QTL analysis of resistance to bacterial wilt caused by Ralstonia solanacearum in potato

Abstract

Ralstonia solanacearum causes bacterial wilt, a soil-borne disease and one of the most important maladies of potato and other Solanaceae crops. We analyzed the resistance of a potato clone to bacterial wilt by quantitative trait locus (QTL) analysis. A resistant diploid potato clone 10-03-30 was crossed with a susceptible diploid clone F₁-1 to generate a diploid, two-way pseudo-testcross F₁ population comprised of 94 genotypes. Dense linkage maps, containing 4,139 single nucleotide polymorphism markers with an average distance of 0.6 and 0.3 cM between markers, were constructed for both parents. The resistance level was evaluated by in vitro inoculation test with R. solanacearum (phylotype I/biovar 4/race 1). Five QTLs (qBWR-1 to -5) were identified on potato chromosomes 1, 3, 7, 10, and 11, and they explained 9.3–18.4% of the phenotypic variance. The resistant parent had resistant alleles in qBWR-2, qBWR-3, and qBWR-4 and susceptible alleles in qBWR-1 and qBWR-5. Accumulation of the resistant alleles in all five QTLs increased the level of resistance compared with that of the resistant parent. This is the first study to identify novel QTLs for bacterial wilt resistance in potato by using genome-wide markers.

Introduction

Bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases in potato, tomato, eggplant, tobacco, and other Solanaceae plants (Hayward 1994). This bacterium invades the plant body through wounded skin layers of the roots, immigrates into the vascular system, and spreads into the wood tissue system. Due to pathogenicity-related factors, such as viscous polysaccharides, bacterial cells themselves, and various enzymes, the conduit part is occluded to reduce the water-passing ability, causing wilting symptoms (Rahman et al. 1999, Wang et al. 2000). R. solanacearum occurs around the world mainly in tropical, subtropical, and temperate regions, with more than 200 plant species recognized as hosts (Denny 2006, Hayward 1991, 1994). The bacterium is soil-borne, which renders its complete removal difficult after the onset of invasion in fields (Hayward 1991, 1994).

R. solanacearum is characterized by diverse phenotypic and genetic variations. According to the host range and physiological traits, it has been classified into five races and six biovars (Buddenhagen et al. 1962, Hayward 1964). The phylogenetic analysis mainly classified it into four phylotypes, depending on the geographical origin: phylotype I (Asia), II (Americas), III (Africa), and IV (Indonesia) (Fegan and Prior 2005, Horita et al. 2014). In Japan, R. solanacearum phylotype I/biovar 4/race 1 and phylotype IV/biovar N2/race 3 are major causal pathogens of bacterial wilt (Horita and Tsuchiya 2001, Horita et al. 2010, Katayama and Kimura 1984). The two phylotypes have been identified at different times and places in potato cultivation fields, with phylotype I being more virulent and mainly detected in the hot season, from September to October, in warm regions of Japan (Horita et al. 2010, Katayama and Kimura 1984, 1986). In culture media, phylotype IV can grow at lower temperatures compared with phylotype I, which may explain why these two phylotypes are detected at different seasons in the fields (Katayama and Kimura 1984, 1986).

Hawkes (1990) described over 200 cultivated and wild potato species. A common potato (Solanum tuberosum) is tetraploid (2n = 4x = 48), whereas diploid (S. phureja) (2n = 2x = 24) to pentaploid (2n = 5x = 60) cultivated species are also known (Hawkes 1990). The wild potato species are mainly diploid (2n = 2x = 24). Tuber-bearing Solanum species are mostly self-incompatible, and S. tuberosum is highly heterozygous with tetrasomic inheritance, a segregation pattern more complex than disomic inheritance (Hawkes 1990).

