UCD LETTUCE BREEDING PROGRAMS...

To monitor pathogen populations to ensure effectiveness of resistance genes in breeding programs.

To identify new genes for resistance in wild germplasm and incorporate them into advanced breeding lines.

To determine the genetic basis of agriculturally important traits, particularly disease resistance.

To advance crisphead and leafy lines with resistance to multiple diseases and good horticultural traits.

To develop and apply molecular markers for increasing the efficiency of breeding for disease resistance.

UCD LETTUCE BREEDING PROGRAMS

OBJECTIVES

SAMPLING OF LDM ISOLATES

or

Participation in the Lettuce Downy Mildew Survey in California and Yuma, Arizona by notifying Aubrey Kenefick when LDM occurs in the field and either:

• Assist UCD employee(s) during monthly field collections or • Send LDM samples to UCD when occurs

Typing Lettuce Downy Mildew Isolates Collected from Field

Increase on cv. Green Towers No known Dm resistance genes

3 to 4 weeks

Inoculation of Differential Series of Resistant Cultivars

4 weeks

Screen for Metalaxyl (in)sensitivity

3 weeks

Test for Mating Type Co-inoculate with B1 and B2

isolates ~4 weeks

Storage - 80 °C long term storage as spore

suspension - 20 °C short term storage as sporulating

seedlings Spore collection for

DNA 3 weeks

Sequence on Illumina

platform ~6 weeks

Sequence analysis in

CLC Workbench

? weeks

?

Annual Frequency of CA Pathotypes

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017

CAVCAVICAVIICAVIIICAIX*vir37*CAnovel

VP groups under nomination (*) do not make up the majority of the CA Novel phenotypes Includes 193 isolates phenotyped by IBEB-participant companies from 2012-17.

Effectiveness of Dm genes 2001 to present

100% susceptible ~50:50 100% resistant

(In)sensitivity of B. lactucae in California and Arizona to mefanoxam (Ridomil)

Sensitive = no sporulation on ≥ 5 ppm at 15 dpi Intermediate = sporulation on 5 or 10 ppm at 15 dpi Delayed insensitive = sporulation on ≥50 ppm at 15 dpi, but not at 7 dpi Insensitive = sporulation on ≥5 ppm at 7 dpi and on 200 ppm at 15 dpi

29%

3%

4%

63%

1%

2016 (N = 111)

38%

62%

2017 (N = 21)

30%

7%

37%

17%

9%

2015 (N = 187)

Sensitive

Intermediate

Delayedinsensitive

Insensitive

Not Tested

Cayla Tsuchida, Aubrey Kenefick

Prevalence of mating types (2006-present)

B1 rare B2 >> B1

O. Ochoa, C. Tsuchida, A. Kenefick

0

20

40

60

80

100

120

140

160

180

200

2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017

# Is

olat

es

Year

No Data

B2

B1

Sexual cycle as a source of variation

Cayla Tsuchida

Genetic potential of sexual reproduction Similarity tree based on virulence phenotypes of progeny and field isolates Field (black text) and progeny isolates of CrA (blue text) and CrB (green text). The common B1 parent (red text) and B2 parents of CrA (purple text) and CrB (orange text) are marked.

Sequence analysis of variation in Bremia lactucae

• 107 isolates sequenced so far, including 28 BL races

• Geographical sampling:

• USA, Europe, Australia

• Temporal series: • 1960’s - 2014

• High level variation in virulence phenotypes

• Clonal lineages with changes in virulence phenotype

Juliana Gil

Isolate Origin Date of isolation

Pathotype/ Race

Mating type Isolate Origin Date of

isolationPathotype/

RaceMating

typeSF5 Finland B1 C90D46 Salinas, CA 1990 CAIIb B2

49.183 Europe C90D55 Salinas, CA 1990 CAIIb B26B F1 IM25R7 x CGI 1979 B2 C91D39 Salinas, CA 1991 CAIV B27C F1 IM25R7 x CGI 1979 B2 C91D49 Salinas, CA 1991 CAIV B2AM Australia C92O47 Santa Maria, CA 1992 CAnovel B2

