The expert consultation on the use of crop wild relatives for pre-breeding in potato was a workshop organized by the Global Crop Diversity Trust in collaboration with CIP and took place from the 22nd – 24th of February 2012.

1. Prebreeding for potato
cultivars improvement
Ewa Zimnoch-Guzowska
IHAR-PIB, Młochów Research Center,
Department of Potato Genetics & Parental Lines,
Poland

2. Department of Potato Genetics & Parental lines
Research Center Młochów,IHAR
• Staff: ∑ 54
Laboratories of:
•Genetics
•Breeding Methodology
•Biotechnology
•Virology
•Phytopathology
• Greenhouses 1600 m2
•11 climatic chambers
• in vitro utilities
• Potato storages

3. 4 experimental fields - 8 ha in total

4. Average yields of potato in Poland (t/ha) in
1996-2010
Late blight drought
25
20
tons/ha
15
10
5
0
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
19
19
19
19
20
20
20
20
20
20
20
20
20
20
20
20

5. Size of potato fields in Poland and their
input in potato acreage (%)
>5 ha
9%
<1 ha
1-5 ha 50%
41%

6. Traits important in potato breeding
Table potato chips French fries Starch potato
Agronomic traits: yield, tuber morphology, starch content, starch
yield, earliness, tuber defects, secondary growth,..
Tuber quality traits: culinary value (taste, texture, smell), skin & flesh
color, discoloration of fresh and cooked flesh, chipping quality (RS
content), TGA, tuber greening, darkening after mechanical damage
Resistance to pathogens and pests: Tolerance to stresses:
Viruses, bacteria, fungi (drought, temperature,
Nematodes, CPB, salinity)

7. Selection scheme
60 000 individuals
1 year
Highly repeatable traits:
morphology,
I phase
monogenic resistances
1-3 year (90%)
Discoloration of tuber flesh
Lower repeatable traits:
II phase Yield & its structure,
culinary values,
multiyear trials- storage diseases,
4-9 year partial resistances;
III phase Specific traits:
TGA,
offical trials chipping, French fries
10-12 year mechanical harvest,
washing, packing
11 – 12 year
1 cultivar registered

8. Bottle necks for progress in breeding
• Narrow range of variation for the trait
• More traits evaluated in selection process – more
difficult to get progress in chosen traits
• Negative correlations among important traits:
A.low reducing sugars (chipping) and yielding ability,
B. resistance to late blight and short vegetation period
C. starch content and tendency to flesh darkening
after mechanical damage

9. Atributes of contemporary breeding
• Search for new sources of desired traits
and their characterization
• Parental line (PL) breeding for parents with
complex of resistances to biotic stresses
and quality traits at 2x and 4x level
• Improvement of selection methods by MAS
in pre-breeding and cultivar breeding
• Use of resources non-crossable to tbr
- protoplast fusion
- cloning genes and cisgenic approach

10. Parental Line (PL) breeding
PL are parental form of new cultivars,
combining several desired traits. PL are
helpfull to reach progress in culinary value,
processing ability, starch content and
resistances to pathogens in bred cultivars
At IHAR prebreeding done on 2x and 4x
level is focused on complex resistance to
viruses, late blight and nematodes in:
• table potatoes
• chipping potatoes
• starch potatoes

11. Scheme of 4x PL breeding today
Resistance
to LB Cooking quality EXTREME
RESISTANCE
TO PVY
Resistance
Moderate
to Processing quality resistance Parental lines
soft rot to PLRV
Resistance
Resistance Starch content to
to & starch yield PCN
PLRV
Earliness
New sources of traits

12. Scheme of PL breeding acc.Swieżynski 1969

13. Combinations of resistance to viruses
(PVY, PLRV, PVM, PVS, PVX) in 250
4x PL
4 viruses 5 viruses
(n=28) (n=15)
1 virus
(n=16)
2 viruses
3 viruses
(n=133)
(n=62)

14. Samples of PL resistant to Late
Blight in foliage and tubers

15. Outputs from 4x Parental Line program
• 277 tetraploid PL
offered to breeders 9 from PL
since 1968 8 Polish
7 Foreign
• 62 cultivars realized in 6
Poland from direct 5
crosses with PL
4
• Significant progress in
3
PVY and late blight 2
resistance due to PL 1
program 0
PVY PLRV Late
blight
Cvs registered in 2009

