Hybrid potato breeding for improved varieties

Full text

http://dx.doi.org/10.19103/AS.2016.0016.04
© Burleigh Dodds Science Publishing Limited, 2016. All rights reserved.
Hybrid potato breeding for improved
varieties
Pim Lindhout, Michiel de Vries, Menno ter Maat, Su Ying, Marcela Viquez-Zamora and
Sjaak van Heusden, Solynta, the Netherlands
1 Introduction
2 The scientifi c basis for hybrid potato breeding
3 The state of the art of hybrid potato breeding
4 Production of and commercialization of hybrid seed cultivars
5 Inbred lines for genetic research
6 Cropping systems based on true seeds
7 Case studies
8 Conclusion
9 Where to look for further information
10 Acknowledgements
11 References
1 Introduction
The cultivated potato, Solanum tuberosum, can be reproduced generatively through
seeds and vegetatively through tubers. This may have evolutionary advantages: seeds
may provide better survival under extreme conditions, such as frost or drought, and can
remain viable in the soil for years. When conditions are mild, tubers survive in a dormant
state for a couple of months. When conditions become favourable again, their fast and
strong sprouting provides a clear competitive advantage over other plants in the same
ecological niche.
In traditional potato breeding, each breeding cycle starts with a cross between
two genotypes, usually tetraploid varieties, followed by many years of selection and
multiplication (see Chapters 2 and 3). The advantage of this approach is uniformity: the
tubers are clones and thus genetically identical. The disadvantage is the low genetic gain in
each lengthy breeding cycle, as the genetic composition of the two parental genotypes is
just reshuffl ed, including alleles which negatively affect plant growth and development. As
a result, potato yield has not signifi cantly been improved over the past century (Douches
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2 Hybrid potato breeding for improved varieties
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et al., 1996; Vos et al., 2015). In addition, the reproduction of seed tubers is less than a
factor 10 per season. It takes many years to build up suffi cient quantities of seed tubers for
commercial production, and the risk of contamination by pathogens increases with each
multiplication step.
True potato seed (TPS) has been promoted as an alternative for seed tubers because
TPS is easy to store and devoid of most soil-borne pathogens. In South Asia, East Africa
and the Andes, TPS is used mainly by subsistent farmers (Almekinders et al., 1996). TPS is
produced by crossing parent plants that have been selected to produce a hybrid variety.
The parents are propagated vegetatively, similar to seed tuber propagation. As the parents
of a TPS variety are heterozygous, all seeds of a TPS cultivar are genetically different. This
results in a highly variable crop that is not acceptable in most markets, such as the high
value markets of Europe and North America.
Since the success of hybrid breeding in corn in the 1930s, breeders have adapted a
hybrid breeding system for many crops (Fig. 1; Crow, 1998; Troyer, 2006; Hua et al., 2003).
Typically, hybrid cultivars produce higher yields and show high crop uniformity (Rijk et al.,
2013). In addition, the breeding system is fast and effi cient and new traits can rapidly be
introduced by marker-assisted introgression.
These advantages are also expected for potato: hybrid potato varieties will be higher
yielding, will need less crop protection chemicals due to disease resistance and will have
better quality for processors and consumers (FAO et al., 2015). A hybrid breeding system
for potato offers two additional advantages: fast multiplication of hybrid seeds and easier
logistics, as clean true seeds can easily be produced, transported and stored (Duvick et al.,
2005).
Figure 1 Fivefold increase in corn yields since the introduction of hybrids (Troyer, 2006).
AQ:
Please
check if ‘is
less than
a factor
10 per
season’ is
OK as is.
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Hybrid potato breeding for improved varieties 3
However, self-incompatibility and inbreeding depression have for long hindered
progress in hybrid potato breeding (De Jong and Rowe, 1971; Charlesworth and Willis,
2009; Jansky et al., 2016). These two limitations have recently been overcome by
introducing a self-compatibility restorer gene and by large-scale and consistent breeding
(Lindhout et al., 2011a).
This chapter describes the scientifi c principles and applied aspects of hybrid potato
breeding. The successful introduction of the principle of hybrid potato breeding was
described in 2011 (Lindhout et al., 2011a). Since then, we have focused on further
developing the potato hybrid breeding system, especially genetic studies to establish
a genetics-driven hybrid breeding system. In a recent paper, Jansky et al. (2016) have
confi rmed the possibilities of such approach.
This is the fi rst publication on the state of the art of a hybrid potato breeding
programme. The authors, all working at Solynta, want to emphasize that scientifi c papers
on this topic are not available yet, and hence we have to rely entirely on the results of
the Solynta breeding and research programme. Still, in presenting these results, we hope
to contribute to a better understanding of the principles and applied aspects of hybrid
potato breeding.
2 The scientifi c basis for hybrid potato breeding
2.1 The principle of hybrid breeding
The basic idea of hybrid breeding is to combine the genes of two parent genotypes,
both of which may harbour suboptimal alleles, resulting in weaker performance. If parents
have different suboptimal alleles, hybrid offspring can show increased vigour and yield,
designated ‘ heterosis ’ , as the suboptimal alleles in one parent may be compensated by
the favourable genes from the other parent (Birchler et al., 2010; Gopal, 2014; Fig. 6). If
the parents are completely homozygous, the resulting hybrid offspring will be partially
heterozygous and genetically uniform. By testing many hybrid offspring under relevant
cultivation conditions, the best combining parents are identifi ed. These are maintained
and propagated in separate groups as ‘ heterotic pools ’ for further breeding (Brown and
Cagliari, 2011).
Thus, hybrid breeding has two distinct processes: development of homozygous parent
lines and production and testing of experimental hybrids.