CG1 Switzerland B2 C98O648 Salinas, CA 1998 CAVII B2CS12 Czech Republic C98O622 King City, CA 1998 CAV B2CS5 Czech Republic C98O622b derived from C98O622 (King City, CA) 1998 CAVI B2CS9 Czech Republic C99S760 Santa Maria, CA 1999 CAV B2IL4 Israel B1 C99S762 Oceano, CA 1999 CAV B2

IM25P11 B2 SSD of IM25 1978 B2 C99S763 Salinas, CA 1999 CAV B2IM25R7 B1 SSD of IM25 1978 B1 C99S764 Salinas, CA 1999 CAV B2

KT2 Germany C01O879 California 2001 CAVIII B2NL1 Netherlands C04O1017 Salinas, CA 2004 CAnovel NTNL6 Netherlands C05R1034 Yuma, AZ 2005 CAnovel B2NL7 Netherlands C06O1181 Salinas, CA 2008 CAnovel B2R60 UK C11O1342 Moss Landing, CA 2011 CAVII B2S1 Sweden C11O1352 California 2011 CAVII NT

SF3 Finland C10D1273 Watsonville, CA 2011 CAnovel B1TV UK C11O1321-B King City, CA 2011 CAnovel B1BL1 BL1 C11O1326 California 2011 CAVIII NTBL2 BL2 C11O1324 California 2011 CAVIII B2BL3 BL3 C11O1343 San Ardo, CA 2011 CAVII B2BL4 BL4 C12O1363 Yuma, AZ 2012 CAVIII B2BL5 BL5 C12O1365 California 2012 CAnovel B1BL6 BL6 C12O1376 King City, CA 2012 CAVII B2BL7 BL7 C12O1385 Salinas, CA 2012 CAIX B2BL10 Netherlands BL10 C12A1407 California 2012 CAVII B2BL12 Netherlands BL12 C12O1377 King City, CA 2012 CAVI B2BL13 Netherlands BL13 C12O1393 California 2012 CAVII B2BL14 Netherlands BL14 C13A1410 King City, CA 2013 CAnovel B2BL15 BL15 C13A1411 King City, CA 2013 CAnovel B2BL16 Netherlands BL16 C13A1412 King City, CA 2013 CAnovel B2BL17 Switzerland 1999 BL17 C13C1414 Castroville, CA 2013 CAIX B2BL18 United Kingdom 1999 BL18 C13A1408a derived from 1408 (Santa Maria, CA) 2013 CAnovel B2BL19 France 1999 BL19 C14C1428 Santa Maria, CA 2014 CAnovel B2BL20 GDR 1999 BL20 C14C1444 Brawley, CA 2014 CAV B2BL21 Italy 1999 BL21 C14C1469 Huron, CA 2014 CAnovel B2BL22 Netherlands 2000 BL22 C14C1460 Yuma, AZ 2014 CAVII B2BL23 France 2001 BL23 C14C1470 Huron, CA 2014 CAnovel B2BL24 Netherlands 2002 BL24 C14C1490 Soledad, CA 2014 CAVII B2BL25 Netherlands 2004 BL25 A14C1474 Yuma, AZ 2014 CAnovel B2BL26 Netherlands 2008 BL26 1474-filtered derived from 1474 (Yuma, AZ) 2014 CAnovel B2BL27 France 2010 BL27 C14C1485 King City, CA 2014 CAnovel B1BL28 France 2011 BL28 C14C1486 King City, CA 2014 CAnovel B2BL29 2013 BL29 C14C1549 California 2014 CAnovel B2BL30 2013 BL30 C15C1689 Watsonville, CA 2015 CAVII B2BL31 2013 BL31 C15C1691 Santa Maria, CA 2015 CAnovel B2

C82P24 Ventura, CA 1982 CAIIa B2 A15C1701 Yuma, AZ 2015 CAVII B2C83M47 Salinas, CA 1983 CAIII B2 C15C1764 San Lucas, CA 2015 CAnovel B2C83M40 Moorpark, CA 1983 CAI B1 C15C1771 Watsonville, CA 2015 CAVII B1C83M42 Salinas, CA 1983 CAI B1 C15C1806 Chualar, CA 2015 CAVII B1+B2C89D36 Fresno, CA 1989 CAIIb B2 C16C1832 Davis, CA 2016 CAnovel B1C90D33 Salinas, CA 1990 CAIII B2

Heterokaryosis is common in Bremia lactucae

8 isolates 8 isolates 3 isolates

0.2 0.8 0.2 0.8 0.2 0.8

Selection will act on populations of nuclei in a coenocytic mycelium rather than on discrete isolates. Explains instability of isolates.