16. Utilization of diploid level in potato
breeding
• Diploid wild and primitive cultivated Solanum species
are sources of new genetic variability
• Disomic inheritance
• Haploidisation of cvs and 4x breeding lines –
production of funcional dH tbr for crosses at 2x
level.
• Utilization of 2n gametes (FDR,SDR) for 4x-2x→4x
crosses = way of transfer of 2x genetic potential
on the 4x level
• Progress in selected traits in 4x progenies from
4x-2x crosses

17. Directions of diploid breeding at IHAR
Młochów
Culinary value Chipping High starch content
tbr, phu tbr, phu, gon chc, yun, ver
Supporting programs
Resistance to viruses Resistance to Resistance to
adg, sto, chc, grl, mga, P. infestans Erwinia spp.
tbr ver, mcd, stn, phu, chc, yun, phu

18. Solanum species utilized in breeding as
sources of resistance
• PVY adg, chc, sto
• PVX adg, acl, chc
• PLRV adg, dms, acl, chc
• PVS adg
• PVA chc, sto
• PVM grl, meg
• Late blight dms, chc, phu,
ver, mcd, mch,rzc,ngr
• soft rot chc, phu, yun
• dry rot chc
• Nematodes grl

19. Solanum species as source of quality
traits
• Lack of darkening of tuber
flesh - phu,ver
• High dry mater content
(starch) - chc, yun, vrn,
ver
• Low accumulation of
reducing sugars after cold
storage - phu, gon, chc
• High culinary value –phu,
gon

20. Interspecific hybrid DG 91-121 selected in
diploid breeding program
Hybrid DG 91-121 (tbr, chc, grl, ver, mcd, phu)
resistant to P. infestans, PVY, PVX, PVM, PLRV, Ro1,
wart, mixed rots, producing 2n male gametes

21. Sexual transfer of diploid potential on the
tetraploid level
• meiotic polyploidization due to 2n DG 81-68, 2n FDR
gametes phenomenon operating in
diploids (FDR or SDR) mainly via 4x-2x
or 2x-4x crosses.
Selected diploids are tested for 2n
gametes production by:
• Frequency of big pollen grains
(indicator of 2n male gamets)
• Test crosses 4x-2x – evaluation
of seed set per berry

22. Selection efficiency in 4x-2x and
4x-4x crosses
Criteria of selection 14%
• Tuber size- 6.5 (in scale 14
1-9)
12
• Tuber weight- 6.5
10
• Tuber yield- 1000 g/plant
• Tuber shape regularity- >7
8
• Darkening after cooking- 8
6
3,3%
• Chip colour- 6,5
4
• Eye deep- 6,5 2
• Lack of secondary growth 0
4x-2x ( N=630)
• Lack of internal tuber 4x-4x ( N=970)
defects

23. Present utilization of diploids in breeding
Over 70 % of IHAR’s 4x parental lines originates
from diploids
Transfer of characters from 2x onto 4x level:
• Resistance to PVM from meg and grl
• Resistance to PLRV from tbr (DW 84 -1457)
• Resistance to soft rot from chc, yun, phu
• Resistance to P. infestans from ver, mcd, phu
• Nondarkening of tuber flesh and good culinary
value
• Cold chipping

24. Development of molecular markers for
marker assisted selection (MAS)
• Mapping the resistance genes to various
pathogens in potato (PVS, PLRV, PVM, PVY, Erwinia
spp., P. infestans)
• Development of PCR- based markers for marker
assisted selection in potato (viral resistances and
P.infestans resistance, nematodes)
PR PS resistant susceptible -
progeny

25. Hot spots of resistance to P. infestans on
potato genetic map
I II III IV V VI
ver, mcd Rpi-blb2
ver, mcd
R2, R2-like, R1
Rpi-blb3,
Rpi-abpt,
Rpi-bst1,
Rpi-mcd1
VII VIII IX X XI XII
R3a, R3b,
ver, mcd Cloned
R5, R6, R7,
Rber, R8, R9,
Rpi-vnt1.1, Rpi-ber1, R10, R11 Used in
Rpi-vnt1.2, Rpi-ber2
cisgenics
Rpi-blb1 Rpi-vnt1.3, Rpi-rzc1
/RB, Rpi-nrs1,
Identified by
Rpi-sto1, Rpi-phu1
Rpi1 IHAR-PIB
Rpi-pta1,
Rpi-mch1 Młochów
Rpi-plt1,
Rpi-mcq1
Rpi-ver1-8

