OILSEED BREEDING AND GENETICS PROGRAM FOR NORTH DAKOTA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1019221
• Project Start Date: Oct 1, 2019
• Project End Date: Sep 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750

Performing Department
Plant Sciences

Non Technical Summary
North Dakota is the leading producer of canola with over 78% of U.S. acreage and produces about 84% of all U.S. canola (1.35 million acres and 2.3 billion pounds with a value of $395 million - 5 yr. average from 2013-2017; USDA-NASS, March 2018). Recently, the construction of two canola based processing plants required to a three-fold increase in the demand for canola in North Dakota alone. To meet the growing demand, it is necessary to improve the genetic potential and adaptation of the crop to ND, and to expand the Northern Plains production region by moving the crop into the more arid western ND and eastern MT and further south into the somewhat warmer central North Dakota. The spring canola varieties grown in this region were developed elsewhere (mostly in Saskatoon, Canada) and might not be optimally adapted to this climate and agronomic region. To facilitate this processing expansion, canola breeding for high seed yield, high oil content and better agronomic traits is needed.North Dakota is the leading producer of flax for oil and food use, with about 92% of the U.S. flax production, 88% of the U.S. acreage, and annually contributes about U.S. $72 million to the national economy (four-year average from 2014-2017; USDA-NASS 2018). However, flaxseed acreage decreased in recent years, making the United States a net importer of flaxseed. In the last ten years, the U.S. produced about 6.9 million bushels of flaxseed and an additional 6.2 million bushels were imported for the crushing industry (USDA, Economic Research Service, 2018). Moreover, flaxseed's popularityas a healthy food continues to develop and expand its value and market. In addition, research with flaxseed as feed for beef animals found that it enhanced carcass value, increased performance and improved cattle health (AmeriFlax). Therefore, the current production shortage and future anticipated demand for flaxseed will expand the acreage in the U.S. and breeding for improved varieties is needed to fulfill the demand.

Animal Health Component

80%

Research Effort Categories

Basic

10%

Applied

80%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	1848	1081	10%
202	1842	1081	10%
203	1848	1081	20%
203	1842	1081	20%
204	1848	1081	10%
204	1842	1081	10%
212	1848	1081	10%
212	1842	1081	10%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 204 - Plant Product Quality and Utility (Preharvest); 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 202 - Plant Genetic Resources;

Subject Of Investigation
1842 - Flax; 1848 - Canola;

Field Of Science
1081 - Breeding;

Keywords

canola

flaxseed

plant breeding

plant genetics

cultivars

breeding methods

plant diseases

molecular markers

Goals / Objectives
1. Develop high seed yielding and high oil content canola and flax germplasm adapted to North Dakota.2. Identify canola and flax germplasm with enhanced disease resistance/tolerance to blackleg and sclerotinia stem rot, and Fusarium wilt and flax rust, respectively.3. Conduct genome-wide association studies on agronomic and disease resistance traits in canola and flax.