Bacterial wilt is one of the most important diseases in potato (French et al. 1998). Although soil disinfection by chemical pesticides is effective, development of resistant varieties has been the most efficient approach for controlling bacterial wilt (Elphinstone 1994, Lebeau et al. 2011). The genetic resistance to bacterial wilt has been reported in various cultivated potato species (including S. tuberosum and S. phureja) and the closely related wild species (including S. chacoense) (Chen et al. 2013, Fock et al. 2000, 2001, Katayama and Kimura 1987, Jaworski et al. 1980, Sequeira and Rowe 1969, Siri et al. 2009, Thurston and Lozano 1968). Quantitative trait locus (QTL) analyses of bacterial wilt resistance in Solanaceae crops were conducted in tomato (Carmeille et al. 2006, Wang et al. 2000, 2013), eggplant (Lebeau et al. 2013), and tobacco (Lan et al. 2014, Qian et al. 2013). In potato, 109 Solanum chacoense-specific simple sequence repeat alleles in 44 somatic hybrids between S. chacoense and S. tuberosum were surveyed, in which three alleles on chromosomes 2 and 9 were significantly associated with the resistance to R. solanacearum (race 1, biovar 3) (Chen et al. 2013). Yanping et al. (2013) employed S. phureja to generate an F₁ mapping population and performed the bulked segregant analysis using sequence related amplified polymorphism markers. Three linkage groups harboring resistance QTLs were identified, but their associated chromosomes are unknown. The heritability of the resistance in potato is low due to its tetraploid nature, and the mode of inheritance of the bacterial wilt resistance is not yet clearly understood (Elphinstone 1994).

A tetraploid breeding clone Saikai 35 carries resistance genes to potato cyst nematode (H1) and Potato virus Y (Ry_chc) and exhibits high level of bacterial wilt resistance (Mori et al. 2012). In the present study, we used a diploid resistant clone induced from Saikai 35 to conduct QTL analysis of the bacterial wilt resistance. The use of a diploid potato for genetic analysis can be more efficient than that of tetraploid and requires smaller populations to detect recessive genes (Ortiz and Peloquin 1994, Peloquin et al. 1990). In addition, the genetic analysis of diploid potato can circumvent the effect of double reduction by tetrasomic inheritance, impeding the estimation of genetic map distance (Ortiz and Peloquin 1994). Dense genetic maps were first constructed for resistant and susceptible diploid parents using 4,139 single nucleotide polymorphism (SNP) markers, which were generated from a 12,808 potato SolCAP SNP array (Bali et al. 2017, da Silva et al. 2017). The present study is the first to detect and map potato QTLs associated with resistance to bacterial wilt by saturated linkage map of genome-wide markers.

Materials and Methods

Plant materials

To facilitate genetic analysis, we used a diploid potato population obtained by crossing a diploid resistant clone and a diploid susceptible clone. The diploid resistant clone was obtained from Saikai 35, a tetraploid breeding clone highly resistant to bacterial wilt (Mori et al. 2012), through parthenogenesis by crossing with the pollen of a haploid inducer S. phureja 460 (=Ivp35) and named 10-03-30 (Fig. 1). This clone also showed high resistance to bacterial wilt in field evaluations conducted over three seasons in Japan and by in vitro inoculation tests. This resistant parent (hereafter, RP) was crossed as the female parent with the susceptible diploid clone F₁-1 (hereafter, SP) as the male parent to generate 94 F₁ plants grown in vitro on the Murashige and Skoog (MS) medium (Murashige and Skoog 1962).

Fig. 1

The pedigree of the segregating F₁ population.

Inoculation and disease resistance test

The in vitro inoculation test was used for resistance evaluation of the F₁ plants because the test is simple and reproducible, and the results were highly correlated with those in the field tests (Habe 2018). The in vitro screening medium, containing 30 mL vermiculite and 20 mL MS liquid medium in a glass tube (40 mm × 130 mm), was sterilized by autoclaving. The in vitro-grown plantlets were cut at nodes below the third or fourth leaf from the apex. The cut stem tips were transplanted onto a screening medium and incubated in a growth chamber at 20°C for two weeks until the inoculation with R. solanacearum to promote rooting. The night-day cycle was 16 h light at 3000–4000 lux and 8 h dark. The R. solanacearum strain MAFF327001 (phylotype I/biovar 4/race 1) (Habe 2018, Horita et al. 2010, Suga et al. 2013) was used in this study for the inoculation test. Phylotype I is distributed widely in Asia (Fegan and Prior 2005). This strain was isolated from potato cultivated in Nagasaki Prefecture, the area with the highest frequency of the bacterial wilt disease in Japan. The MAFF327001 strain was grown at 30°C on 2,3,5-triphenyltetrazolium chloride solid medium (Kelman 1954). The white fluidal colonies were transferred into casamino acid-peptone-glucose medium (Hendrick and Sequeira 1984). The concentration of inoculum suspension was determined by measuring OD at 600 nm and adjusted to 10⁸ colony-forming units mL⁻¹. One milliliter of bacterial suspension was poured into each screening medium. The incubation temperature after the inoculation was 28°C. Ten plantlets per genotype were treated as one replicate, and three replicates were prepared for the experiment.

