Farmer supported research on lentil pre breeding: What has been achieved so far? How does the funding model work and is it sustainable?

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2 Farmer supported research on lentil pre breeding: What has been achieved so far? How does the funding model work and is it sustainable? Albert Vandenberg Department of Plant Sciences/Crop Development Centre University of Saskatchewan International Workshop on the Promotion of Public Private Partnerships for Pre breeding 2 4 February 2015

3 Spectrum Specialty Seeds Inc. Welcome to the Fourth Dimension P-P-P-P Tecomango Inc. Vive le vice président!! Personal Public Private Partnerships..living the dream??

4 A short history of producer involvement 1972 Crop Development Centre established at University of Saskatchewan Dr. Al Slinkard (lentil breeder) hired from University of Idaho there were 2 or 3 lentil producers growing and unadapted common variety from the USA first lentil varieties were plant introductions Laird and Eston these have become trade names not much breeding, mostly ALL aspects of agronomy, weed control

5 A short history of pulse producer involvement in the 1980s first Ph.D. student in genetics and breeding 1983 Saskatchewan Pulse Crop Development Board (SPCDB) established by provincial legislation after lobbying by growers Mandatory levy of 0.5% of farm sales remitted to SPCDB by purchasers of all pulse crops 10,000 ha each of pea and lentil total amount approximately $200,000 /yr used to fund research grants based on priorities set by growers and their advisors SPCDB and advisors meet 10 times per year, receive no compensation or travel costs for the next 15 years funding from 1983 to 1991 spent on agronomy and breeding to support production grants were matched, if possible, by asking anyone who looked like they had money 1980s was a drought cycle combined with low prices about half the farmers left the land

6 1991 Hybridization begins in earnest (I was hired) serious lentil breeding started SINGing begins (Systematic Integration of New Germplasm) using cultivated germplasm first strategy for increasing diversity 1990s first biotic stresses appear ascochyta blight development of imidazolinone tolerance with (Cyanamid) BASF 1995 farmers double the levy to 1% to accelerate pace of research in all pulses, especially genetics and marketing efforts governments in Canada implement 20% budget cuts!! 1997 first red lentils cultivars released Variety Release Program initiated

7 Lentil Production (tonnes) World Levy doubles in 1995 MORE research and development needed Canada unwinds crop subsidies, and a lot more Canada Ascochyta is joined by anthracnose Year

8 Variety Release Program 1997 The First PPP Producer Public Partnership Breeder seed of pulses produced in LARGE quantities Released only to Select growers at cost of production No royalties paid seed price at local demand Applicable to lentils and all other pulses (pea, chickpea, dry bean, faba bean) Highly controversial at farm, seed industry and university level!!

9 The Price of PPP Participation 1997 UofS gave commercialization rights (in Canada) to the SPCDB organization for 20 years No other investors could be identified!! Designed the Variety Release Program breeder seed distributed to Select growers (approximately 100 qualified) All breeding programs programs supported by the levy except for some professional salary Canada did not sign UPOV 91 SPCDB agreed to double research funding. Dedicated pulse pathologist hired in 1998 Second breeder in 1999.(now up to 4) FULL PARTICIPATORY BREEDING!!

10 Linkages Expanded Needs Quality Visual, Nutritional, Processing Agronomy and Weed Control Genomics Breeding and Genetics Marketability Nutrition and Health Pathology

11 Many flavours & colours of lentil whole and split

12 Lentil Production (tonnes) World Canada Year

13 Main Drivers of Successful Crop Development in Saskatchewan where 50% of the world s lentils are now produced Appropriate germplasm development system Research and development funding system Participation by producers, who also became exporters Good relations with a patient provincial government Long term vision, extension of basic farming principles Collapse of cereal based subsidy and low price system Focused research priorities with short, medium and long term goals Overdelivery of research as a strategy Freedom to operate (university system?) and anticipate future needs

14 Main Sources of funding for lentil breeding and research 1 Contract with lentil growers for breeding 2 NSERC matching funds for research program 3 Provincial government for research projects 4 Various funding agencies for linked projects 5 Royalty income from seed companies outside Canada 6 Royalty income from BASF (herbicide tolerance)

15 The inevitable 30 year march of biota living on lentil Disease Affected Tissues Pathogen Onset Ascochyta blight leaf, pod, stem Ascochyta lentis 1980s Anthracnose stem Colletotrichum truncatum 1990s Gray mold foliage, all Botrytis cinerea late 90s White mold stems, pods Sclerotinia sclerotiorum late 90s Stemphylium blight stems, leaves Stemphylium botryosum 2005 Fusarium rot roots, canopy Fusarium avenaceum droughts Aphanomyces roots, canopy Aphanomyces euteiches 2010 Rust canopy Uromyces viciae fabae not yet Orobanche spp. plant O. crenata; O. foetida not yet

16 2001 was a pivotal year first grant for using crop wild relatives in breeding to investigate anthracnose resistance Small startup grant to find disease resistance for anthracnose started interspecific hyridization as soon as possible Most early work done by graduate students highly motivated Use parallel research to integrate commercially relevant strategies Key to success: Undersell, overdeliver and understand economic value