Project Methods
Procedures:Objective 1: Develop high seed yielding and high oil content canola and flax germplasm adapted to North Dakota.Canola: Crosses and reciprocal crosses will be made between winter-type and spring-type canola B. napus cultivars in a greenhouse. Each of the four types of F1 will be advanced to the F1:2 generation in the next greenhouse cycle. In the following summer, about 100 seeds per F2 family will be planted at Prosper, ND. A total of 500 F2:3 lines will be selected and will be grown in the winter nursery in Temuco, Chile to generate F3:4 populations. The self-pollinated F4 seeds from the 500 families will be grown in the following year as individual rows in a single-row nursery at Prosper, and will be advanced to the F4:5 generation. Simultaneously, early generation testing will be conducted on the 500 F4 lines in full plots (5ft x 15ft) in an augmented trial with replicated checks at Prosper and Carrington, North Dakota. Data will be collected on seed yield, oil content, fatty acid composition, maturity, lodging, and other agronomic traits. About 100 (20%) lines (F4:5) will be selected from the trials and will be sent to the winter nursery in Chile for generation advancement (F5:6). The F6 seeds from self-pollinated plants will be grown in individual rows in a canola single-row nursery at Prosper and will be advanced to the F6:7 generation. Simultaneously, a yield trial will be conducted at three North Dakota locations using a RCBD with two replications. The lines will be screened for blackleg and sclerotinia stem rot resistance in a greenhouse. Data on seed yield, seed oil, fatty acid composition, flowering, maturity, lodging, and other agronomic traits will be collected. A total of 25 (25% from the trial) F6-7 lines will be selected based on seed yield and other traits for the following year's wide-area yield trial. The 25 selected F7 lines will be advanced to the F7-8 at the winter nursery. The self-pollinated F8 lines will be advanced to F8:9 in a canola single-row nursery at Prosper. Simultaneously, a yield trial will be conducted at six locations with three replications in North Dakota. A disease trial for both sclerotinia stem rot and blackleg will be conducted in the field at Fargo employing artificial inoculation. Data on seed yield, oil content, fatty acid composition, flowering, maturity, lodging, and other agronomic traits will be collected. A total of 6-9 (≈30%) lines will be selected based on seed yield, oil content, disease resistance, desired agronomic traits and breeder's impression.The best performing lines will be released as varieties, and Ogura-CMS and Restorer systems will be introgressed to develop inbred lines for hybrid production. The inbred lines will be shared with commercial companies under a Material Transfer Agreement (MTA) for hybrid production and joint release in North Dakota.Flax: Fifty crosses will be made among selected parents. Each of four F1 seeds will be planted and F2 seeds will be bulked to generate 50 F2 families. The 50 F2 families will be planted as hill plots in the field and will be bulk harvested as 50 F3 families. Again, the 50 F3 families will be planted as single rows in the following year. About 100 individual plants from each F3 family will be pulled and threshed for the following year's field evaluation. About 5,000 (50 x 100) F4 families will be planted as short rows in a single location. Agronomic potential compared to check varieties will be assessed visually. About 500 F4 will be selected and advanced to the F5 generation. The 500 F5 entries will be planted as a preliminary yield trial (PYT) in full plots (5ft x 8ft) using augmented design with replicated check cultivars at Fargo. Agronomic scores and seed yield will be recorded and about 100 entries will be selected. The selected entries will be advanced to the F6 generation. The 100 F6 entries will be evaluated in an intermediate yield trial (IYT) at three locations using randomized complete block design (RCBD) with three replications. Data on plant height, days to flowering, days to maturity, agronomic score, Fusarium wilt score, flax rust score, seed yield, and seed oil content will be recorded. About 30 entries will be selected and advanced to the F7 generation. In the F7 and later generations (F7-F10), yield testing will be conducted at multiple locations in an advanced yield trial (AYT) and universal regional nursery (URN) using RCBD with 3-4 replications. All lines in the multiple location nurseries will be evaluated for plant height, days to flowering, days to maturity, agronomic score, Fusarium wilt score, flax rust score, seed yield, and seed oil content. In this final stage of evaluation (F7-F10), the lines will also be included in Research Extension Center trials, North Dakota Flax Variety trials. The best performing inbred lines will be increased, named and released. Objective 2: Identify canola and flax germplasm with enhanced resistance/tolerance to blackleg and sclerotinia stem rot, and Fusarium wilt and flax rust, respectively.Canola disease screening: A total of 500 publicly available canola germplasm accessions and breeding lines will be evaluated against sclerotinia stem rot and blackleg. The best accessions resistant/tolerant to the diseases with other agronomic traits will be used in the crossing program to develop multi-parent advanced generation inter-crosses (MAGIC) populations. The eight accessions (e.g. A,B,C,D,E,F,G,H) will be crossed in four pairs to create four (AB, CD, EF, GH) F1 seeds. These F1 seeds will be inter-crossed (AB x CD, EF x GH) to make 4-way (ABCD and EFGH) F1 seeds. Again, each 4-way F1 will be inter-crossed (ABCD x EFGH) to make 8-way (ABCDEFGH) F1 seeds. A total of 1,000 8-way F1 seeds will was grown and will be advanced to the F6 generation by single seed descent to create the MAGIC population (Huang et al. 2012).In the flax breeding program, the breeding lines will be screened against Fusarium wilt and flax rust. All of the breeding lines used in the URN, AYT and IYT, and susceptible checks will be planted in replicated trials at NDSU Plot# 30 for Fusarium wilt screening. The disease scoring will be conducted according to Rashid and Kenaschuk (1993). Flax rust resistance screening will be conducted on the same breeding lines (URN, AYT and IYT) in a greenhouse. The best accessions of both crops (resistant/tolerant to the respective diseases) will be used in a crossing program to develop MAGIC populations.Objective 3: Conduct Genome-Wide Association Study on agronomic and disease resistance traits in canola and flax.In canola, 366 publicly available germplasm accessions were used for genotyping-by-sequencing (GBS), and 42,575 single nucleotide polymorphism (SNP) markers were obtained association. In flax, the GBS was conducted on 377 diverse flax germplasm, and 28,266 SNP markers were identified. Phenotypic data on breeding lines and world collections of canola and flax germplasm for agronomic traits will be collected from field trials conducted at multiple locations and multiple years. The same germplasm panel will also be used to screen for resistance/tolerance to sclerotinia stem rot and blackleg (canola), and Fusarium wilt and flax rust (flaxseed) under greenhouse and field conditions. PROC GLM and PROC MIXED (SAS, SAS Institute) will be used for data analysis. The mean data will be used for GWAS using TASSEL 5.0 (Bradbury et al., 2007). Principal components (PC) that account for 25% and 50% of the cumulative variations will be used in the regression model to control population structure. Six regression models (naïve, PC25%, PC50%, Kinship, Kinship+PC25%, and Kinship+PC50%) will be used to determine the best model for further analysis. The most significant SNP markers will be determined based on the 0.1 percentile tails of an empirical distribution obtained from 10,000 bootstraps (Mamidi et al., 2014).