Multiple SNP profiles: Diploid profile “Triploid” profile “Tetraploid” profile

But measurements with flow cytometry => nuclei all same size Single sporing and sequencing sexual and asexual progeny => heterokaryosis not polyploidy

Kyle Fletcher, Lien Bertier

Variation in Mitochondria of B. lactucae

Frank Martin

Data used for distance matrix Phenotype Mitochondrial

Mito

chon

dria

l Phy

loge

ny

P

heno

type

Phy

loge

ny

Western US Europe Australia

Distantly related Closely related Cayla Tsuchida

Future Prospects & Long-term Plans For Lettuce Downy Mildew

All known and many new Dm genes being sequenced. More Dm genes being deployed. ⇒ Selection will increase pathogen diversity -> more virulence phenotypes. ⇒ More opportunities to detect variants in pathogen. Presence of both mating types in EU and CA => big increase in variation. Need continued monitoring for variation. Pathogen sequencing ~expensive but highly informative. Diagnostic molecular markers for races and pathotypes. Aim 1 to 2 day turn-around, sequencing of novel phenotypes. Diagnostic marker for fungicide (Ridomil) insensitivity. Real-time distribution & access to data over www. New data will require new criteria for declaring new BL races or Pathotypes. New strategy for classifying variation in B. lactucae and resistance in lettuce.

QTLsChr. position

/MRCSource species &

cultivar/ID Notes

qDMR2.1 Chr2 L. sativa , Iceberg Field Resistance. Identified in Grand Rapids x Iceberg.

qDMR5.1 Chr5 L. sativa , Iceberg Field Resistance. Identified in Grand Rapids x Iceberg.

qDMR9.1 MRC9A L. sativa , Grand Rapids Field Resistance. Identified in Grand Rapids x Iceberg.

qDMR2.2 MRC2 L. sativa , La Brillante Seedling and adult plant resistance. Identified in Salinas 88 x La Brillante.

qDMR4.1 Chr4 L. sativa , La Brillante Seedling and adult plant resistance. Identified in Salinas 88 x La Brillante.



qDMR9.2 MRC9A L. sativa , La Brillante Seedling and adult plant resistance. Identified in Salinas 88 x La Brillante.

qDMR7.2 Chr7 L. saligna , CGN05271 Seedling and adult plant resistance. Identified in CGN5271 x L. sativa cv. Olof.


qDMR9.3 MRC9A L. saligna , CGN05271 Seedling and adult plant resistance. Identified in CGN5271 x L. sativa cv. Olof.

qDMR3.1 MRC3 L. saligna , CGN05271 Seedling and adult plant resistance. Identified in CGN5271 x L. sativa cv. Olof.




Sequential designations 1 to 52 Original sources Current type cultivars Diagnostic isolates Primary citations for source genetics linkage Proposal for future Dm nominations

Parra et al. (2016) Euphytica 210:309-326

Iceberg & Leafy types introgression programs for LDM resistance Advanced breeding lines

Intermediate breeding lines

Early breeding lines

Source Donor species Lettuce type StatusPI491000 L. saligna Green Leaf Testing for R CA isolates05G1411 L.serriola Green Leaf Testing for R CA isolatesISR-380 L. serriola Romaine BC5