17 Second Main Breeding Strategy for Long Term Yield Enhancement SCREAMing Systematic Creation of Really Exotic Abnormal Material otherwise known as pre breeding using crop wild relatives

18 Looking for genes in wild species Courtesy: A. Tullu Outdoor Anthracnose Race Ct1/Ct0 L. ervoides L. lamottei Indoor Anthracnose Race Ct0 L. nigricans Ascochyta blight L. erv L. ode L. lam L. ori Anthracnose L. tomentosus L. odemensis L. orientalis L. nig L. tom Tullu et al., Genetic Resources and Crop Evolution 53: Tullu et al., Genetic Resources and Crop Evolution 57:

19 Lens culinaris x Lens ervoides hybrids Resistance to anthracnose no resistance was accessible in cultivated lentil Transfer of resistance to ascochyta blight confirmed Transfer of resistance to stemphylium documented Evidence for transfer of tolerance to Aphanomyces root rot Evidence for transfer of tolerance to Orobanche spp. Rust resistance? Will be checked Fusarium resistance? Can be checked. Some evidence for substantial yield increase

20 Genetic Resources in Development using wild species of lentil Gene Pool Based on new GBS data to be published early in orientalis tomentosus odemensis lamottei ervoides nigricans Intra specific F4 F4 F4 F5 F10 F2 Inter specific F10 F3 F6 F2 F11 possible?

21 LR 59 and LR 26 screening for BIOTIC AND ABIOTIC stress resistance Location Stemphylium blight Orobanche crenata Drought Spain x U Leon, Cordoba (rust?) Morocco x X ICARDA Egypt x? ICARDA Lebanon x? ICARDA Turkey x Harran U, Dicle U Saskatoon x X U of S Bangladesh x BARI/ICARDA West Bengal X X IIPR/ICARDA MP X X (RR) IIPR/ICARDA Bihar X IIPR UP X IIPR Ethiopia x ICARDA (also rust) CONFIRMED TENTATIVE Funded by Global Crop Diversity Trust

22 A little know fact: Vavilov married his Ph.D. graduate student Elena Barulina who published her thesis on the collection of lentils, including wild lentils, in Afghanistan Lentils in Canada

23 Crop MT/yr % Growth/ 40 yr Protein % Soybean Common bean Chickpea Pea Cow pea Lentil Pigeon pea Faba bean People 109???

24 28 lentil breeding programs in the world 1 of them supplies 50% or world lentils CDC has 100% market share in Canada, more than 50% in USA, unknown amount in RUK Genome Canada application in development to understand the genomics/genetics of lentil in major climatic zones photoperiod, temperature responses GCDT project will be the international engine for introgression into the various adaptation pools

25 Other grants received from outside agencies: Global Crop Diversity Trust, Provincial Governments, NGOs, others. Germplasm exchange and support of collaborators Lentil Breeding/Pre Breeding (Variety Development) Lentil Pre Breeding Genetics (Research, Publication) Grants

26 If you want to save a species, make sure people want to eat it L. nigricans L. ervoides L. culinaris

27 Will support for pre breeding from farmers last? Is farmer support still needed if we sign UPOV 91? How do we share the biological and economic wealth we create? Lentils in Canada

28 Tecomango

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30 LR 75 RIL Lens culinaris x Lens orientalis

31 Yield increase rate is about 3% per year million ha 2015 estimate 1.5 million ha Crop planted in May harvested August

32 Frequency of lines Ascochyta resistance gene transferred to cultivated gene pool? LR-59 (Eston L. ervoides L01-827A) % Eston 21% L01 827A 12% Transgressive S 66% Transgressive R Successful gene transfer from L. ervoides Disease severity % Courtesy: Ehsan Sari 32

33 LR-26 screening for stemphylium blight in Bangladesh 35 Eston 32 ILL 8006 Frequency IG ILL Disease severity (%) ILL 5888 LR-26 lines Courtesy: Rajib Podder IG ILL 5888

34 Orobanche species.

35 Orobanche spp. Courtesy: Beybin Hacikamiloglu

36 Courtesy: Beybin Hacikamiloglu Parents of LR 59

37 Field evaluation of LR 59 interspecific RILs in Kiziltepe, Turkey, 2013 LR 59 = Eston x L01 827A % of plants infected by Orobanche crenata 50,0 45,0 40,0 35,0 30,0 25,0 20,0 15,0 10,0 5,0 0,0 Data courtesy of Beybin Hacikamiloglu, Harran University (unpublished) Note: similar results obtained for LR 26 = Eston x IG 72815

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39 Weed Control Systems Herbicide registrations Herbicide tolerance (various strategies)

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41 KASP assay results for imi-tolerance gene BCF2 individuals F1 individuals! TO THE FIELD FOR AGRONOMIC EVALUATION

42 Rapid Generation Technology (breed for speed) Average timelines in days for lentil generation cycle using rapid generation technology. D0 D2 3 D3 4 D31 33 D37 39 D45 46 Seeding Germination Transfer to substrate Flowering Pod development Embryo rescue 6 generations/year in controlled environments (NOT doubled haploidy)