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The main target audiences were canola and flax growers, canola industry such as Cibus, Cladbeck Consulting, Proseed who were providing canola cultivars in this North Central region of USA. Changes/Problems: Weather is always a problem for field crop production. Planting time is often delayed because of early rain. Similarly, storm or rain also delayed the harvesting. Excessive rain, flood, drought, heat during crop growing time significantly affect the crop production. In 2020, due to severe rain and flooding at Prosper and Casselton the yield trials were severely affected and could not grow for authentic data. Moreover, the experimental site at Carrington for flax was heavily affected by root-rot disease, and the experiments were abounded. Due to COVID-19 pandemic situation, travelling to Minot, Dickinson, Williston and Hettinger was restricted and could not take breeder's impression data. However, we got the agronomic and seed quality data from respective agronomists. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Presentation was given to AmeriFlax board on current flax activities and research on Wednesday October 28, 2020. Presentation was given to stakeholder's (Northern Canola Growers' Association) annual meeting on current canola activities and research on Thursday November 05, 2020. A presentation was given to SaskFlax Board Meeting on current NDSU flax activities and research program on Friday June 26, 2020. What do you plan to do during the next reporting period to accomplish the goals? I will continue to talk to the canola growers to understand their concern and to report their questions. Research emphasis will be given to increase the genetic diversity in canola, and to develop high seed yield, high oil content, biotic and abiotic stress tolerant canola germplasm adapted to North Central region of the USA. I will continue to talk to the flax growers and AmeriFlax board to understand their concern and to report their questions. Research emphasis will be given to increase genetic diversity in flax, develop high seed yield, high oil content, biotic and abiotic stress tolerant flax germplasm adapted to North Central region of the USA.