The resistance level was represented by the disease index (DI) measured 20 days after inoculation using a 0–4 scale on the basis of the extent of stem wilting: 0 (no symptoms), 1 (up to 25%), 2 (26–50%), 3 (51–75%), and 4 (76–100% of the stem wilted) (Habe 2018) (Fig. 2).

Fig. 2

Plants with the disease index score 0 (no symptom) and score 4 (76–100% wilted) in the in vitro potato resistance assay for Ralstonia solanacearum. Caps were removed for photography.

SNP genotyping

Approximately 100 mg of fresh leaves were collected from in vitro plantlets of the F₁ plants, and total DNA was extracted using CTAB-LiCl method (Sul and Korban 1996). DNA concentration was measured by the Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). One microgram of dried DNA from each sample was sent to GeneSeek (Neogen Corporation, Lincoln, NE, USA) to obtain data from the 12K potato V2 SNP array (Bali et al. 2017, da Silva et al. 2017). The obtained microarray intensity data were analyzed using GenomeStudio Polyploid Clustering Ver 1.0 (Illumina Inc., San Diego, CA, USA) with default parameters and cluster distance of −0.07. After the analysis, all genotype calls were manually checked for accuracy; inaccurate data were treated as missing data.

Linkage map construction

The obtained 12K SNP data were filtered by excluding poor quality SNPs. Thus, the SNPs lacking parental genotypes were excluded, and those that segregated into two clusters of parental types were selected. In addition, SNPs with week signals (Norm R < 0.2) or with >20% missing data were also excluded. As both diploid parents were highly heterozygous, the segregating population was regarded as a two-way pseudo testcross population (Grattapaglia and Sederoff 1994). The two parental maps were constructed using Carthagene Ver 1.0 (Givry et al. 2005) with logarithm of odds (LOD) score of 10 and the maximum genetic distance between markers of 100 cM, with the latter being calculated based on Kosambi function (Kosambi 1944). The two-way pseudo testcross strategy described in Iwata et al. (2016) was employed for linkage analysis. During the mapping process, SNPs that were differently located from or unanchored in Potato pseudomolecules (PMs) v. 4.03 (Sharma et al. 2013) were excluded.

QTL analysis

QTL analysis was conducted on a backcross design by regarding the F₁ population as a backcross population in QTL Cartographer version 2.5 (Wang et al. 2005) with backcross mode and using composite interval mapping, which is specifically designed to reduce background noise that can affect QTL detection. Parameters of the analysis were set for model 6 with window size of 2 cM, probability for “into” of 0.05, and probability for “out” of 0.05. A LOD threshold for QTL detection was calculated by permutation tests with 1,000 repetitions to control for genome-wide error rate of 5%. QTL analysis was separately performed for each of the two parental linkage maps. Linkage maps and QTL positions were drawn by MapChart 2.30 (Voorrips 2002).

Statistical analysis

All statistical analyses, excluding QTL analysis, were done in Rcmdr package (Fox 2005) of R version 3.3.3. (R Core Team 2017). Analysis of variance (ANOVA) was used to assess the difference between each QTL, in which the data of the three replicates obtained from the in vitro tests were used as a responsive variable.

Results

Evaluation of the resistance to bacterial wilt

In vitro inoculation tests were conducted for 10 plantlets per genotype with three replications. ANOVA test confirmed no significant difference among the three replicates (P = 0.1797). Thus, the DIs of the triplicates could be averaged and used as a DI of each genotype. The DI of the RP was 0.60 and that of the SP was 2.77. The DI of F₁ ranged from 0.17 to 3.63; it was lower in some F₁ plants than in RP, but higher in other F₁ plants than in SP (transgressive segregation). The F₁ plants’ DIs were distributed normally, and their mean value was 1.69 (Fig. 3).

Fig. 3

Distribution of the disease index (DI) after inoculation with Ralstonia solanacearum in a segregating population, which consisted of 94 genotypes from the cross between a resistant parent (RP) and a susceptible parent (SP). The number of resistant quantitative trait loci (QTLs) possessed by each F₁ plant is represented by a color density in each bar. The numbers in the legend indicate the mean of disease index for each group.