CGN5309 L. serriola Green Leaf BC5

CHEC-023 L. serriola Iceberg BC4



CHEC-147 L. saligna Romaine BC3

CHEC-101 L. saligna Romaine BC2

CHEC-136 L. saligna Red Leaf BC3

RUSS-635 L. serriola Red Leaf BC3

KYRGY-237 L. serriola Romaine BC3

GEOR-301 L. serriola Romaine BC3

GEOR-289 L. serriola Romaine BC3

GEOR-297 L. serriola Red Leaf BC3

GEOR-292 L. serriola Green Leaf BC3

GEOR-299 L. serriola Iceberg BC3







05G1421 L.serriola Butterhead BC3

CHEC-158 L. saligna Green Leaf BC2

KYRGY-247 L. serriola Butterhead BC2



CGN13330 L. saligna Romaine BC1

RUSS-653 L.serriola Butterhead BC1

GEOR-282 L. serriola Butterhead BC1

GEOR-284 L. serriola Red Leaf BC1

GEOR-288 L. serriola Iceberg BC1

CGN5157 L. saligna Green Leaf BC1

ARM09-158 L. serriola crisphead BC1



CGN5301 L. saligna romaine F1

Maria-Jose Truco Pauline Saunders

• Phenotyping in greenhouse sick plots • 96 RILs, 6 plants/rep, 3 reps • Phenotype: whole plant root discoloration stem culture on PDA for clean roots

Analysis of PI251246 x Armenian L. serriola RIL for Verticillium resistance

• Phenotypic analysis done at Davis and USDA, Salinas (German Sandoya)

• Genotyping by GBS Maria-Jose Truco Pauline Saunders

Resistance to Verticillium race 2

2 populations: PI251246 x Arm L. serriola: UCD trial & USDA trial PI171674 x PI204707: USDA trial

Major QTL in LG6 detected in both populations and trials qVER

T3.1

qVERT4.1

qVERT4.2

qVERT6.1

qVERT6.1

qVERT6.1

LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9

qVERT8.1

qVERT8.1

.2

~22%

Maria José Truco German Sandoya

Fusarium trial summer 2016

Tested uniformity of resistant selections 54 lines selected for uniformity Collaboration with Tom Gordon Re-tested 54 in Fusarium-infected field 45 homozygous resistant 9 segregated Seed increase of 45 selected lines Molecular marker genotyping Anticipated release in early 2017

Segregating population from USDA Collaboration with Jim McCreight

King Louie x Autumn Gold 90 F2:3 families Good correlation between trials

Maria José Truco

Maria José Truco

QTLS for resistance to Fusarium on King Louie x Autumn Gold F2 population

1 2 3 4 5 6 7 8 9

Davis 2016

USDA 2010 USDA 2012

Different resistant QTLs were observed in the three field trials

QTLS FOR RESISTANCE TO FUSARIUM RACE 1 IN LINKAGE GROUPS 1, 2, 3,& 4 LG1 LG2 LG3 LG4

POPULATIONS Salinas x L. serriola (RIL) WGS F1 ValxSal x Salinas (RIL) 384 SNP panel Red Tide x Lolla Rosa (F2) GBS Lolla Rosa x Salinas (F2) GBS Salinas x Green Towers (F2) GBS King Louie x Autumn Gold (F2) GBS

Maria José Truco

RESISTANT QTL ALLELES Valmaine, Green Towers & King Louie Lolla Rosa Salinas Autumn Gold

QTLS FOR RESISTANCE TO FUSARIUM RACE 1 IN LINKAGE GROUPS 5, 7, 8, & 9 LG5 LG6 LG7 LG8 LG9

Maria José Truco

POPULATIONS Salinas x L. serriola (RIL) WGS F1 ValxSal x Salinas (RIL) 384 SNP panel Red Tide x Lolla Rosa (F2) GBS Lolla Rosa x Salinas (F2) GBS Salinas x Green Towers (F2) GBS King Louie x Autumn Gold (F2) GBS

RESISTANT QTL ALLELES Valmaine, Green Towers & King Louie Lolla Rosa Salinas Autumn Gold

Head firmness Tipburn Incidence Fresh Plant Weight Leaf Serration Tipburn Severity Miguel Macias Gonzalez

4 QTLs detected in LGs 2, 5, 7, and 8 (2, 5, and 8 previously ID in EMP x ELD)

QTL mapping of Tip Burn in Salinas x Calicel RIL population

Fine mapping of tipburn QTL in Yuma and Salinas Yuma

DDDDD DDDEE DEEEE EEDDD EEEED EEEEE HHDDD

Haplotype Tipburn incidence by haplotype D = El Dorado allele (resistant); E = Emperor allele (susceptible) Seven recombinants (DDDEE, EEDDD, & HHDDD) have been sequenced A genomic region with 21 genes associated with the resistance has been identified