2.2 Diploids are more effi cient than tetraploids for hybrid
breeding
Homozygous diploids are faster to generate than homozygous tetraploids. For instance,
seven generations of selfi ng are required to obtain 50% homozygous loci starting from a
tetragenic tetraploid heterozygote (carrying four different alleles). The same homozygosity
level is reached starting from a heterozygous diploid by only one generation of selfi ng
(Haldane, 1930; Fig. 1). For this reason, hybrid potato breeding is more effi cient at the
diploid level.
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4 Hybrid potato breeding for improved varieties
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2.3 Generation of homozygous diploid potato genotypes via
haploidization and via inbreeding
Haploid genotypes can be generated from an egg cell (gynogenesis) or from pollen,
often via anther culture (androgenesis). In potato, some haploids have been generated
by anther cultures. The resulting haploids were converted into diploids by chromosome
doubling. However, the resulting homozygous diploids were very weak and sterile (van
Breukelen et al., 1977; Uijtewaal et al., 1987b), hampering their usage in breeding.
Haploidization has been more successful in crossable species like S. chacoense
(Cappadocia, 1990; Phumichae et al., 2005; Phumichae and Hosaka, 2006) and S. phureja
(Chani et al., 2000).
A reason for the failure to produce vigorous doubled haploids may be the transition
to complete homozygosity in one step. Inbreeding depression may be so severe that
homozygous plants are too weak to survive. Repeated selfi ng, on the other hand, might
lead to a more gradual improvement of homozygosity. However, in potato, inbreeding is
seriously limited by self-incompatibility, which prevents self-fertilization. Still some rare
examples of homozygous diploid plants have been generated by inbreeding but again the
homozygous diploid plants always showed a strong inbreeding depression, which limited
their usage in breeding (De Jong and Rowe, 1971; Charlesworth and Willis, 2009).
2.4 Large genetic variation in potato causes inbreeding
depression
The tetraploid and outcrossing nature of commercial potato is likely responsible for the
large genetic variation. In a study on the allelic composition of 800 genes in 83 potato
cultivars, an average frequency of 3,2 alleles per locus within a genotype was identifi ed
(Uitdewilligen et al., 2013). Among the 83 cultivars, often more than ten alleles per locus
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
012345678910
homozygocity level
generations of selfing
diploid AB
tetraploid AAAB
(digenic simplex)
tetraploid AABB
(digenic duplex)
tetraploid AABC
(trigenic)
tetraploid ABCD
(tetragenic)
Figure 2 The theoretical increase in homozygosity in diploids and tetraploids through inbreeding,
adjusted from Haldane et al. (1930).
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Hybrid potato breeding for improved varieties 5
were observed. In addition, the frequency of single nucleotide polymorphisms (SNPs)
in potato is 1 in 15 – 30 base pairs (PGCS, 2011; Visser et al., 2014). This means that
the genetic distance between the two sets of chromosomes within one diploid potato
genotype is four times larger than the genetic distance between the genomes of man and
chimpanzee (CSAC, 2005).
The high frequency of allelic variation has the inevitable consequence that ‘ weak alleles ’
that have a negative effect on plant fi tness are maintained. Such alleles remain hidden in
the large buffer of four genomes, but reveal themselves upon inbreeding when the chance
for homozygosity increases. This is even more manifested at the diploid level where the
homozygosity level more rapidly increases upon inbreeding (Fig. 2).
This large genetic variation is also helpful for breeding as it forms a genetic reservoir of
useful genes. It is a challenge to identify alleles that contribute most to plant performance.
As potato has 39 000 genes, the identifi cation and usage of the ‘ best alleles ’ per locus,
including interactions between them (epistasis), will gradually take place over decades of
research and breeding (PGSC, 2011). Corn may serve as a good example, whereby, after
a century of dedicated breeding by numerous breeders worldwide, a genetic gain of over
1% per year is still achieved (Troyer, 2006).
2.5 Crossable diploid species and tetraploids increase genetic
reservoir for diploid breeding
The potato germplasm available for breeding comprises many species, including diploid
species (Jansky and Peloquin, 2006). These have been used as source to introduce
resistance genes into cultivated germplasm. Breeders often use diploid potato to rapidly
combine favourable traits that can be introduced into the tetraploid germplasm by direct
crossings, bridge crossings or via chromosome doubling (De Mainea, 1982; Chauvin et al.,
2003). The diploid breeding programme at Wageningen University (Hutten, 1994) has
generated donor lines that harbour the most important traits for potato breeding (Table 1).
Additional diploid germplasm is available from potato gene banks and public research
institutes such as University of Wisconsin-Madison, United States; Potato Germplasm
Enhancement Laboratory, Japan; Gene Bank at Gatersleben, Germany and International
Potato Centre, Peru.
Another source of diploid germplasm is tetraploids that can be prickle pollinated to
generate diploid offspring, designated dihaploids (Uijtewaal et al., 1987a). A collection of
dihaploids obtained from one tetraploid harbours the full set of genes from the tetraploid
and can be exploited in a diploid potato breeding programme.
In conclusion, the large genetic variation in potato and in its wild relatives, combined
with the technology to switch between ploidy levels, provides a tremendous wealth of
germplasm available for diploid hybrid breeding.