Linkage map construction

After filtering the 12,808 SNPs, we identified 4,139 SNPs that were polymorphic between RP and SP and used them to create genetic maps (Table 1). In RP, 1,476 SNP loci were heterozygous (AB genotype), while these SNPs were homozygous (AA genotype) in SP and segregated among the F₁ plants to AA (SP type) and AB (RP type) genotypes. In linkage analysis, these SNP loci were mapped to 422 positions, spanning over 12 RP chromosomes. This map is referred to as RP map hereafter. In SP, 2,663 SNP loci were heterozygous and segregated among the F₁ plants; these SNP loci were homozygous in RP. These were mapped to 475 positions on 12 SP chromosomes; the map is denoted as SP map hereafter. The total length of the RP map was 948.2 cM, and that of the SP map was 828.4 cM. The average distance between SNPs was 0.6 cM in the RP map and 0.3 cM in the SP map, respectively. Distorted segregation was observed in the RP chromosome 12 and in the SP chromosomes 1, 2, 11, and 12 (data not shown).

Table 1 The number of segregating single nucleotide polymorphisms (SNPs) derived from a resistant parent (RP) and a susceptible parent (SP) and their number of mapped positions and map lengths

Chromosome	Genetic map of RP			Genetic map of SP			Total number of SNPs	Total number of map positions
	No. of SNPs	No. of mapped positions	Map length (cM)	No. of SNPs	No. of mapped positions	Map length (cM)
1	239	47	110.5	381	57	80.2	620	104
2	50	20	50.0	324	44	78.4	374	64
3	95	26	83.6	309	39	88.8	404	65
4	135	35	90.2	163	42	69.1	298	77
5	91	28	73.7	217	37	69.6	308	65
6	107	41	73.0	186	35	59.9	293	76
7	121	34	79.3	191	39	81.0	312	73
8	118	27	70.8	111	29	50.5	229	56
9	200	65	85.0	108	38	69.4	308	103
10	77	28	77.0	179	35	62.0	256	63
11	136	36	68.9	238	30	58.8	374	66
12	107	35	86.2	256	50	60.7	363	85
Total	1476	422	948.2	2663	475	828.4	4139	897

QTL analysis

QTL analysis was performed for the DIs of the F₁ population using the RP and SP maps. According to the permutation tests with 1,000 repetitions, the LOD threshold at the 5% significance level was 2.87 for the RP map and 2.80 for the SP map. At these threshold levels, five QTLs (qBWR-1, qBWR-2, qBWR-3, qBWR-4, and qBWR-5) on chromosomes 1, 3, 7, 10, and 11 were detected using the composite interval mapping method (Table 2, Fig. 4). The QTL with the highest effect was qBWR-3, identified using the RP map, and it explained 18.4% of the total variance. At the SNP locus (solcap_snp_c2_4555) nearest to the maximum LOD of qBWR-3, the mean DIs of the RP-type and SP-type genotypes were significantly different at 1.40 and 2.01, respectively. The other four QTLs were detected using the SP map. The maximum LOD score and explained variance of each of these QTLs ranged from 3.26 to 5.56 and from 9.3 to 15.6%, respectively, in the RP- and SP-type genotypes. The mean DIs among F₁ plants with the RP-type genotype and those with the SP-type genotype at the SNP loci nearest to the maximum LODs were significantly different at 5% level, except for the SNP (solcap_snp_c2_37816) of qBWR-1 (Table 2). As expected, the RP-type genotypes were more resistant than the SP-type genotypes at qBWR-2, qBWR-3, and qBWR-4. However, at qBWR-1 and qBWR-5, the SP-type genotype was more resistant than the RP-type genotype.

Table 2 Quantitative trait loci for bacterial wilt resistance detected in the F₁ population of a resistant (RP) and a susceptible (SP) parent

