Establishment of wild pea Pisum fulvum chromosome segment substitution lines in cultivated P. sativum genetic background, as a tool to study domestication and to broaden genetic diversity.

Petr Smýkal Department of Botany Palacký University in Olomouc, Czech Republic

(*1822 - †1884)

J.G.Mendel and pea diversity exploration

8. February/8. March

1865

Lack of wild accessions in legume germplasm collections

Total numberwild species landraces

breeding

linesvarieties

bean Phaseolus 261 963 2 39 4 10

soya Glycine 229 444 6 17 7 13

groundnut Arachis 128 435 3 31 10 4

chickpea Cicer 98 313 1 50 7 6

pea Pisum 94 001 2 19 8 17

cowpea Vigna 65 323 3 40 4 2

lentil Lens 58 405 3 36 4 3

faba Vicia 43 695 1 32 9 7

pigeonpea Cajanus 40 820 3 49 13 2

lupine Lupinus 38 050 18 27 12 6

bambara Vigna 6 125 1 79 2 1

medic Medicago 91 922 47 6 7 6

clover Trifolium 74 158 53 5 3 6

vetch Vicia 38 460 25 23 3 3

grasspea Lathyrus 26 066 25 21 1 1

trefoil Lotus 8 128 52 6 4 5

1 041 345

Percentage

49248

16910 51450

12396

4980

all wild (2.5%, 1863) 11938

commercial varieties (34%)

breeding lines (13%)

landraces (38%)

mutant stock (2%)

RILs (3.7%)

P. subsp. elatius (0,42%)

"P.humile/syriacum" (1.2%)

"P. transcaucassicum, asiaticum" (0.2%)

P. abyssinicum (0.36%)

P. fulvum (0.46%)

uknown

Pea accessions stratification from 75 324 accessions with known passport data (total 98,000 acc.)

Smýkal P, Coyne C, Redden R, Maxted N (2013) Peas. Chapter 3. In: Singh M, Bisht IS editors, “Genetic and Genomic Resources for Grain Legume Improvement.” Elsevier Insights, London. Pp. 41-80.

Pea germplasm diversity

Jing et al. (2010) BMC Evol. Biol., Smýkal et al. (2012) Plant Genetic Resources

4,538 Pisum accessions held in six European Genebanks

wild Pisum

Pisum genus diversity and phylogeny

Jing et al. (2010) BMC Evol. Biology

Pisum sativum subsp. sativum Pisum fulvum

Pisum sativum subsp. elatius Pisum abyssinicum

Pisum genus geographical distribution variety of habitats and bioclimatic parameters

Secondary genepool Pisum fulvum

P. sativum subsp. abyssinicum

Primary genepool Pisum sativum subsp. sativum subsp. elatius Tertiary genepool

Vavilovia formosa

Pea genepools

Smýkal et al. (2014) Critical Reviews in Plant Sciences

Domestication and selection

Wild species

wild species domestication modern crop

How to reverse the domestication bottleneck

Crop x wild species

Use of crop wild relatives to broad diversity

Jing et al. 2010

P. fulvum WL2140 P. elatius L100

Pea genome – large (4.6 Gbp) and rich in repetitive DNA sequences

9.09 pg DNA/2C haploid genome size of 4.6 Gbp repetitive DNA (70-80%)

Macas et al. (2006) BMC Genomics

Smýkal et al. (2012) Agronomy DPMI

Parent B

Parent A

Principle of introgression lines (backrosses)

I II III IV V VI VII

Linkage group position of used molecular markers

AA212 16

Accox 30

6572

-90

-102

-120

-130

-5

-21

-30

-58

-84

-100

-158

-10

-21

-45

-55

-70

-80

-92

-125

-149

-180

-262

-18-25

-45

-63

-72-79

-120

-180

-19

-30

-55-61

-86

-109

-135

-180

-188

-5

-19

-33

-45

-53

-80

-117

-125

-163

-7

-18

-30

-59

-76

-100

-120

-134

-145

-300

-250

-200

-150

-100

-50

0

0 1 2 3 4 5 6 7 8

Selected 28 microsatellite and 44 gene-specific markers cover all seven linkage groups of pea 4.6 Gbp genome at 2 to 82 cM spacing, with mean of 15.4 cM, delimiting 78 chromosomal regions

Bin-approach

CSSL Finder visualization of genotyped P. fulvum x P. sativum BC3S4 104 lines (unordered)

CSSL Finder selected subset of 49 lines (of 104 analyzed) as sufficiently representative

Issue of nuclear-cytoplasmic conflict

F2 segregation of WL1238 x WL2140 (P.sativum x P. fulvum) 9 albinotic : 31 green

Heritability of mtDNA (cox1) and cpDNA (rbcLA) in F2 plants

cox-1/PsiI rbcLA/AspLEI

WL1238(P.sativum) x WL2140(P.fulvum) WL1238(P.sativum) x WL2140(P.fulvum)

WL1238 x WL2140 WL1238 x WL2140

WL2140 x WL1238

WL2140 x WL1238

WL2140 WL1238

WL2140 x WL1238

WL2140 x WL1238

WL2140 WL1238

Mitochondria Inheritance Chloroplast Inheritance

Selective elimination of cpDNA from P. fulvum P. Smýkal

The heterozygozity was detected by molecular markers in 533 (8%) cases while recurrent Pisum sativum parent in 4552 (69%) and introgressed segments of P. fulvum in 1551 (23%) of 30 cM in average. Taking in account the detection limit achieved with 72 markers for 7LG and spacing total length of 1389 cM of Pisum sativum genome with average spacing 15 cM, the average size of introgressed Pisum fulvum fragmets was estimated to be 40 cM. The were 1.2, 1.3, 2.3, 1.3, 0.8, 1.1 and 1.5 segments per LG. There were 5 to 14 segments per line, with mean of 9.6

Summary of current genotyping

Height (cm) Number of nodes Number of pods

Example of ILs used for gene identification Case of pod dehiscence

Wild pea = pod dehiscent

Cultivated pea = pod indehiscent

• Preliminar phenotyping for 14 traits showing transgression and novel traits.

• Further backcrosses is being undertaken to obtain „single“ fragment lines.

• Upon suffficient homozygozity, lines will be genotyped by medium to high density within LEGATO project partners.

• Such lines will allow phenotypic characterization of unlimited number of target traits, which, coupled together with higher density markers, will provide means for QTL and gene identification and subsequent incorporation in desired genotypes ultimately leading to better performing commercial pea varieties.

• In parallel the series of lines is being established with Pisum sativum subsp. elatius (L100) and P. sativum cv. Cameor parents.

Conclusions and prospects

Kosterin O. Institute of Cytology and Genetics, Novosibirsk, Russia Burstin J., Aubert G. INRA, Dijon, France Gyorgy Kiss AMBIS Ltd., Hungary Peter Winter GenXPro, Germany

Financial support acknowledgement: This work was supported by ME10062 project and current research received funding from the European Community's Seventh Framework Programme (FP7/ 2007-2013) under the grant agreement n°FP7-613551, LEGATO project (2014-2017).

J.G.Mendel W.Bateson Watson/Crick human genom

1865 1900 1902 1915 1942 1953 1970 1973 1983 2000

Sutton-Bovery Maxam/Gilbert/Sanger Morgan Avery

Ch.Darwin McClintock

1944/DNA GMO PCR gene 1859

An International Consortium for Pea Genome Sequencing