2.6 Diploid potato may perform equal to tetraploids
Most important food crops such as rice, corn and soybean are diploid. Sugar beet cultivars
were initially tetraploid, then triploid and since 2000, all new cultivars are diploid. In potato,
it has long been assumed that tetraploids outperform diploids (Rowe, 1967; Hutten et al.,
1994). Occasional observations have contradicted this assumption: Progeny of diploid
potato USW4 with S. chacoense M6 produced large tubers and high yield (Lipman and
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6 Hybrid potato breeding for improved varieties
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Zamir, 2007; Jansky et al., 2014). Uijtewaal et al. (1987b) observed that heterozygous
diploid potato outperformed all homozygous di- and tetraploid derivatives. These
results from potato and from other crops support the expectation that diploid potato will
eventually replace tetraploid potato for commercial usage.
3 The state of the art of hybrid potato breeding
In 2008, Solynta started its research by making crosses between diploid potato germplasm,
obtained from a pre-breeding programme from Wageningen University (Rutten, 1994),
and a homozygous accession of the wild species S. chacoense , carrying the dominant self-
compatibility controller gene Sli (Hosaka and Hanneman, 1998a,b; Phumichai et al., 2005;
Lindhout et al., 2011a). The F
1 plants were extremely vigorous and about half of them
produced many berries upon self-pollination. This was considered a major breakthrough
as these were, to our knowledge, the fi rst vigorous, self-compatible diploid potato plants
ever obtained. These F
1 plants were highly heterozygous. The fi rst generation after selfi ng
(designated F
2 ) should harbour at least 50% homozygous loci. As many of these loci might
Table 1 The Sli -gene donor, designated DS and 16 diploid potato
germplasm, designated D1 – D16, used for hybrid breeding at
Solynta. The trait abbreviations are according to Hutten (1994)
Abbreviation Short description
DS Sli- gene donor
D1 Early (maturity), long, Y , Qcook
D2 Early, Y, Qcook
D3 R3 , H1 , Gpa2 , RXadg , Y (yellow fl esh)
D4 Grp1, early, long (shape), Ro1 ( H1 ?)
D5 Early, long, Y , Qfry
D6 Long, Y , Qfry, H1 , Qcook
D7 Early, long, Y , H1 , Qcook, Zep (orange fl esh)
D8 Early, y (white fl esh)
D9 Qstarch, Y
D10 Wild species hybrid: phyt avl
D11 Wild species hybrid: phyt rch
D12 Round (shape), Qcook, Qfry
D13 Early, round, Zep , Y , Spectacled, Qcook, blue
anthocyans
D14 Wild species hybrid: phyt tar
D15 Wild species BC1: early, phyt vnt1 , round, Y , H1
D16 Early, round, y (white fl esh)
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Hybrid potato breeding for improved varieties 7
harbour ‘ weak alleles ’ , it was expected that F
2 plants would be too weak to survive. We
also made BC
1 populations by backcrossing to the S. chacoense parent.
As expected, the F
2 and BC
1 showed weak growth and many died in the fi eld. However, a
number of plants survived and 10% of the surviving F
2 plants even proved self-compatible
(Lindhout et al., 2011a). We generated the second inbred generation, designated F
3 and
made crosses between self-compatible individual F
3 plants. These inbred plants were
tested with SNP markers to confi rm their genetic identity as real inbreds. These results
indicated that breeding hybrid potato was now feasible (Lindhout et al., 2011b).
The weak plant performance, the poor tuber quality and the low yield of the inbreds
was not only due to inbreeding depression, but was also caused by the wild S. chacoense,
a species that hardly produces tubers. So, by this approach, we not only started a hybrid
potato breeding system, but also the process of domestication of a new ‘ diploid potato ’
based on an interspecifi c cross of diploid S. tuberosum and the wild species S. chacoense.
We hypothesized that developing vigorous inbred lines is challenging as for each of the
39 000 loci the most favourable alleles should be identifi ed and combined. Unfavourable
genes with large effects on plant performance are identifi ed easily and hence selecting
increased plant performance is initially easy and fast. Undesired characters from the
S. chacoense parent such as abundant stolons, small leaves and twisted, small and low-
yielding tubers were removed in a few breeding generations.
Sli is a dominant gene (Phumichai et al., 2005; Phumichai and Hosaka, 2006). However,
the successful expression of this gene requires a vigorous plant that is fertile and supports
self-pollination. Often these criteria are not met. Therefore, the frequency of self-
compatible plants is usually lower than expected based on a monogenic trait. Inbreeding
depression is exhibited as weaker plant growth upon higher generations of inbreeding
(Fig. 3). As a consequence, the self-compatibility level tends to decrease upon further
selfi ngs. The fi rst inbred lines obtained by Solynta, containing over 95% homozygous
Figure 3 Inbreeding in the diploid Solynta germplasm in winter 2014 – 15. The Fx indicates x-1 generation
of selfi ng after the last cross was made. Data are averaged over the complete trial consisting of over
5000 plants. The scale of plant vigour ranges from 1 = very weak via 3 = average to 5 = very strong.
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8 Hybrid potato breeding for improved varieties
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loci, hardly produced progenies and the seedlings were extremely weak. Therefore, new
crosses were made between F
3 and F
5 inbred lines and selfi ng was started again from
these F
1 ’ s to continue selecting parent lines that combine benefi cial traits. As a result,
performance of the inbreds improved over the following generations (Fig. 4).
Genoty
p
e
Lady Roset ta
Felsina
Lady Ana
Mozart
Hybrid A
Hybrid B
Hybrid C
Hybrid D
Hybrid E
Hybrid F
Hybrid G
Hybrid H
InbredA
Inbred B
Inbred C
Inbred D
Inbred E
Inbred F
Inbred G
Inbred H
Tuber Yield (gr/plant)
0
200
400
600
800
1000
Checks
Hybrids
Inbreds
LSD α=0,05
Figure 5 Hybrid performance of the fi rst diploid experimental potato hybrids. Seed tubers were
harvested from greenhouse-grown plants, raised in the winter from tubers (checks) and seedlings
(hybrids and inbreds). The seed tubers were planted on 8 May 2015 in a trial fi eld on sandy soil in
Wageningen and harvested on 17 September 2015.