Impacts
What was accomplished under these goals? We have done genetic diversity studies on Flax (350 germplasm) and Canola (373 germplasm) using SNP markers of globally-distributed germplasm accessions. Released the 1st conventional canola (non-GMO) "NDOLA-01" in North Dakota. Released "ND Hammond" brown-seeded flax cultivar in 2019. Developed Public-Private partnership research with Cibus, Caldbeck Consulting (Kentucky) for the development of joint-hybrids or OPs of canola. Identified seven potential flax advanced breeding lines. The advanced breeding lines out-performed all varieties grown in North Dakota in the 2020 North Dakota Canola Variety Trial (3rd party evaluation) at Langdon, Carrington, Minot, Dickinson and Williston. Identified three potential non-GMO (conventional) canola breeding lines. The advanced breeding lines were out-performed over many commercial canola hybrids (Langdon, Carrington, Williston) under 2020 North Dakota Canola Variety Trial (3rd party evaluation). Identified several USDA-ARS plant introduction (PI) lines resistant/tolerant to blackleg, sclerotinia and stem rot diseases. The PI lines will be used by the breeding program to introgress the resistant/tolerant genes into elite breeding lines. 2020 canola breeding research activities: In the 2020 summer testing program, a total of 1,550 (Breeding lines + F2) germplasm were evaluated in 2,751 plots at 6 locations in North Dakota. The canola plot testing locations were distributed across the state, including, Fargo, Prosper, Carrington, Minot, Williston and Hettinger of North Dakota. In Joint-hybrid program with Cibus, 12 hybrids including four commercial hybrid checks were evaluated in a randomized complete design with three replicatesat Prosper, Carrington, and Osnabrock. Wide area yield trial (Conventional) for OP lines: Thirty-six OP lines including four commercial hybrid checks were evaluated in a randomized complete design with three replicatesat Prosper, Carrington, Minot, Osnabrock, Williston and Hettinger. Advanced yield trial (Conventional) for OP lines: Sixty-four OP lines including four commercial hybrid checks were evaluated in a randomized complete design with three replicatesat Prosper, Carrington, Osnabrock, and Minot. Early generation testing (Conventional) for OP lines: Three hundred and twenty-four OP lines including two replicated hybrid checks were evaluated in an augmented design at Prosper, Carrington, and Osnabrock. Single-row breeding nursery for OP lines: A total of 1,092 single rows of B. napus, were planted in a single-row breeding nursery at Prosper. About 500 breeding lines were self-pollinated for generation advancement by using micro-perforated selfing bags and bamboo sticks in the nursery. Greenhouse activities: We usedthree greenhouse rooms in the new AES greenhouse complex for our breeding program. Crossing, backcrossing and selfing are conducting in the greenhouse. We growthree crop cycles in the greenhouse per year. A total of about 10,000 plants were planted and harvested in three growing cycles. Seed quality analysis: Northern Crops Institute (NCI), Fargo, ND has modern equipment and lab facilities for seed quality analysis. All canola entries samples were analyzed by NIR for the seed oil, seed protein and other fatty acid contents such as, palmitic acid, stearic acid, oleic acid, linolenic acid, linolenic acid. We are also calibrating seed oil content using our NMR. Field data: The data on seed yield, seed moisture at harvest, early vigor, days to flowering, relative maturity, standability, lodging, breeder's impression were taken at locations. Sclerotinia: One hundred and fifty-two spring type breeding lines and germplasm accessions were screened against Sclerotinia stem rot disease in the field at Carrington and Osnabrock. Data were collected from42 infected plants per accession. Shattering: One hundred and fifty spring type breeding lines and germplasm accessions with three replications were evaluated for shattering tolerance at Fargo, Carrington and Osnabrock. Data on 180 pods per accession were recorded. 2020 flax research activities are as follows: Uniform Regional Nursery (URN) (F8 generation): 36 entries were evaluated in Fargo, Casselton and Carrington. The URN had 3 replicates,4 rows per plot, and 8ft plot each. The plots were harvested using Wintersteiger combine of canola breeding program. Advanced yield trial (AYT) (F7 generation): 36 entries were evaluated in Fargo, Casselton and Carrington. The AYT had 3 replications, 4 rows per plot, and 8ft plot each. The plots were harvested using Wintersteiger combine of canola breeding program. Intermediate yield trial (IYT) (F6 generation): 100 entries were evaluated in Fargo, Casselton and Carrington. The IYT had 3 replications, 4 rows per 8 ft plot. The plots were harvested using a Wintersteiger combine. Preliminary yield trial (PYT) (F5 generation): 753 entries were evaluated in Fargo. The PYT was planted with replicated checks and2 rows per 8 ft plot.The plots were harvested using a Suzue 2 row binder. Short-row generation (F4 generation): 2,632 short rows (7ft each) were planted in Fargo, and 600 rows were selected based on visual agronomic scores and harvest using a Suzue 1 row binder for 2021 PYT. F3 Bulk population (F3 generation): 96 populations were planted in Fargo. 5,000 individual plants were pulled and threshed for planting the F4 short-row generation in 2021. F2 Bulk Populations (F2 generation): 272 crosses were made and the entire populations were harvested to plant the F3 bulk populations in 2021. The breeding lines in the URN, AYT and IYT were screened for Fusarium wilt in our historical Plot#30 on the NDSU campus. The same germplasm are screened against flax rust in greenhouse. The following are plans for the winter greenhouse 2020-2021: 8 brown-seeded cultivars/advanced lines, CDC Neela, CDC Glas, Webster, NDFB13, NDFB24, NDFB30, NDFB40, NDFB957, will be crossed with each other. 