Miguel Macias Gonzalez

DDDDD DDDEE DEEEE EEDDD EEEED EEEEE HHDDD Salinas

Collaboration with Matt Colgan & Chi Nguyen

LiDAR with 32 near-infrared lasers images canopy at 720,000 points/sec -> plant height and volume. Multispectral RGB + NIR cameras measure surface reflectance -> leaf area and growth rate. 94 RILs Grand Rapids x Iceberg, 16 cultivars 3 replicates, 3 nitrogen levels (30, 110, 230 lbs/acre) Four drone flights: July 11, 17 days after planting, 2 before thinning July 15, 2 days after thinning August 1, stand count measured August 22, 2 days before harvest Harvested August 24. Phenotypes measured on harvested heads: head weight, dry weight, N, P, K content. Correlation between drone and ground data. Already genotyped. QTL analysis.

Miguel Macias-Gonzales et al. Funded by NIFA SCRI

Variation in head weight for Grand Rapids, Iceberg & 6 RILs NUE experiment Salinas 2016.

Average plant weight (g) plotted against each nitrogen treatment LN = Low Nitrogen, MN = Middle Nitrogen, HN = High nitrogen

Treatments provided nitrogen stress. Miguel Macias Gonzalez

Data from Blue River collaboration • Blue River has recently delivered estimates of

biomass derived from remote sensing data collected from drone.

• Data currently being analyzed to see how well correlated with ground data.

• Data will be used to conduct QTL analysis to test if the QTL identified by the drone and ground data are the same.

1

2

3

4

5

6

7

8

9

Genomic Distribution of Disease Resistance, Developmental & Physiological Phenotypes

cloned targeted for cloning

Disease Resistance Phenotypes Downy Mildew Lettuce Mosaic Virus Anthracnose Root aphid Plasmopora lactucae-radicis Corky root Fusarium Verticillium Die Back LDM Field Resistance Developmental Phenotypes Bolting, Flowering Leaf shape Heading Side shoots Rosette Spines Capitulum shattering Physiological Phenotypes Seed Thermotolerance Tip Burn Heat Tolerance Shelf Life Biomass

Genotyping by Sequencing (GBS)

Maria Jose Truco, Lien Bertier & Adolfo Vargas

Cross generation size Traits Collaboration Analysis StatusRed Tide x Lolla Rosa F2:3 112 Fusarium resistance UCD Analyzed

Lolla Rosa s Salinas F2:3 96 Fusarium resistance UCD Analyzed

Salinas x Green Towers F2:3 96 Fusarium resistance UCD Analyzed

Salinas x Calicel RIL7 382 Tip Burn UCD AnalyzedPI251246 x Armenian L. serriola RIL7 193 Verticillium resistance UCD Analyzed

PO141674 x PI204707 189 Verticillium resistance USDA,G. Sandoya AnalyzedGrand Rapids x Iceberg RIL7 94 LDM field resistance & NUE/WUE USDA, I. Simko Analyzed

Grand Rapids x Salinas RIL7 110 LDM field resistance UCD Analyzed

Iceberg x PI491224 RIL7 157 LDM field resistance UCD AnalyzedReine des Glaces x Delsay 128 Xanthomonas resistance INRA (France), B. Maisonneuve Analyzed

Reine des Glaces x EruptionRIL6

163Lettuce drop resistance, bacterial leaf spot resistance, morphology

USDA, R. Hayes Analyzed

RH08-0111 x PI491108-1 F2 350 XCV resistance to strain BS3127 USDA, R. Hayes Waiting for data

Vanda x Gisele F2 150 Fusarium resistance UNB (Brazil), C. Cabral Analyzed

PI171674 x Salinas F2 150 Verticillium resistance USDA,G. Sandoya AnalyzedSalinas x L. serriola , core pop. RIL7 317 Domestication traits UCD Analyzed

King Louie x Autumn Gold F2 84 Fusarium resistance USDA, J. McCreight Analyzed

Ninja x Valmaine F2 130 Effector response UCD Analyzed

PI358001-1 x Tall Guzmaine F2 163 Bacterial Leaf Spot resistance UFL, German Sandoya to be Sequenced