Figure 4 Examples of Solynta diploid germplasm. Plants were raised from seedlings and grown in the
greenhouse in the summer season of 2014 (left panel) and 2015 (middle and right panel). They were
among the best genotypes in a breeding programme comprising over 15 000 plants.
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Hybrid potato breeding for improved varieties 9
In mature hybrid breeding systems, parents of the hybrids are selected based on general
combining abilities (GCA), whereby molecular markers can be used to better predict the
breeding value of the parents (Tobias et al., 2009). As no historic data were available on
combining abilities of diploid potato parents, the selection of the fi rst parents was based
on the performance of the parents themselves.
Following the predictions of good performing inbred lines, crosses between these lines
were made resulting in 45 potato hybrids which were tested in the fi eld together with 20
inbred lines in two replicates of two plants per plot. The yields varied from 83 to 580 g/
plant (Fig. 5). Thirteen hybrids scored higher than any of the inbreds, also outperforming
the check variety Lady Anna, while some also showed a strong heterosis for yield (Fig. 6).
The trials are being repeated in 2016 by using seed tubers raised from the fi eld.
In addition, a new series of 216 experimental hybrids was generated in the winter-season
2014 – 15, mainly based on F
6 parent lines. The hybrid seeds were sown immediately after
harvest and ten seedlings per hybrid were transplanted into the fi eld in June, 2 months
later than the usual seed tuber plantings. Still the yield of some hybrids was higher than
500 g/plant and the tubers were similar in size and shape to commercial seed tubers.
The results of the fi rst experimental hybrids illustrate the potential of diploid hybrid
potato varieties. As the fi rst hybrids were randomly made without any a prior information
about the combining abilities of the parents, it is expected that the next series of hybrids
based on the results of these fi eld trials will perform better and may show a stronger
overlap with commercial controls.
4 Production of and commercialization of hybrid seed
cultivars
The production of hybrid potato seeds is mainly done by hand pollinations. The seed
yield per plant varies from hundreds to many thousands of seeds. Each successful hand
pollination generates a berry with 50 – 150 seeds and each plant produces 5 – 50 berries. This
Figure 6 Example of heterosis in diploid hybrid potato. The plants were from the same trial as in
Figure 5. At the left the female F
3 parent and at the right the male F
5 parent, while the hybrid is in the
middle.
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10 Hybrid potato breeding for improved varieties
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is similar to other vegetable Solanum crops, like tomato and pepper, where commercial
seed is produced by hand pollination.
The emasculation of the fl owers is a time-consuming part of the hand pollinations.
Male sterility systems have been reported that make emasculations redundant and hence
reduce seed production costs (Li, 2008). Alternatively, functional male sterility may be
used, whereby pollen is prevented to land on the stigma of the same fl ower as the exerted
stigma may be manually pollinated by pollen of the male parent before the own pollen
may reach the stigma (L ö ssl et al., 2000; Abrol et al., 2012).
The transport of potato seeds over the world is very restricted. This is because the
dominant breeding systems are based on the production of seed tubers and hardly any
rules are in place for potato seeds. As a consequence, most countries consider potato
seeds as belonging to the highest risk classes. This is remarkable as seed tubers may
harbour any of over 200 species of pathogens that attack potato (Delleman et al., 2004).
In contrast, only six pathogens are seed-borne (Solomon-Blackburn and Barker, 2001).
These are fi ve viruses and a viroid, which are absent in major potato-growing regions like
the Netherlands. So, potato seeds are very safe and regulations will need to be adjusted
accordingly.
The registration process for breeders ’ rights poses a similar situation: in countries
which are members of the International Union for the Protection of new Varieties of Plants
(UPOV), the path to commercialization of a hybrid starts with the registration process to
obtain breeders ’ rights, which is based on seed tubers. So, protection by breeders ’ rights
of hybrid potato seed cultivars is not yet possible. The European Union (EU) is adjusting
the legislation process, but it may still take several years before this is established. Other
non-UPOV countries will likely follow later.
5 Inbred lines for genetic research
Inbred lines allow the generation of mapping populations such as F
2 , BC
1 and BC
2 . As
the parents have limited allelic variation, the signal-to-noise ratio is much higher than
in studies with heterozygous tetraploid populations. Moreover, putative quantitative trait
loci (QTL) can effectively be confi rmed in dedicated populations that are selected to
segregate for the loci under investigation, and are fi xed for other regions on the genome
(Wang et al., 2008; Schmalenbach and Pillen, 2009; Fu et al., 2010). In addition, new
genetic populations can be generated, which are very powerful for quantitative genetic
studies, like nearly isogenic lines (NILs), recombinant inbred lines (RILs) and libraries of
introgression lines (Young et al., 1988; Paran et al., 1995; Jeuken and Lindhout, 2004;
Finkers et al., 2007; Zhang et al., 2005; Chen et al., 2010; Viquez et al., 2014).
Genetic studies in potato have been done at the tetraploid level and at the diploid level.
Tetraploids may support simple genetics like the mapping of resistance genes (Solomon-
Blackburn and Barker, 2001), but quantitative studies are less reliable as the genetic
noise of the numerous highly heterozygous loci is high (unexplained error). Genome-
wide association studies (GWAS) at the tetraploid level will always generate hundreds of
potential leads, but only a few hits may be meaningful (Li et al., 2010; D ’ Hoop et al., 2014).
