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TRANSCRIPT
International Workshop on the Promotion of Public‐Private Partnerships for Pre‐breeding
2 – 4 February 2015
Farmer supported research on lentil pre‐breeding: What has been achieved so far? How does the funding model work and is it sustainable?
Albert VandenbergDepartment of Plant Sciences/Crop Development CentreUniversity of Saskatchewan
Tecomango Inc.
Spectrum Specialty Seeds Inc.
Welcome to the
Fourth Dimension…
P-P-P-P
PersonalPublic
Private Partnerships
…..living the dream??Vive le vice‐président!!
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 commonvariety from the USA
• 1978‐80 ‐ first lentil varieties were plant introductions – Laird and Eston‐ these have become “trade names”
• 1972‐1982‐ not much breeding, mostly ALL aspects of agronomy, weed control
A short history of pulse producer involvement in the 1980s…..
‐ 1983 ‐ 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
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 researchin all pulses, especially genetics and marketing efforts
‐ governments in Canada implement 20% budget cuts!!
1997 ‐ first red lentils cultivars released‐ Variety Release Program initiated
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Lentil Production (tonnes)
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Canada
‐ Levy doubles in 1995
‐ MORE research and development needed
‐ Canada unwinds crop subsidies, and a lot more
‐ Ascochyta is joined by anthracnose….
Variety Release Program – 1997The 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!!
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 levyexcept 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!!
Breeding and Genetics
Quality – Visual, Nutritional, Processing
Genomics
Nutrition and Health
Pathology
Marketability
Agronomy and Weed Control
Linkages
Expanded Needs
Many flavours & colours of lentil – whole and split
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Lentil Production (tonnes)
World
Canada
Main Drivers of Successful Crop Development in Saskatchewanwhere 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
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)
Disease Affected Tissues Pathogen OnsetAscochyta 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
The inevitable 30 year march of biota living on lentil
• 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
2001 was a pivotal year – first grant for using crop wild relatives in breeding to investigate anthracnose resistance
Second Main Breeding Strategy for Long Term Yield Enhancement
SCREAMingSystematic Creation of Really Exotic Abnormal Material
otherwise known as pre‐breeding using crop wild relatives
Looking for genes in wild species
L. erv L. ode
L. lam L. ori
L. nig L. tom
L. ervoides L. lamottei
L. tomentosus
L. nigricans
L. odemensis L. orientalis
Ascochyta blight
Anthracnose
Indoor‐ Anthracnose Race Ct0Outdoor‐ Anthracnose Race Ct1/Ct0
Courtesy: A. Tullu
Tullu et al., 2010. Genetic Resources and Crop Evolution 57: 1053‐1063.Tullu et al., 2006. Genetic Resources and Crop Evolution 53: 111‐119.
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
Genetic Resources in Development using wild species of lentil
orientalis tomentosus odemensis lamottei ervoides nigricans
Intraspecific
F4 F4 F4 F5 F10… F2
Interspecific
F10 F3 F6 F2 F11 possible?
1 1 2 2 3 4
Based on new GBS data to be published early in 2015….
Gene Pool
LR‐59 and LR‐26 screening for BIOTIC AND ABIOTIC stress resistance 2014‐2017
Location Stemphyliumblight
Orobanchecrenata Drought
Spain x U Leon, Cordoba (rust?)Morocco x X ICARDAEgypt x? ICARDALebanon x? ICARDATurkey x Harran U, Dicle USaskatoon x X U of SBangladesh x BARI/ICARDAWest Bengal X X IIPR/ICARDAMP X X (RR) IIPR/ICARDABihar X IIPRUP X IIPREthiopia x ICARDA (also rust)
CONFIRMEDTENTATIVE
Funded by Global Crop Diversity Trust
Lentils in Canada
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…
Crop MT/yr% Growth/
40 yrProtein
%
Soybean 276.4 618 43Common bean 23.1 91 23Chickpea 13.1 82 23Pea 11.0 5 23Cow pea 5.7 419 23Lentil 4.7 351 23Pigeon pea 4.1 141 23Faba bean 3.4 ‐16 30People 109 ???
• 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
Lentil Breeding/Pre‐Breeding(Variety Development)
Lentil Pre‐BreedingGenetics (Research, Publication)
Grants
Other grants received from outside agencies: Global Crop Diversity Trust, Provincial Governments, NGOs, others ….
Germplasm exchange and support of collaborators
L. nigricans L. ervoides L. culinaris
If you want to save a species, make sure people want to eat it…
Lentils in Canada
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 economicwealth we create?
Tecomango…
LR‐75 RIL – Lens culinaris x Lens orientalis
• Yield increase rate is about 3% per year• 2014 – 1.1 million ha• 2015 estimate ‐1.5 million ha • Crop planted in May – harvested August
Ascochyta resistance gene transferred to cultivated gene pool?
LR-59 (Eston× L. ervoides L01-827A)• 1%‐ Eston• 21%‐ L01 827A• 12%‐ Transgressive S• 66%‐ Transgressive R
• Successful gene transfer from L. ervoides
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2
4
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14
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Freq
uenc
y of
line
s
Disease severity %
Courtesy: Ehsan Sari
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5
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15
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35
8
23
32
2320
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Freq
uenc
y
Disease severity (%)
IG 72815
Eston
ILL 5888
ILL 8006
LR-26 screening for stemphylium blight in Bangladesh
ILL 5888
LR-26 lines
IG 72815 ILL 5888Courtesy: Rajib Podder
Orobanche species….
Courtesy: Beybin Hacikamiloglu
Orobanche spp.
