Carolina, from both new germplasm and pea selections in 2017 that were advanced another generation, were planted in the fall of 2018 for the subsequent cycle of evaluation and selection.
In 2018-19, bioinformatic analysis of the assembly results indicated the need for further sequencing runs and, preferably, utilization of long-read sequencing technology in addition to short read ones for proper assembly of a draft genome.Amr Ibrahim, Ph.D., at Noble Research Institute, conducted RNA sequence analysis for genes expressed in root, leaf, flower, stem, seed and seed coat of hairy vetch. Analysis of data is in progress for identification of genes differentially expressed in soft and hard seeds of hairy vetch. We used assembled hairy vetch transcriptome in conjunction with assembled genome for identification of hairy vetch HS1 and qHS1 genes. Both hairy vetch HS1 and qHS1 genes, previously identified to be related to hardseededness inMedicago truncatulaand soybean, will be knocked out by targeted genome editing and evaluated for seed softness. As we only recovered partial sequences of HS1 and qHS1 genes from the assembled hairy vetch genome and transcriptome, we worked on cloning the full length of these genes. We determined the complete sequence of the HS1 gene and its corresponding mRNA. Sequencing of the full length of the qHS1 gene is still in progress.
In parallel, we worked in development of a hairy vetch transformation and regeneration system that is a prerequisite to any gene editing attempts for producing soft-seeded cultivar(s). We tested different explants, including leaf, shoot tip, root, and immature seed for tissue culture response as well as Agrobacterium-mediated transformation. We were able to induce callus from young leaf, shoot tip and root, but none of them could be regenerated into plant. We examined a variety of medium types with a series of hormone combinations for tissue culture response. Callus can be induced but not regenerable under a series of hormone gradients (BAP and Kinetin). AlteRNAtively, cotyledons from soaked mature seeds have also been tested as explant through direct organogenesis. We were able to obtain regenerated shoots and plants, but no positive transgenic plants have been obtained.
For performing hairy the vetch genome editing experiments, we will use the currently available CRISPR/Cas9 system vectors. Meanwhile, we worked in developing a new RNA virus-based vectors for expression of the CRISPR/Cas9 system components (e.g., Cas9 protein and guide RNA(s)) in plant cells. These vectors, if working, will be tested as a transient DNA-free approach for genome editing in cover crops. We constructed and tested three different vectors for expression of CRISPR/Cas9 components. All three vectors were functional as evident by detectable expression of inserted GFP sequence. Consistent expression of full-length Cas9 protein from these vectors could not be achieved (likely due to the big size of the Cas9 protein). Modification and optimization of these vectors is underway for proper expression of the Cas9 protein.
In the 2018-19 season, seed collected by Twain Butler’s team at Noble Research Institute from year one of the advanced germplasm of winter survivor selections (66 lines) were planted again at the Red River Ranch near Burneyville, Oklahoma, on Oct. 21, 2018. The species represented in these advanced evaluations and their corresponding number of entries, (shown in parenthesis), areBrassica napus(22),B. rapa(11),B. juncea(20),B. nigra(5), andRaphanus sativus(9). Where seed quantities allowed, seed of these advanced selections were also distributed to the FFAR grant collaborators in Nebraska, Missouri, North Carolina and Maryland for potential autumn 2018 planting. After weather-related failures during the 2017-18 season, a repeat planting of a total of 833 GRIN accessions ofB. oleraceawere planted Oct. 19 and Oct. 20, 2018, along with standard check varieties (Dwarf Siberian Kale and Impact Forage Collard obtained from Green Cover Seeds, Inc. and Allied Seed Company, respectively). Additionally, a total of 144 GRIN accessions of chicory (Cichorium intybus) were planted Oct. 30, 2018, along with the standard check variety Commander (PGG Wrightson Seeds). This trial planting is located near Ardmore (south-central), Oklahoma. Data collection on the existing survivors from all current trials is ongoing.
In addition to evaluation and selection in breeding nurseries for each crop, Advanced Line Trials (ALTs) were initiated for each crop. In the fall of 2018, bulk synthetics (for hairy vetch and crimson clover) or selected lines (for winter pea) from each site were planted at North Carolina State; Beltsville, Maryland; Noble Research Institute; and other collaborating sites in the LCCB network. The advanced line trials will be evaluated in 2019 for performance in a broader range of geographic areas as a step toward release of commercial cultivars adapted for use as cover crops.
The USDA-ARS (Beltsville, Maryland), North Carolina State University (Raleigh), University of Missouri (Columbia) and University of Nebraska (Lincoln) sites also participated in advanced line trialing during the 2018 harvest season. One hundred small grain genotypes (75 rye, 15 triticale, 6 wheat and 4 oat) and 66 brassica genotypes (21 entries of brown mustard, 16 entries of rapeseed, 5 entries of black mustard, 12 entries of field mustard and 12 entries of radish) and 41 entries of annual legumes (16 entries of hairy vetch, 18 entries of winter pea and 7 entries of crimson clover) will be evaluated for vigor, winter hardiness, heading and flowering dates, plant height, biomass and disease.