More accurate and reliable quantitative studies were done at the diploid level, initially by
crossing heterozygous parents (Prasher et al., 2014) and, more recently, in a diploid F
2
population (Endelman and Jansky, 2016).
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Hybrid potato breeding for improved varieties 11
The possibility of using self-compatible, homozygous inbred lines for genetic studies
offers three powerful advantages:
1 Only one allele per homozygous locus is present.
2 Only two alleles per heterozygous locus are present.
3 Backcrosses and selfi ngs are feasible for confi rmation studies.
For more than fi ve decades, mutant studies have uncovered new alleles of important
genes and have helped to confi rm or determine the function of genes. Although advanced
technologies like the CRISPR/Cas system (Belhaj et al., 2013) are likely to replace the
methods by which mutants are made, mutants will remain powerful tools to discover
unknown phenotypic traits or to study induced alleles that also have the advantage to be
free of deregulation rules.
5.1 The fi rst completely homozygous self-compatible diploid
inbred line in potato
Most Solanum species that are crossable with cultivated potato are self-incompatible. An
exception is S. chacoense (Hosaka and Hanneman, 1998a; Hawkes, 1990; Jansky et al.,
2014) and introducing the Sli- gene from S. chacoense into cultivated diploid potato
resulted in fertile self-compatible offspring (Lindhout et al., 2011a). After several rounds
of inbreeding, highly homozygous self-compatible inbreds were generated. The level of
homozygosity was assessed by using SNP markers to investigate the effects of inbreeding
(Fig. 7). A strong correlation was observed between the overall level of homozygosity and
reduced self-compatibility. By new series of crosses, selections and selfi ngs, the agronomic
87%
88%
89%
90%
91%
92%
93%
94%
95%
96%
97%
98%
99%
100%
Homozygosity percentage
Plants of research lines
Homozygosity level of four research lines
BC1F6
BC1F7
Figure 7 The increase of the level of heterozygosity in the offspring (BC
1 F
7 ) of four BC
1 F
6 plants
obtained by selfi ng of the BC
1 (D1 × F
1 [DS × D1]) TQ: Please check the usage of brackets is as per
style. (Table 1). The width of the bar indicates the number of plants in the progeny within the class with
plants of the same homozygosity level. The calculations were based on a total of 150 markers.
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12 Hybrid potato breeding for improved varieties
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performance of the inbred lines continuously improved. After six generations of selfi ng,
a homozygous self-compatible line was generated with only 1 out of 150 SNP markers
still heterozygous (Fig. 8). Genotyping by sequencing techniques make it now possible to
check the level of homozygosity in more detail.
5.2 Genetic studies in segregating diploid F
2 populations
Recently, Endelman and Jansky (2016) published the fi rst results of a mapping study in an F
2
population of diploid potato. This was based on a cross between the doubled monoploid
potato DM1-3 and M6, which is an S
7 inbred line derived from the self-compatible wild
relative S. chacoense . A single F
1 plant was then self-pollinated and an F
2 population of
109 genotypes was grown, genotyped (>2200 SNPs) and phenotyped. Meijer et al. (2016)
analysed an F
2 population (108 markers) based on a cross between two clones, namely DS
(a homozygous diploid S. chacoense clone containing the self-incompatibility overcoming
Sli- gene) and D2 (a partly heterozygous diploid S. tuberosum clone; see also Table 1).
The results of both studies are comparable: tuber shape is associated with a region on
chromosome 10, fl esh colour with a region on chromosome 3 and tuber and pigment colour
on chromosomes 2, 10 and 11. These QTLs were identifi ed at the same loci as described
in literature (van Eck et al., 1993, 1994). In both studies, additional QTLs were identifi ed.
Furthermore, there is an overwhelming reservoir of potential useful QTLs in the potato
germplasm (Bradshaw et al., 2007) and thus also in the dihaploids that can be made. Such
QTLs can now be more reliably studied at the diploid level and this will ultimately lead
to the identifi cation of the underlying genes. A limited subset of the diploid germplasm
may already harbour many important traits for potato breeding (Table 1). Relevant genes
01 02 03 04 05 06 07 08 09 10
9.4
11 12
Figure 8 The fi rst essentially homozygous self-compatible diploid potato. The position of the SNP
markers is based on the published sequence (PSGC, 2011). The 12 vertical bars indicate the 12
chromosomes. Red bars indicate homozygous D1, blue bars indicate homozygous DS (Table 1). The
green line on chromosome 11 indicates a heterozygous scored marker.
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Hybrid potato breeding for improved varieties 13
for these traits can be introgressed into vigorous and fertile diploid genotypes to develop
progenies with maximum genetic fi xation to minimize genetic noise. Such genotypes are
unique materials for further detailed genetic studies.
5.3 Marker-assisted backcrossing
Another application for inbred lines in potato is introgression breeding. This requires
knowledge of diagnostic markers for the gene of interest, preferably inside the gene,
markers for the recurrent parent genome and a self-compatible homozygous backcrossing
parent. There are dozens of well-studied resistance genes in potato that can be used
for introgression breeding. This paves the way for a marker-assisted backcrossing (MAB)
programme in potato (Frisch and Melchinger, 2005).
To this end, homozygous inbred lines are crossed with a diploid donor carrying a specifi c
gene of interest. In two backcrosses, NILs can be generated by selection with diagnostic
markers for the gene of interest and against markers in its fl anking regions, combined
with selection for markers well distributed over the potato genome (whole background
selection). Such NILs can harbour over 98% of the recurrent genome in combination with
the specifi c gene. Both parents of a hybrid may have an introgressed gene, resulting in a
double stack hybrid. To introgress specifi c genes in a homozygous parental line will take
2 – 3 years. These MAB programmes are routinely used in other crops and are also feasible
in potato (Mallick et al., 2015; Jeong et al., 2015).