8 yellow-seeded cultivars/advanced lines, Gold ND, CDC Dorado, CDC Bright, Tamp 202, NDFY68, NDFY81, NDFY101, NDFY594, and NDFY603 will be crossed with each other. Development of a MAGIC population for brown-seeded flax is in progress. Development of MAGIC population for yellow-seeded flax is in progress. 2020 Flax plot testing locations under NDSU Oilseed breeding program were Fargo, Casselton, and Carrington. Greenhouse activities: We have been using one greenhouse roomin the new AES greenhouse complex, and two rooms in the NDSU-USDA greenhouse complex. Crossing, backcrossing and selfing are done in the greenhouse. We are growing three crop cycles in the greenhouse per year. Seed oil content is analyzed using our NMR.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Hoque A, Fiedler JD, and Rahman M (2020) Genetic diversity analysis of a Flax (Linum usitatissimum L.) global collection. BMC Genomics, 21: 557. DOI: 10.1186/s12864-020-06922-2. (Corresponding and contributing author; Role: Principal Investigator).
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Arifuzzaman M, Horvath D, and Rahman M (2020) Genome wide association mapping and candidate gene mining for root architectural traits in rapeseed/canola (Brassica napus L.) at late growth stage. Euphytica 216: 164. DOI: https://doi.org/10.1007/s10681-020-02700-z. (Corresponding and contributing author; Role: Principal Investigator).
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Vollmer J, Johnson B, Deckard E, and Rahman M (2020) Evaluation of simulated hail damage on seed yield and agronomic traits in canola (Brassica napus L.). Canadian Journal of Plant Science, https://doi.org/10.1139/CJPS-2020-0036 (Corresponding and contributing author; Role: Principal Investigator).
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Vollmer J, and Rahman M (2020) Effect of Simulated hail damage manifested by stem cutoff on seed yield and agronomic traits in Brassica napus L. Journal of Crop Improvement. 34(4): 470-485, DOI: 10.1080/15427528.2020.1737295. (Corresponding and contributing author; Role: Principal Investigator).
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Arifuzzaman M, Horvath D, and Rahman M (2020) Transcriptome Analysis Suggests Cytokinin and Gibberellin Signaling May Account for Differences Between Spring and Winter Canola (Brassica napus L.) Root development. Journal of Plant Biology. https://doi.org/10.1007/s12374-020-09270-6. (Corresponding and contributing author; Role: Principal Investigator).
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Horvath DP, Zhang J, Chao WS, Mandal* A, Rahman M, Anderson JV (2020) Genome wide association studies and transcriptome changes during acclimation and deacclimation in divergent canola varieties. International Journal of Molecular Sciences, 21: 9148; doi:10.3390/ijms21239148. (Contributing author; helped data interpretation and reviewed manuscript, Role: Committee member).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Hoque* A, and Rahman M (2020) Genetic diversity analysis of a canola (Brassica napus l.) global collection. An abstract for 112th Annual meeting of the North Dakota Academy of Science (Virtual), December 04, 2020 (Corresponding and contributing author; Role: Principal Investigator).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Roy J, Mendoza LEDR, and Rahman M (2020) Genome-Wide Association Study Identifies Genomic Regions Involved in Resistance Against Sclerotinia Stem Rot in Brassica napus (L). An abstract for 2020 International Annual Meetings organized by ASA-CSSA, November 8-11, 2020, in Phoenix, Arizona, USA (Virtual). (Corresponding and contributing author; Role: Principal Investigator).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Roy J, Mendoza LEDR, and Rahman M (2020) Association Mapping Study of Sclerotinia Stem Rot Resistance in Rapeseed/Canola Based on Petiole Inoculation Technique. An abstract for 2020 International Annual Meetings organized by ASA-CSSA, November 8-11, 2020, in Phoenix, Arizona, USA (Virtual). (Corresponding and contributing author; Role: Principal Investigator).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Shaikh TM, Rahman M, Anderson JV, Chao WS, and Horvath DP (2020) Loci and markers identification associated with freezing tolerant trait in Camelina sativa. An abstract for Plant Biology 2020 Worldwide Summit, organized by American Society of Plant Biologists, during July 27-31, 2020, Washington DC (Virtual). (Contributing author; Role: Principal Investigator).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mandal A, Rahman M, Chao W, Anderson J, Horvath DP (2020) Increasing freezing tolerance in winter canola by reducing deacclimation impact on freeze survival. An abstract for Plant Biology 2020 Worldwide Summit, organized by American Society of Plant Biologists, during July 27-31, 2020, Washington DC (Virtual). (Contributing author; Role: Principal Investigator).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Roy J, delRio L, Chittem K, and Rahman M (2020) Genome-Wide Association Study for Sclerotinia Stem Rot Resistance in Brassica napus L. An abstract for National Sclerotinia Initiative, 18th Annual National Sclerotinia Initiative Meeting, during January 22-23, 2020 at Fargo, ND, USA. (Corresponding and contributing author; Role: Principal Investigator).