26. Selection of 4x resistant forms possesing the
Rpi-phu1 gene using markers GP94 or phu6
IX
2.7 GP39
GP94 (6.4 cM) ( Śliwka et al., 2007)
B7
12.3
PR PS resistant susceptible -
progeny
Rpi-phu1
6.4
3.4 GP94
GP129
4.9
CP135
PCR marker phu6 based on know
21.7
gene sequence (Świątek et al., 2011)
+ + + + + + + + - - - - C
S1D11
506
396
phu6 344
298

27. Genes for resistance to viruses, map location,
markers for MAS at IHAR
Gene Chromosome Marker Author
PVY
Rysto =Ry-fsto XII GP122564 Flis
1,2cM et al., 2005
Ny-1 IX SC895 Szajko
0,5cM et al.,2008
PVM
Rm XI GP250510 0,8cM Marczewski
Gm IX SC 878885 2,0cM et al., 2006
PLRV
PLRV.1 XI Nl271164 Marczewski
et.al., 2001
PLRV.4 XI UBC864816 6,0cM Marczewski
et. al., 2004
PVS
Nsadg VIII SC 811454 0,8cM Marczewski
et. al., 2002

28. Presence of the GP122564 marker for
Rysto gene of extreme resistance to
PVY in 4x clones
L S 2 3 4 5 6 7 8 9 10 11 12 13 14 15
bp
564 bp
500
300
100
L-DNA Ladder, S: cv Bryza (-), 2: cv Ursus (+); 3-15 – tested 4x clones

29. Scheme of screening 4x Parental Lines for
multiple resistance to pathogens -introduction
of MAS
Year/# Name Phenotypic Phenotypic + MAS
I 1st year
5000 seedlings PVM, PVYNWi, PVX PVX
II 1st tuber PVYN-Wi, I late blight, late blight
700 progeny
III 2nd tuber PVYN-Wi, PLRV, PVM, Ry-fsto, Rpi-phu1,
100 progeny II late blight, I wart, H1, Rm, Ns
I Ro1, I wart
IV 3rd tuber PVS, PVM, PLRV, late II wart
30 progeny blight field, I tuber PLRV
blight, II wart, IIRo1
V 4th tuber PVYNTN PLRV, soft rot
10 progeny PLRV, soft rot

30. Asexual sources of genetic variation
1. Somatic hybridization
2. Cisgenesis

31. Somatic hybridiation
Protoplast fusion is a potentialy supporting
technique in modern potato breeding:
• to combine non-crossable
dihaploids tbr with diploid
Solanum species or hybrids,
•to combine various Solanums
with different EBN level
• to obtain autofused forms
with enhanced ploidy level
(2x → 4x)
• to obtain intergeneric
hybrids

32. Somatic hybrids between
S. michoacanum (1 EBN) resistant to LB, useful for
cold chipping (4oC) and S.tuberosum hybrid DG 81-68
mch
mch DG 81-68
mch (+) DG 81-68

33. Transfer of P.infestans resistance from S.
michoacanum (1 EBN) into S.tuberosum
Protoplast fusion
2x mch
population
autofusion of
2x mch(+)2x tbr
2x mch
BCs mapping cloning
Rpi-mch1 Rpi-mch1
MAS
Potato cultivar cisgenesis

34. Somatic hybrids S. nigrum (+) Zel 1136

35. Gene technology in potato: Cisgenesis
• Unlike in transgenesis, genes are only transferred
between closely related organisms
• Because cisgenesis could be achieved through
conventional breeding it should not face as much
criticism as GM through transgenesis.
• Cisgenesis has the advantage over conventional
breeding that it can produce new cultivars more quickly

36. Resume- future directions for potato
breeding for poor and rich
• Tolerance to drought, high temperature
• High adaptability
• Resistance to bacterial diseases
• Low input cultivars – resistant to pathogens
and pests,
• Quality in table potatoes- strategic food
• Organic (eco-) potato
• Potato as biofactory (GM)
• Nisha markets (baby potato)

37. Thank you,