6 Cropping systems based on true seeds
The production of commercial tubers in most parts of the world starts with seed tubers.
These have a large reservoir of nutrients for the growing shoots, allow a rapid initial plant
growth and fast leaf coverage of the soil, which is one of the most critical factors for potato
yield. In contrast, potato seeds are extremely tiny, about 2500 seeds per gram. As a result,
during the fi rst period after germination the young seedlings are very vulnerable for abiotic
stresses like drought, frost and heat. Field emergence has been reported between 50%
and 80% with acceptable tuber yields under different experimental conditions (El-Bedewy
et al., 1994; Renia, 1995). However, without a protective environment, the risk is very high
that an emerging seedling will not survive, even when it is pelleted or primed.
This chapter describes alternative strategies to circumvent the exposure of week
seedlings to harsh conditions.
6.1 Production of seedling tubers in greenhouse
Seedling tubers can be produced under greenhouse conditions by sowing in a medium
with suffi cient water supply and at optimum germination temperature of 15 – 20°C (Struik
and Wiersema, 2012). As soon as seedlings reach 5 – 10 cm in length, they are transplanted
in pots. The desired tuber size, the available space in the greenhouse and the length of
the growing period will determine the pot size, nutrient supply, day/night temperature and
light regime (for a detailed protocol see Struik and Wiersema, 2012). Densities of 80 – 170
plants/m
2 are common in greenhouses (Lommen, 1995; Tierno et al., 2014). There is a
trade-off between number of tubers, size of tubers, planting density and time to harvest.
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14 Hybrid potato breeding for improved varieties
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Depending on the exact conditions, end-product requirements and production costs,
the optimal combination is chosen. Alternatively, hydroponic techniques are available,
whereby the roots are grown in a dark, humid and soilless environment in two layers, one
for nutrient uptake and the other layer for producing mini-tubers (Lommen, 2007). The
seedling tubers are picked at regular intervals and the total yield per plant may reach
dozens of tubers. The soilless culture assures clean seed tubers. The conditions and
picking regime are set to have optimal numbers and tuber sizes.
6.2 Production of seedlings for commercial crop
Greenhouse-grown seedlings can also be used to start the cultivation of a commercial
crop. This system is equivalent to the one used for lettuce, leek, cabbage and onion
(Leskovar et al., 2014). Technically, commercial potato production from seedlings is
feasible and maximizes the benefi ts of true seeds. When potato transplants are grown
as a ware potato crop, a whole new cultivation system must be developed. Important
elements are: transplanting systems, plant spacing including ridging or bedding, the use
of soil coverage, weed control, irrigation and harvesting methods. Further mechanization
and dedicated cultivation systems will be optimized for cropping systems that start with
potato seedlings (Roy et al., 2015). In Kenya, tuber yields of 30 tonnes/ha were obtained,
whereby seedling transplants were used as starting material for a commercial cultivation
(Muthoni et al., 2014). This already represented 50% higher yield than average in Kenya
(Wang ’ om and van Dijk, 2013).
6.3 Seedling tubers as starting materials for a commercial crop
Seedling tubers are equivalent to mini-tubers that are produced from tissue culture,
which is routinely done to start a new multiplication round with clean basic seeds (Amin
et al., 2014). They are certifi ed as G1 material. The great advantage of seedlings are the
reduced costs, compared to in vitro grown plants, and the fl exibility to start the production
whenever and wherever needed, as seeds can easily be stored and transported. As the
cost to produce mini-tubers from in vitro grown plants is very high, in the present potato
system at least three rounds of fi eld multiplications are needed to dilute these high costs
over many seed tubers. For a hybrid seed system, the cost of producing seedling tubers
is much lower and hence fewer propagation rounds are needed. Therefore, seedling
tubers should be multiplied only 1 year and then released to commercial farmers. Such a
system also fi ts better to the fast introduction of new cultivars, which is typical for a hybrid
breeding system.
6.4 Production of seed tubers from seedlings in fi eld
At present, the production of seedling tubers is mostly done under tropical conditions.
Seeds are sown in a simple greenhouse or in the fi eld under plastic cover with plant
densities of 80 – 100 plants/m
2 (Kumar, 2014; Struik and Wiersema, 2012; Fig. 9). When the
seedlings have reached 5 – 10 cm in length, they are transplanted to the fi eld, in ridges,
at a defi ned plant density. Additional hilling will increase the number of seed tubers per
plant (Wiersema, 1986). To decrease the risk of root damage, the complete substrate is
transplanted with the seedling. Plantlets need some time to adapt to outdoor conditions
before transplanting directly to the soil (Gopal, 2004). In South Asia, transplants are
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Hybrid potato breeding for improved varieties 15
planted on the sides of the ridges to benefi t from the shade and higher soil humidity. The
planting distance may be adjusted to compensate for a shorter growing season compared
to seed tubers, if appropriate. The cultivation conditions are similar to traditional potato
cultivation systems. In Egypt, seed tuber yields of 40 – 60 tonnes/ha were obtained in such
a system, based on tetraploid TPS populations (El-Yazied et al., 2004).
Seedlings are more sensitive to frost and drought than seed tubers. Thus, transplanting
is done in a frost-free season and with irrigation. Compared to the traditional systems of
producing seed tubers, whereby the tubers are planted far before the last night with frost,
the length of the growing season of transplants may be 2 months shorter. In addition, the
plant development may be further delayed due to a transplanting shock and weak initial
growth. Hence, tuber numbers are lower and tuber sizes are smaller compared to seed
tuber grown plants, causing severe yield reductions. Plant density may be increased to
compensate for these reduced yields per plants.