New sources of resistance effective against most isolates of Bremia lactucae (downy mildew) being sequenced

25 UCD releases 1986-2012 3 advanced breeding lines 40 differential cultivars

Lorena Parra

UC

0710

5

UC

0710

6

UC

0710

7

UC

0710

8

UC

0220

2

UC

0220

3

UC

0220

4

UC

0220

5

UC

0220

6

UC

1210

0

UC

1210

1

UC

1210

2

UC

1210

3

DM gene new new new new new new new new new new new new new

CAV(C04?1017) - - - - - - - - - - - - -CAVI(C05R1034) - - - - - - - - - - - - -CAVII(C98O648) - - - - - - - - - - - - -CAVIII(C01O879) - - - - - - - - - - - - -CAIX(C11O1327) - - - - - - - - - - - - -CAVIII-879 - - - - - - - - - - - - -CAVII-1435 - - - - - + - - - - - - -CAIIa-P24 - - - - - + - - - - - - -CAIII-M47 - - - - - + - - - - - - -CAV-1452 - - - - - + - - - - - - -Novel-1557 - - - - - + - - - - - - -Novel-1481 - - - + - + - - - - - - -Novel-1324 - - - - - + - - - - - - -Novel-1326 - - - + - + - - - - - - -Novel-1485 - - - - - - - - - - - - -Novel-1622* + + - + + + + + + + - + -Novel-1690* + + - + + + + + + + - + -novel-1549 - - - - - + - - - - - - -C15L1744 - (+) - + - + - - - - - - -C15L1746 - - - - - + - - - - - - -C15L1745 - - - - - + - - - - - - -C15L1742 (pi) - (+) (+) + - + - - - - (+) - -C15L1743( ice) - - - - - + - - - - - - -C15L1749 - - + + - - - - - - + + -C15L1752 + - + + - (+) - - - - (-) + -

A) Chromosome 1 for line UC02-105. The introgression is located in the site corresponding to MRC1. B) Chromosome 2 for line UC12-101. The introgression is located in the site corresponding to MRC2. C) Chromosome 3 for line UC12-103. The introgression is located in the site corresponding to MRC3.

Breeding lines showing potential introgressions of regions encoding the Major Resistance Clusters (MRC) on chromosomes 1, 2 and 3.

C

MRC3

Whole genome sequencing approach to identify chromosome segments introgressed from wild species during breeding for DM resistance.

Lorena Parra

PI491224 Romaine type

Highly susceptible

Iceberg Batavia type

Highly resistant in the field

160 RILs

x

self 7x

F1

F2

Identification of major QTLs that control field resistance in lettuce Collaboration with Ivan Simko

00.5

11.5

22.5

33.5

IP-1

68IP

-ICE

IP-1

28IP

-175

IP-1

78IP

-227

IP-1

97IP

-201

IP-2

07IP

-136

IP-1

98IP

-210

IP-1

66IP

-211

IP-1

09IP

-232

IP-2

25IP

-221

IP-2

19IP

-170

IP-2

46IP

-204

IP-1

91IP

-213

IP-1

47IP

-230

IP-1

12IP

-217

IP-1

26IP

-193

IP-1

69IP

-250

IP-2

05IP

-192

IP-1

35IP

-145

IP-2

02IP

-214

IP-1

06IP

-153

IP-2

43IP

-183

IP-1

74IP

-165

IP-2

40IP

-259

IP-2

03IP

-187

IP-1

59IP

-140

IP-1

25IP

-244

IP-P

IIP

-167

ICExPI DM severity in the field at harvest maturity

0102030405060

0 0.33 0.66 1 1.33 1.66 2 2.33 2.66 3 3.33

DM scores (average reps)

Lorena Parra

Identification of major QTLs that control field resistance in lettuce: Genotyping and QTL detection

So far, 1 QTL detected in Chromosome 5, that is co-located with qDMR5.1 previously detected in a RIL population originating from Iceberg x Grand Rapids

Lorena Parra

Overview of the L. sativa cv. Salinas genome A. Number of scaffolds in 1 Mb

intervals. Blue, SOAPdenovo scaffolds; Red, HiRise superscaffolds.