Parents of LR‐59
Courtesy: Beybin Hacikamiloglu
0,0
5,0
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Field evaluation of LR‐59 interspecific RILs in Kiziltepe, Turkey, 2013LR‐59 = Eston x L01‐827A
% of p
lants infected by
Orobanche
crenata
Note: similar results obtained for LR‐26 = Eston x IG 72815
Data courtesy of Beybin Hacikamiloglu, Harran University (unpublished)
Weed Control Systems
• Herbicide registrations
• Herbicide tolerance(various strategies)
KASP assay results for imi-tolerance gene
BCF2 individuals
TO THE FIELD FOR AGRONOMIC EVALUATION
F1 individuals!
Rapid Generation Technology (breed for speed)
6 generations/year in controlled environments (NOT doubled haploidy)
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
Nutritional Traits of Interest for Lentil
• Polyphenolics (PhD)• Folates (PDF)• Iron (PhD)• Zinc (MSc)• Selenium (multi)• RFOs (PhD• Phytates, et cetera• Proteins (PDF)
Two Main Breeding Strategies for Long Term Yield Enhancement
Sequencing – LOTS and LOTS and then MORE
Sequencing – LOTS and LOTS and then MORE
Assembly – LOTS of Computing Power
Assembly – LOTS of Computing Power
Annotation and
Validation
Annotation and
ValidationUSE ITUSE IT
Genome Sequencing and AssemblyCDC Redberry
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
UofS, UC Davis, ICARDA
Sequencing – LOTS and LOTS and then MORE
Sequencing – LOTS and LOTS and then MORE
Assembly –LOTS of
Computing Power
Assembly –LOTS of
Computing Power
Annotation and
Validation
Annotation and
ValidationUSE ITUSE IT
USE IT
Genome Sequencing and AssemblyCDC Redberry
Lentil Genome Sequencing Update
Kirstin Bett1, Larissa Ramsay1, Crystal Chan1, Andrew Sharpe2, Douglas Cook3, R. Varma Penmetsa3, Peter Chang3, Clarice Coyne4, Rebecca McGee4, DorrieMain5, Albert Vandenberg1
LenGen
GOAL: to establish a lentil genomic reference sequence (CDC Redberry)Focused on a full representation of lentil gene-spaceAnchoring of gene-space 'islands' into pseudomoleculesusing high-density mappingGene prediction and annotationCharacterizing diversity in lentil germplasmsharing of data globally
January 2015 Status Report
Genomic sequencing more or less complete
Pre-release v0.7 now available for queryingHigh-density mapping using GBS underway on several RIL populationsRe-sequencing of diversity panel about to start (~400 lines)
Number of scaffolds 72,451Total size 2.3 GbMax scaffold length 842 KbN50 value 9,289N50 size 66 Kb
Look for Sequences of your Favourite Gene
BLAST against the current Lentil Genome Build
Expand summary to see Alignment
BLAST Hits for multiple Query Sequences
• 1.3 Gb of unique reads from 1st set of PacBio sequencing @ WSU• Has the potential to be useful but need way more reads – which are
coming
PacBio reads
3 Mapping Populations Sequenced using GBS
LR18 – CDC Robin x 964-46a145 individuals + 2 parents18 SE HiSeq 2500 Lanes~9X mean loci coverage per individual
LR03 – ILL 1704 x ILL 7537108 individuals + 2 parents7 SE HiSeq 2500 Lanes
LR86 – L. culinaris x L. orientalis94 individuals7 SE HiSeq 2500 Lanes
LR18 SNP Markers
110,541 Polymorphic Loci MarkersFixed and different between two parental genotypes
51,440 markers called in 90% of non-parental genotypesAcross 14,918 v0.7 scaffoldsTotal of 967MB
13824 scaffolds have <10 markers 1094 scaffolds with >= 10 markers
Contain 16,570 markersTotal of 186MB
0 10 20 30 40 50
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Average Length of Scaffold with x Markers
Num Markers on Scaffold
Aver
age
Leng
th o
f Sca
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(kb)
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040
080
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Total Length of Scaffolds with >=x Markers (MB)
Num Markers on Scaffold
Tota
l Len
gth
of S
caffo
lds
(MB)
186MB
LR-18 SNP Markers - Mapping
MSTMapPlaced into 7 linkage groups15,268 markersTotal of 182MB
Next step is to integrate with the existing LR-18 map
LG1 LG2 LG3 LG4 LG5 LG6 LG7
Num. Markers
2404 1199 1537 3789 3101 2001 1237
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:
http://olomouc.ueb.cas.cz/
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 progress
chromosomeschromatids clumps
2 2 2
1
Genome dissection by chromosome sortingJ. Dolezel, D. Edwards
GBS in Lens spp.
• GBS protocol of Elshire et al. (2011)
• PstI and MspI cuts• 48 individuals, duplicated
• Indexed with barcodes and pooled for IlluminaPE sequencing
• Read mapping and SNP calling using in‐house pipeline and v0.6 genome build
• Filtering
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
Many Helpful Collaborators, Students, Technicians, Colleagues …here is a small sample from a long list…..
University of SaskatchewanNeha VermaMelissa WongLacey SandersonCarolyn Caron Bunyamin Tar’anCarmen BreitkreutzPulse Crop Field Crew
NRC-SaskatoonKevin KohJacek NowakJanet CondieCarling Clarke
UC DavisNoelia Carrasquilla Garcia
Thank you !
Lentils in Canada
Lentils in Canada
Lentils in Canada
International Workshop on the Promotion of Public‐Private Partnerships for Pre‐breeding
2 – 4 February 2015
Lentils in Canada