7 Case studies
7.1 Combatting Phytophthora infestans
Late blight, caused by the oomycete Phytophthora infestans , was responsible for the Irish
famine of 1845 – 47 (Fry, 2008). All potato cultivars were susceptible to the disease and
suffered severe yield losses that led to food shortage. Since then, breeders have selected
cultivars with fi eld resistance and from the early twentieth century onwards, have introduced
specifi c R -genes, often sourced from wild relatives. However, cultivar Pentland Dell, which
Figure 9 The fi rst seedlings of the fi rst diploid hybrid potato hybrids in Democratic Republic of Congo.
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16 Hybrid potato breeding for improved varieties
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carries three R- genes, already turned out susceptible to a new virulent race of the fungus
even before its widespread use and just 4 years after its introduction (Malcomsum, 1969).
At present, the optimal way to control P. infestans is a combined approach of clean seed
tubers, clean soils, early and preventive chemical protection and destruction of crop debris
after harvest. The global annual cost of cultivation measures and yield loss is estimated at
US$5 billion (Duncan, 1999).
P. infestans reproduces mainly clonally. With hundreds of billions of spores produced per
hectare in an infected crop (Skelsey et al., 2010) and a mutation rate of 1:10
9 , mutations
in any given gene of P. infestans are likely to occur in a disease-infected fi eld. Sexual
recombination, combined with the redundancy of several effectors that are recognized by
R -genes, explains why P. infestans easily mutates effector genes and develops virulence
(Jiang and Tyler, 2012).
As a result of the high genetic variation in potato, dozens of resistance sources have
been identifi ed and are available for breeding (Park et al., 2009). Whereas single genes
are easily defeated by virulent races to P. infestans , combinations of R -genes are more
effective, although the Pentland Dell case indicates that a more dynamic approach may
be needed (Niks et al., 2011).
Ideally, isogenic cultivars are developed that only differ in the combination of R -genes.
This would allow to deploy the most suitable cultivar, dependent on the epidemiology
of P. infestans . However, the introduction of one gene from a wild related species into a
tetraploid cultivar by traditional breeding already takes several decades, and to combine
different R -genes in a breeding programme is simply too complicated.
Since 1990, many R -genes to P. infestans have been mapped and cloned (Ballvora et al.,
2002; Huang et al., 2005; Park et al., 2009). These all belong to the so-called class of ‘ NBS/
LRR genes ’ and have a cytoplasmic interaction with effector genes of P. infestans , resulting
in defence responses that block the growth of the pathogen (Jones and Dangl, 2006). A
genetic modifi cation (GM) approach to develop a series of isogenic cultivars with different
R -genes from crossable species is being pursued (Haverkort et al., 2016, Jacobsen and
Schouten, 2008). These so-called cisgenic plants might fall under the highly costly and
complex GM legislation, which would hamper their commercial opportunities.
The hybrid breeding system offers a clear path towards resilient resistance to P.
infestans : R -genes can be stacked in a potato hybrid via marker-assisted introgression
(Park et al., 2009). Two genes can be combined via the two parents in 2 – 3 years, and
additional R -genes can be added within a year to generate multi-stack resistance hybrids.
In this way, series of R -gene isogenic hybrids can be generated as a dynamic resource to
select the best combination of R -genes to protect the crop against the prevailing races of
P. infestans .
7.2 Hybrid potato breeding for East Africa
Hybrid potato cultivars will bring great benefi ts, not only to modern commercial farmers
in the developed world, but they may even have a greater social impact in tropical regions
where the population rely on potato as a major source of energy and nutrition (FAOstat,
2016). It is very challenging to start an initiative to develop hybrid potato for these regions
(Thomas-Sharma et al., 2016; Kumara et al., 2015). When legal and physical protection of
the breeding germplasm is not secured in these regions, the development of inbred lines
and the hybrid crosses are done elsewhere. The implementation of hybrid potato cultivars
in these regions requires considerable investments and strong cooperation of committed
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Hybrid potato breeding for improved varieties 17
public and private partners. As an example, the implementation of hybrid potato cultivars
for East Africa is briefl y described below.
The highlands of East Africa are traditionally important production areas for potato
because the crop is an important component of the local diet (Table 2). However, yields
remain very low (Table 2). A range of traditional varieties is used from local sources
(Kaguongo et al., 2008) as well as improved material from the International Centre for
Potato (CIP). Seed tubers are produced by farmers and storage conditions are far from
optimal (Kaguongo et al., 2008; Gildemacher et al., 2009). Janssens et al. (2013) concluded
that bacterial wilt, lack of clean seed tubers and poor storage are the most prominent
production constraints. Gildemacher et al. (2009) showed that only 3% of the seed tubers
sold were free of viruses.
True hybrid potato seeds are devoid of contaminating pathogens and therefore offer
an excellent opportunity to potato production improvement in East Africa. Such hybrids
should be attuned to the needs of the farmers, who grow their crop at the typical local
conditions like a short growing cycle of 90 – 100 days, high temperatures and tuber
development under short days. The prerequisites for establishing dedicated hybrid potato
breeding system for East Africa are:
• A (private) organization executing a breeding programme tailored to the needs of
the region.
• Secured supply of hybrid seeds for the region.
• Regulations supporting imports and exports of seeds, seed tubers and commercial
tubers.
• Formal registration system for breeders ’ rights protection.
• Local organizations testing new experimental hybrids.