B. Chromosomal pseudomolecules. Dark areas indicate 63% of genome positioned and oriented accurately.

C. Gene density.

D. Repeat density.

E. Density of SNPs used for genetic map construction.

F. Size of tandem gene arrays. Black blocks show MRC regions.

Colored lines in center link syntenic blocks of at least five genes derived from whole genome triplication.

Reyes-Chin-Wo et al. Nat. Comm. 2017

Total genome size = 2.7 Gb 88% assembled 65% repeated elements 97% genetically validated N50 = 6.5 Mb (Dovetail)

International Lettuce Genomics Consortium (ILGC) March 2015 - 2018

Objectives • To generate a high quality lettuce genome reference sequence with a

minimal number of ordered scaffolds that span the nine lettuce chromosomes.

• To map qualitative and quantitative traits of agricultural importance relative to candidate genes that can be used as markers for marker-assisted selection.

• To assess the allelic diversity of candidate genes across the lettuce breeding gene pool.

• To generate genetic resources that will be the foundation of future lettuce improvement.

Support: Seventeen breeding companies and TKI, The Netherlands. Two components: UC Davis: Richard Michelmore, PI. WAU/PRI/CGN: Eric Schranz & Sander Peters, PIs.

• Scaffold N50 = 295 Mb, >99% of assembly. • Nine largest superscaffolds colinear with 9

chromosomal LGs. • Another 19 smaller scaffolds = LG fragments;

some may be centromeric.

Chromosomal superscaffolds of lettuce

Reyes-Chin-Wo, Michelmore, et al.,

Sequencing of Diversity Analysis of Lactuca spp. Batch Number

Accessions Depth SNP/Accession* Status

L. serriola reference 1 serriola 36x 8.8 M Distributed to ILGC

L. sativa de novo assemblies 6 sativa 15x? 1.4 M Distributed to ILGC

Low pass diversity panel 20 sativa 2 serriola

~10x 1.1 M Distributed to ILGC

RNA-seq data (Collaboration with Hanhui Kuang)

144 sativa 29 serriola 4 saligna 4 virosa

98 ??

NA NA Being genotyped

Introgression sources 1 sativa 1 serriola 6 saligna

19 Unknown

NA NA Being genotyped

Extended diversity panel (Collaboration with Ivan Simko)

87 sativa 2 serriola

~25x NA Sequenced & genotyped

Total 236 sativa 35 serriola 10 saligna

4 virosa + 98?

20.1 M polymorphic

positions

* High quality SNPs: 20+ reads across panel with 10+ reads representing the alternate allele. Each accession should have 5+ reads to call a genotype. Dean Lavelle, many collaborators

Zooming below 500 Kb SNPs are displayed individually by accession

Below 100 bp genotype is displayed as SNP per accession

Medium & High Resolution Display of SNP Data

Sebastian Reyes Chin-Wo

Targets for CRSIPR/Cas Gene Editing in Lettuce • Targeted gene knockouts (NHEJ-dependent)

Candidate genes from genetic mapping: • NCED4, ERF1: germination thermosensitivity • Vert1: Verticillium wilt resistance (race 1) • Dm genes: Downy mildew resistance (Brett Pike) • cor: Bacterial corky root resistance • Tipburn QTL: Physiological tissue breakdown (Miguel Macias) • Lobe: leaf shape (Dean Lavelle) • XTH: Cell wall biosynthesis genes (Anabelle Damerum)

Other: • Phytoene desaturase: Chlorophyll biosynthesis, KO reporter • DsRed: Fluorescent KO reporter • Rib discoloration, PPO: Physiological disorder • mlo homolog: Powdery mildew resistance • Dmr6: Downy mildew resistance (susceptibility gene)

• Allele replacements & gene insertions (HDR-dependent) – Dm genes, stacks at a single genomic locus – ALS, ESPS: Herbicide resistance – Leaf color: Anthocyanin biosynthesis genes, GOF reporters – Allele replacements for precise backcrossing and stacking (Vert1, Xar1, Trv1, Dm#s, …)