QTL	Chr.	Map	Position (cM)	Max. LOD (cM)	Max. LOD score	Explained variance (%)	Mean disease index (SE) at the SNP locus nearest to the max. LOD position
							SNP	Position (cM)	Resistant genotype	RP-type genotypea	SP-type genotypea	P valueb
qBWR-1	1	SP	76.8–81.2	79.1	4.09	11.0	c2_37816	79.1	SP-type hetero	1.75 ± 0.09 (n = 50)	1.60 ± 0.12 (n = 42)	0.067
qBWR-2	3	SP	11.8–18.0	15.0	5.56	15.6	c2_50637	15.0	RP-type homo	1.49 ± 0.08 (n = 56)	1.98 ± 0.12 (n = 38)	<0.005
qBWR-3	7	RP	14.2–26.3	25.3	5.33	18.4	c2_4555	25.3	RP-type hetero	1.40 ± 0.11 (n = 46)	2.01 ± 0.11 (n = 45)	<0.001
qBWR-4	10	SP	6.6–11.9	8.8	5.54	15.5	c2_32779	8.8	RP-type homo	1.46 ± 0.08 (n = 47)	1.91 ± 0.12 (n = 45)	0.007
qBWR-5	11	SP	31.6–37.0	36.0	3.26	9.3	c2_12333	35.0	SP-type hetero	2.17 ± 0.15 (n = 20)	1.54 ± 0.08 (n = 72)	<0.001

^a  The mean of disease indices and the number of individuals were calculated excluding individuals with at least one missing data at each SNP marker.

^b  Mann-Whitney U test was performed on the mean of the disease indices between individuals of RP- and SP-type genotypes.

Chr., chromosome; SNP, single nucleotide polymorphism; LOD, logarithm of odds.

Fig. 4

Five quantitative trait loci (QTLs) detected on susceptible parent (SP) chromosome 1 (qBWR-1), SP chromosome 3 (qBWR-2), resistant parent (RP) chromosome 7 (qBWR-3), SP chromosome 10 (qBWR-4), and SP chromosome 11 (qBWR-5). The solid box and the loci of bold letters represent a QTL region. The order of the markers located at the same loci follows that of the physical position in Potato pseudomolecules v. 4.03 (Sharma et al. 2013).

The ANOVA of all detected QTLs as factors indicated significant effects of each QTL, as well as of the interactions among qBWR-1, qBWR-3, and qBWR-5 (Table 3); that is, the resistance effect of qBWR-1 was possible only if both qBWR-5 and qBWR-3 were resistant genotypes (p < 0.10). If genotypes at either qBWR-3 or qBWR-5 were susceptible genotypes, qBWR-1 did not show the resistant effect (Table 4). Thus, an epistatic effect of qBWR-1 to both qBWR-3 and qBWR-5 was disclosed.

Table 3 Analysis of variance representing a significant interaction among three quantitative trait loci (QTLs)

QTL	Df	F value	P value
qBWR-1	1	12.87	<0.001
qBWR-2	1	22.88	<0.001
qBWR-3	1	14.58	<0.001
qBWR-4	1	20.01	<0.001
qBWR-5	1	17.69	<0.001
qBWR-1 × qBWR-3 × qBWR-5	1	3.97	<0.047

Table 4 Interaction among three quantitative trait loci (QTLs) (qBWR-1, qBWR-3, and qBWR-5)

qBWR-3 (c2_4555)	qBWR-5 (c2_12333)	qBWR-1 (c2_37816)a		P valueb
		SP type (resistant)	RP type (susceptible)
RP type (resistant)	SP type (resistant)	1.12 ± 0.10 (n = 15)	1.35 ± 0.11 (n = 21)	0.072
	RP type (susceptible)	1.76 ± 0.16 (n = 5)	2.01 ± 0.26 (n = 3)	0.653
SP type (susceptible)	SP type (resistant)	1.79 ± 0.24 (n = 14)	1.91 ± 0.09 (n = 18)	0.143
	RP type (susceptible)	2.47 ± 0.32 (n = 6)	2.28 ± 0.35 (n = 6)	0.748

^a  The mean of disease indices and the number of individuals were calculated excluding individuals with at least one missing data at each SNP marker.

^b  Mann-Whitney U test was performed on the mean of the disease indices between individuals of the resistant parent (RP)-type and the susceptible parent (SP)-type genotypes.

In the F₁ population, the mean DI decreased by increasing the number of resistant alleles at the five QTLs in a single individual: the mean DIs was 2.83 for F₁ plants with no resistant QTL, 2.41 for those with one resistant QTL, 2.13 for those with two QTLs, 1.54 for those with three QTLs, 1.17 for those with four QTLs, and 0.43 for those with five QTLs (Fig. 3).