Such breeding programme can only become sustainable if the complete downstream part
of potato food chain is also well organized. This includes the following:
• Production systems of disease-free seed tubers from seedlings.
• An effi cient supply system for farmers to obtain hybrid cultivars.
• Effi cient farmers ’ cropping systems to produce high quality potato tubers for the
target markets.
• Effi cient logistics to transport farm produce to consumers and processors.
Table 2 Potato area and production in six East-African countries in 2014
(FAOstat, 2016)
Country Area (000 ha) Production (000 tonnes) Yield (tonnes/ha)
Burundi 24.4 181.2 7.4
Kenya 115.6 1626.0 14.1
Rwanda 166.4 2225.1 13.4
Tanzania 211.5 1761.0 8.3
Uganda 39.0 188.0 4.8
Ethiopia 67.4 921.8 13.7
AQ: We
have
changed ‘T’
to ‘tonnes’
in “Table
2”. Please
check if this
is correct?
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18 Hybrid potato breeding for improved varieties
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• A well-developed consumer market.
• A business plan over the whole potato chain, whereby all stakeholders benefi t.
Solynta is already testing the fi rst experimental hybrids in the highlands of the Albertine
Rift in Ituri Province, Democratic Republic of Congo. These are experimental hybrids
derived from the European hybrid breeding programme. At a local farm, established by
the Lake Albert Foundation, seeds have been sown and seedlings have been transplanted
into the fi eld (Fig. 9). These hybrids are evaluated in good cooperation with local farmers
and the results are shared with the Solynta breeding programme. These data are used
to adjust the selection of inbred lines to the needs in East Africa and to continuously
generate new experimental hybrids, which will be tested at the Congo farm again. This
iterative and interactive process may already select the fi rst dedicated hybrids for East
Africa in 2 – 3 years.
This breeding programme is accompanied by research on cropping systems for the
region. In addition, training programmes for research institutions, agronomists, local staff
and interested farmers in the regions will make the farmers ’ communities and relevant
institutions familiar with the new concepts of hybrid potato cultivars. Also market studies
are needed to identify and secure stable and sustainable markets for the farmers ’ potato
products. This market may comprise other countries in the Great Lakes Region (South
Sudan, Uganda, Burundi and Rwanda).
The support for this programme by national authorities and development agencies –
also in the neighbouring countries like Uganda – is required, but it will take time before
the concept of hybrid potato cultivars is fully understood and accepted.
8 Conclusion
Since the fi rst crosses in 2008, the Solynta research efforts have been focused on the
development of a hybrid potato breeding system. This research has now reached the stage
where hybrid potato breeding will become reality. This has recently been supported by
two leading potato breeding companies in EU, KWS and HZPC, who have also expressed
their conviction that hybrid potato breeding will be future main breeding system (KWS,
2016). In addition, 21 leaders in the industry and potato science in the United States have
expressed their opinion on ‘ Reinventing potato as a diploid inbred line-based crop ’ with a
scientifi c base for diploid hybrid potato breeding (Jansky et al., 2016).
We have made great advances in the development of useful homozygous inbred lines
and the fi rst fi eld evaluations of experimental hybrids have shown the potential of hybrid
cultivars to harvest heterosis.
The technologies to develop new cropping systems adapted to various climate zones
and agronomic practices, which allow the production of commercial seed tubers from
seedlings, are already available.
We envision that future hybrid potato varieties, similar to modern tomato hybrids, will
harbour up to 15 resistance genes. In addition, the lifetime of new potato hybrid cultivars
will be reduced to less than 5 years as is the experience in sugar beet, where the lifetime
of new cultivars is only 2 – 3 years since the fi rst diploid hybrids have been introduced into
the market.
Our inbred lines will also be of great value for research purposes as they allow the
development of sophisticated populations that are very helpful in genetic and genomic
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Hybrid potato breeding for improved varieties 19
studies as has been shown in other crops. This will give a boost in the exploration and
exploitation of the genetic variation in the potato germplasm.
The self-compatible potato inbred lines will greatly stimulate quantitative research on
the genetic control of complex traits such as interaction with mycorrhiza, biotic stresses,
nutrient uptake, nutrition value and processing quality.
The application of hybrid potato breeding technology will not be restricted to the most
advanced research groups in the public or private institutions. New players in the scientifi c
and applied breeding fi eld of the potato business will arise and new cooperations will
be established to take full advantage of the hybrid breeding technology in science and
product development.
Finally, hybrid potato breeding will require the skills of the breeder as well as the
knowledge and tools of scientists. As a result, a new generation of potato breeding teams
will be established combining the skills of the breeder with the in-depth knowledge of
plant scientists.
9 Where to look for further information
This chapter describes the fi rst implementation of a hybrid potato breeding strategy
and the progress that is made since 2008. We direct the interested reader to the papers
of Almekinders (1996, 2009) to learn about ‘ conventional TPS ’ . As a textbook on plant
breeding, we suggest Brown and Cagliari (2011), while more advanced potato genetics
and genomics is found in Bradeen and Kole (2011). The history and mechanism of
Phytophthora attack is well described by Fry (2008). Finally, FAO (FAOstat, 2016) statistics
provide numerous data on potato cultivation.
Leading centres of research include Wageningen University in The Netherlands, James
Hutton Institute in the United Kingdom, the International Potato Centre in Peru, Wisconsin
University and Michigan State University both in the United States, whereby the most
recent paper of Jansky et al. (2016) can be considered a must for anybody interested in
hybrid potato breeding.
10 Acknowledgements
We are grateful to Jan Leemans and Herman Fleer for critically reading and reviewing this
manuscript.
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