43 Nutritional Traits of Interest for Lentil Polyphenolics (PhD) Folates (PDF) Iron (PhD) Zinc (MSc) Selenium (multi) RFOs (PhD Phytates, et cetera Proteins (PDF)

44 Two Main Breeding Strategies for Long Term Yield Enhancement

45 Genome Sequencing and Assembly CDC Redberry UofS, UC Davis, ICARDA Sequencing LOTS and LOTS and then MORE Assembly LOTS of Computing Power Annotation and Validation USE IT NGS: Illumina, 454, PacBio Paired ends Long mate pairs Very long reads Server with 1TB RAM 300 TB storage Database with genotypic and phenotypic data (KnowPulse ) 2015

46 Genome Sequencing and Assembly CDC Redberry Sequencing LOTS and LOTS and then MORE Assembly LOTS of Computing Power Annotation and Validation USE IT USE IT

47 Lentil Genome Sequencing Update Kirstin Bett 1, Larissa Ramsay 1, Crystal Chan 1, Andrew Sharpe 2, Douglas Cook 3, R. Varma Penmetsa 3, Peter Chang 3, Clarice Coyne 4, Rebecca McGee 4, Dorrie Main 5, Albert Vandenberg 1

48 LenGen GOAL: to establish a lentil genomic reference sequence (CDC Redberry) Focused on a full representation of lentil gene-space Anchoring of gene-space 'islands' into pseudomolecules using high-density mapping Gene prediction and annotation Characterizing diversity in lentil germplasm sharing of data globally

49 January 2015 Status Report Genomic sequencing more or less complete Number of scaffolds 72,451 Total size 2.3 Gb Max scaffold length 842 Kb N50 value 9,289 N50 size 66 Kb Pre-release v0.7 now available for querying High-density mapping using GBS underway on several RIL populations Re-sequencing of diversity panel about to start (~400 lines)

50 Look for Sequences of your Favourite Gene Expand summary to see Alignment BLAST against the current Lentil Genome Build BLAST Hits for multiple Query Sequences

51 PacBio reads 1.3 Gb of unique reads from 1 st set of PacBio WSU Has the potential to be useful but need way more reads which are coming

52 3 Mapping Populations Sequenced using GBS LR18 CDC Robin x a 145 individuals + 2 parents 18 SE HiSeq 2500 Lanes ~9X mean loci coverage per individual LR03 ILL 1704 x ILL individuals + 2 parents 7 SE HiSeq 2500 Lanes LR86 L. culinaris x L. orientalis 94 individuals 7 SE HiSeq 2500 Lanes

53 LR18 SNP Markers 110,541 Polymorphic Loci Markers Fixed and different between two parental genotypes 51,440 markers called in 90% of non-parental genotypes Across 14,918 v0.7 scaffolds Total of 967MB scaffolds have <10 markers 1094 scaffolds with >= 10 markers Contain 16,570 markers Total of 186MB Average Length of Scaffold (kb) Total Length of Scaffolds (MB) Average Length of Scaffold with x Markers Num Markers on Scaffold Total Length of Scaffolds with >=x Markers (MB) 186MB Num Markers on Scaffold

54 LR-18 SNP Markers - Mapping MSTMap Placed into 7 linkage groups 15,268 markers Total of 182MB LG1 LG2 LG3 LG4 LG5 LG6 LG7 Num. Markers Next step is to integrate with the existing LR-18 map

55 BAC sequencing Test run of 24 BACs Unfortunately fragment sizes were small so it was tricky to assemble BUT Pretty solid BAC to scaffold relationship in many cases And then there are the repeats:

56 Genome dissection by chromosome sorting J. Dolezel, D. Edwards Chromosome genomics for lentil (n = 7) Simplification of genome analysis Improvement of genome assembly Preliminary results indicate separation of 6 chromosome types in pairs and 1 single chromosome type Assignment of chromosome to the peaks in chromatids chromosomes progress clumps

57 GBS in Lens spp. GBS protocol of Elshire et al. (2011) PstI and MspI cuts 48 individuals, duplicated Indexed with barcodes and pooled for Illumina PE sequencing Read mapping and SNP calling using in house pipeline and v0.6 genome build Filtering

58 Consequences Many previous publications on wild species report misclassified germplasm Casts some doubt on previous reports of interspecific hybrids Will save time and effort in future

59 Many Helpful Collaborators, Students, Technicians, Colleagues here is a small sample from a long list.. Thank you! University of Saskatchewan Neha Verma Melissa Wong Lacey Sanderson Carolyn Caron Bunyamin Tar an Carmen Breitkreutz Pulse Crop Field Crew NRC-Saskatoon Kevin Koh Jacek Nowak Janet Condie Carling Clarke UC Davis Noelia Carrasquilla Garcia

60 Lentils in Canada

61 Lentils in Canada

62 Lentils in Canada

63 International Workshop on the Promotion of Public Private Partnerships for Pre breeding 2 4 February 2015

64 Lentils in Canada