Discussion

This study analyzed the segregating diploid population of potato obtained by crossing two heterozygous parental lines. In full-sib population from two heterozygous parents, segregations of two genotypes (AA:AB) to four genotypes (AC:AD:BC:BD) can be expected, which complicates genetic analysis. In this study, to simplify the genetic analysis, the population was treated as a “two-way pseudo-testcross” population (Grattapaglia and Sederoff 1994), by using only polymorphic loci that were heterozygous in one parent and homozygous in the other parent. The simple association between phenotype and allele revealed the regions associated with the bacterial wilt resistance by evaluating the segregation of two genotypic classes (AA vs. AB).

The RP 10-03-30 of F₁ population is a diploid clone derived from a tetraploid clone Saikai 35 (Mori et al. 2012), which was obtained by breeding S. phureja and S tuberosum, and the SP F₁-1 is an interspecific hybrid between S. chacoense and S. phureja (Hosaka and Hanneman 1998). Some clones of both S. chacoense and S. phureja are popular sources of bacterial wilt resistance (Chen et al. 2013, French et al. 1998). In addition, some cultivars of S. tuberosum were confirmed to be resistant to bacterial wilt (Jaworski et al. 1980, Katayama and Kimura 1987). The bacterial wilt resistant QTLs in F₁ population likely originated from S. chacoense, S. phureja, and S. tuberosum.

As mentioned above, bacterial wilt resistance of potato is controlled by QTLs. It was assumed that S. phureja has three major resistance genes (Rowe and Sequeira 1970), whereas the resistance of S. tuberosum is encoded by a recessive resistance gene (Katayama and Kimura 1987) and related to plant heat tolerance (Tung et al. 1990a). In contrast, bacterial wilt resistance in S. phureja, S. chacoense, and S. tuberosum has been reported to be affected by a specific strain of R. solanacearum, the potato genotype, and environment interaction (Tung et al. 1990b, Watanabe et al. 1999); thus, bacterial wilt resistance in potato is a complex genetic process (Patil et al. 2012). The locus position of two genes responsible for resistance to R. solanacearum (race 1, biovar 3) was presumed to be on chromosomes 2 and 9 of S. chacoense, respectively (Chen et al. 2013). It is hardly clarified in other gene locus positions.

The bacterial wilt resistance derived from RP and SP was quantitatively inherited to the F₁ population (Fig. 3), which is in agreement with previous reports (Katayama and Kimura 1987, Rowe and Sequeira 1970, Tung et al. 1990a, 1990b, Watanabe et al. 1992, 1999). The QTL analysis using composite interval mapping detected five resistance QTLs (Table 2, Fig. 4). In the F₁ population, the RP-type genotypes were more resistant than the SP-type genotypes at qBWR-2, qBWR-3, and qBWR-4, whereas the SP-type genotypes were more resistant at qBWR-1 and qBWR-5. These results suggest that RP was heterozygous with resistant allele and susceptible allele at qBWR-3, homozygous with a resistant allele at both qBWR-2 and qBWR-4, and homozygous with a susceptible allele at both qBWR-1 and qBWR-5, whereas SP was homozygous with a susceptible allele at qBWR-3 and heterozygous at other QTLs. The two QTLs (qBWR-2 and qBWR-4) were speculated to exhibit the possibility of recessive inheritance (Table 2). The detected QTLs qBWR-1 and qBWR-5 (chromosome 1 and 11) in SP were derived either from one of its parents, S. chacoense 525-3 or S. phureja 1.22, or from both. In contrast, Chen et al. (2013) reported that the resistant genes derived from S. chacoense were on chromosomes 2 and 9. Because the S. chacoense genotype and the biovar of R. solanacearum used in the present study differ from those in previous studies, it is difficult to discuss which origin contributed to the resistance detected in our study. Alternatively, if we assume that the resistance reaction was fully derived from the RP, the RP had three QTLs promoting the resistance and two QTLs suppressing the resistance. However, considering that some of the F₁ plants were more susceptible than the SP or more resistant than the RP (Fig. 3), the former explanation stating the presence of resistant genes in both parents would be more probable.

The detected QTLs explained 9.3–18.4% of the variance and no major QTL was detected. However, the tendency to improve the degree of individual resistance was confirmed as the number of QTLs increased—one F₁ plant had five resistant alleles and DI of 0.43, and thus it was more resistant than the RP (DI = 0.60) (Fig. 3). Bacterial wilt resistance in potato population could be improved by accumulating the resistance of different origin (Tung et al. 1990a), which is evident in our study. In addition, the interaction between QTLs contributed to the higher resistance to bacterial wilt (Tables 3, 4). Tung et al. (1990a) proposed the possibility of combining ability (effect caused by the combination of mating parents) affecting resistance levels to bacterial wilt, which may be explained by the genetic interaction between QTLs suggested in this study.