Done Underway Imminent

Lien Bertier

Future Prospect: Resistance Gene Stacking

Use CRISPR/Cas9 to integrate known genes within an existing cluster: multiple R genes effective against all known pathotypes and multiple viral, bacterial, fungal, oomycete diseases as well as insect pests and nematodes Needs cloned genes for resistance to each disease Complement with HIGS genes against multiple pathogens Inherited as single Mendelian unit (recombination repressed) Herbicide resistant gene (e.g. ALS) as selectable marker for locus Gene stack expanded as more resistance genes become available Genes replaced when overcome by changes in the pathogens

Dm## HIGS## Dm## ALS Dm## FUS## Ve1

CURRENT ‘BOOM-AND-BUST’ CYCLE OF DISEASE CONTROL The Agricultural Consequences of Pathogen Evolution

CULTIVATION OF RESISTANT CULTIVAR

BREEDING OF RESISTANT CULTIVAR

PATHOGEN CHANGES TO RENDER RESISTANCE INEFFECTIVE

SUSCEPTIBLE CULTIVAR

GERMPLASM SCREENS CROSSES

(Suneson, 1960)

Equilibrium in favor of pathogen

SLOWED ‘BOOM-AND-BUST’ CYCLE OF DISEASE CONTROL Containment of Pathogen Evolution

CULTIVATION OF RESISTANT CULTIVAR

BREEDING OF RESISTANT CULTIVAR

PATHOGEN CHANGES TO RENDER RESISTANCE INEFFECTIVE

SUSCEPTIBLE CULTIVAR

GERMPLASM SCREENS CROSSES, EDITING Rapid introgression

Heterogeneous deployment of R genes in space & time

Maximize evolutionary hurdle to virulence Multiple modes of action

Gene stacks

• Continued reduced monitoring of downy mildew,

• Backcrossing different sets of new resistance genes into all types

• Germplasm asymptomatic to an isolate of Verticillium race 2

• Genetic basis of resistance to:

Downy mildew , Verticillium wilt race 1, Fusarium wilt race 1,

Big Vein, Botrytis bottom rot, leaf shape, bolting, (tipburn)

• Chromosome scale genome assembly with genes for

disease resistance and horticultural traits

• Lettuce genome sequence refinement continuing

• Molecular markers linked to these resistances & candidate genes

• Molecular markers for corky root resistance, Verticillium race 1 resistance,

leaf shape, bolting, heading, tipburn …..

SUMMARY

BREEDING GOALS • CONTINUE MONITORING ISOLATES OF DOWNY MILDEW • GERMPLASM COLLECTION & SCREENS FOR VERTICILLIUM RESISTANCE • INTROGRESSION OF MULTIPLE DISEASE RESISTANCE GENES • RELEASE OF DISEASE RESISTANT LINES • GENETICS OF DISEASES AND HORTICULTURAL TRAITS • MARKER DEVELOPMENT & MARKER-ASSISTED SELECTION

GENETIC GOALS • MORE POPULATIONS (USDA SCRI FUNDED)

Mapping of phenotypes relative to candidate genes •SEQUENCING OF GENOME (INT. LETTUCE GENOME CONSORTIUM FUNDED) More genotypes More complete gene catalog • SEQUENCING DOWNY MILDEW (CONSORTIUM FUNDED)

Tools for selection of specific Dm genes • DATABASE CURATION (USDA SCRI FUNDED)

Integration of breeding, genetic, and molecular data

OBJECTIVES 2017-2018

UC Davis Lettuce Genetics and Breeding Group Richard Michelmore Maria Jose Truco Pauline Sanders Keri Cavanaugh Cayla Tsuchida Juliana Gil Sebastian Reyes Chin-Wo Dean Lavelle Miguel Macias Gonzalez Kyle Fletcher Lien Bertier Alex Kozik Huaqin Xu Lorena Parra German Sandoya Lien Bertier Lin Zhang Aubrey Kenefik

COLLABORATORS Ivan Simko Richard Smith Mike Cahn Steve Koike Krishna Subbarao Steve Klosterman Kent Bradford Tom Gordon Sylvie Jennie Beiquan Mou Jim McCreight FUNDING CLGRB USDA NIFA SCRI CDFA Specialty Crops Program Int. Lettuce Genome Consortium Bremia Diversity Consortium

THANKS TO THE CALIFORNIA LEAFY GREENS RESEARCH BOARD FOR SUPPORT

UCD LETTUCE BREEDING PROGRAMS...

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