A study on the resistance of the model plant Arabidopsis thaliana to bacterial wilt revealed that receptor protein kinase gene CLAVATA 1 (CLV 1) enhanced resistance to bacterial wilt (Hanemian et al. 2016). CLV 1 plays a crucial role in the regulation of stem cell homeostasis at the shoot (Clark et al. 1993, 1997) and root apical meristems (Stahl et al. 2013). The SNP solcap_c2_50637 (Chromosome 3, 15.0 cM) closest to qBWR-2 (Fig. 4) is included in CLV 1 (PGSC0003DMG400016685), and it was assumed that CLV 1 and qBWR-2 were correlated. Tomato is a related crop species with almost identical chromosome synteny to that of potato (Tanksley et al. 1992). In tomato, two major QTLs, Bwr-6 on chromosome 6 and Bwr-12 on chromosome 12, were identified for resistance to phylotype I and phylotype II strains of R. solanacearum (Wang et al. 2013). The QTLs on chromosomes 1, 3, 7, 10, and 11, detected in the present study, were differently located from tomato QTLs. Five hot spots for resistance genes have been identified on potato and tomato chromosomes 5 (two hot spots), 9, 11, and 12 (Gebhardt and Valkonen 2001). qBWR-5 was identified in a region very close to or within the resistance cluster on chromosome 11, which also harbors the resistance genes to potato wart (Sen1; Obidiegwu et al. 2015), Potato virus Y (Ry_adg; Hämäläinen et al. 1997), and white cyst nematode Globodera pallida (GpaXI^l_tar; Tan et al. 2009). The other four QTLs were not specifically located within these resistance gene clusters. However, qBWR-1 was located in the proximity of the potato wart resistance gene (Rse-1b; Obidiegwu et al. 2015), and qBWR-2 and qBWR-3 were positioned in the vicinity of resistance QTLs to Globodera rostochiensis (Gro1.4 and Gro1, respectively; Barone et al. 1990, Kreike et al. 1996). Because the genes resistant to various pests and diseases are also clustered in potato, new resistant genes may be located in the proximity of QTLs reported here. These results may facilitate the identification of other candidate genes and molecular functions of resistance QTLs.

In conclusion, we identified and mapped five novel QTLs for bacterial wilt resistance; our study is the first to conduct QTL analysis of the bacterial wilt resistance in potato by genome-wide markers. In general, there is a possibility that QTLs are linked to undesirable agronomic traits or they are difficult to transfer into cultivars (Denny 2006). The resistant parent used in the present study was 10-03-30, a diploid of Saikai 35. Saikai 35 is a breeding clone carrying potato cyst nematode resistance (H1) and Potato virus Y resistance (Ry_chc) genes and bacterial wilt resistance (Mori et al. 2012), and it has been used as breeding material. Recently, Saikai 35 was used as a female parent to breed a new variety, ‘Nagasaki Kogane’, which has H1 and Ry_chc genes and exhibits a high level of resistance to bacterial wilt (Sakamoto et al. 2017). ‘Nagasaki Kogane’ is equivalent to Saikai 35 in the mean tuber weight and marketable yield which are one of the most important traits in economical potato production. This cultivar has high starch content among cultivars implemented in double cropping practice and does not have a red tuber eyes possessed by Saikai 35, which is recognized to be a poor trait in regions with double cropping pattern (Mori et al. 2012, Sakamoto et al. 2017). This implies that the QTLs found in the present study are not linked with serious genetic defects. However, it is unknown whether the resistance of Saikai 35 can be fully explained by the five QTLs, and it is necessary to confirm whether these QTLs function similarly in the tetraploid genetic background. For potato breeding, a discovery of these novel QTLs is an important step toward development of molecular markers for bacterial wilt resistance.

Author Contribution Statement

IH designed and executed the study, prepared all tables and figures, and wrote the manuscript. KM completed the test design, supervised the experiments, and corrected the manuscript. TN supervised the test design. MY contributed to the test design and data analysis and corrected the manuscript. TH supervised the linkage analysis and QTL analysis.

Acknowledgments

We thank Dr. Kazuyoshi Hosaka, Obihiro University of Agriculture and Veterinary Medicine, for improving an earlier version of the manuscript. This research was funded by Nagasaki Prefecture.

Literature Cited

•  Bali,  S.,  V.  Sathuvalli,  C.  Brown,  R.  Novy,  L.  Ewing,  J.  Debons,  D.  Douches,  J.  Coombs,  D.  Navarre,  J.  Whitworth et al. (2017) Genetic fingerprinting of potato varieties from the northwest potato variety development program. Am. J. Potato Res. 94: 54–63.
•  Barone,  A.,  E.  Ritter,  U.  Schachtschabel,  T.  Debener,  F.  Salamini and  C.  Gebhardt (1990) Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis. Mol. Gen. Genet. 224: 177–182.
•  Buddenhagen,  I.W.,  L.  Sequeira and  A.  Kelman (1962) Designations of races in Pseudomonas solanacearum. Phytopathology 52: 726.
•  Carmeille,  A.,  C.  Caranta,  J.  Dintinger,  P.  Prior,  J.  Luisetti and  P.  Besse (2006) Identification of QTLs for Ralstonia solanacarum race 3-phylotype II resistance in tomato. Theor. Appl. Genet. 113: 110–121.
•  Chen,  L.,  X.  Guo,  C.  Xie,  L.  He,  X.  Cai,  L.  Tian,  B.  Song and  J.  Liu (2013) Nuclear and cytoplasmic genome components of Solanum tuberosum + S. chacoense somatic hybrids and three SSR alleles related to bacterial wilt resistance. Theor. Appl. Genet. 126: 1861–1872.
•  Clark,  S.E.,  M.P.  Running and  E.M.  Meyerowitz (1993) CLAVATA1, a regulator of meristem and flower development in Arabidopsis. Development 119: 397–418.
•  Clark,  S.E.,  R.W.  Williams and  E.M.  Meyerowitz (1997) The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell 89: 575–585.
•  da Silva,  W.L.,  J.  Ingram,  C.A.  Hackett,  J.J.  Coombs,  D.  Douches,  G.J.  Bryan,  W.  De Jong and  S.  Gray (2017) Mapping loci that control tuber and foliar symptoms caused by PVY in autotetraploid potato (Solanum tuberosum L.). G3 (Bethesda) 7: 3587–3595.
•  Denny,  T. (2006) Plant pathogenic Ralstonia species. In:  Gnanamanickam,  S.S. (ed.) Plant-associated bacteria, Springer, Dordrecht, pp. 573–644.
•  Elphinstone,  J.G. (1994) Inheritance of resistance to bacterial diseases. In:  Bradshaw,  J.E and  G.R.  Mackay (eds.) Potato genetics. CAB international, Wallingford, pp. 429–446.
•  Fegan,  M. and  P.  Prior (2005) How complex is the Ralstonia solanacearum species complex. In:  Allen,  C.,  P.  Prior and  A.C.  Hayward (eds.) Bacterial wilt disease and the Ralstonia solanacearum species complex. APS Press, St. Paul, pp. 449–461.
•  Fock,  I.,  C.  Collonnier,  A.  Purwito,  J.  Luisetti,  V.  Souvannavong,  F.  Vedel,  A.  Servaes,  A.  Ambroise,  H.  Kodja,  G.  Ducreux et al. (2000) Resistance to bacterial wilt in somatic hybrids between Solanum tuberosum and Solanum phureja. Plant Sci. 160: 165–176.
•  Fock,  I.,  C.  Collonnier,  J.  Luisetti,  A.  Purwito,  V.  Souvannavong,  F.  Vedel,  A.  Servaes,  A.  Ambroise,  H.  Kodja,  G.  Ducreux et al. (2001) Use of Solanum stenotomum for introduction of resistance to bacterial wilt in somatic hybrids of potato. Plant Physiol. Biochem. 39: 899–908.
•  Fox,  J. (2005) The R Commander: A basic-statistics graphical user interface to R. J. Stat. Softw. 14: 1–42.
•  French,  E.R.,  R.  Anguiz and  P.  Aley (1998) The usefulness of potato resistance to Ralstonia solanacearum, for the integrated control of bacterial wilt. In:  Prior,  P.,  C.  Allen and  J.  Elphinstone (eds.) Bacterial wilt disease. Springer, Berlin, pp. 381–385.
•  Gebhardt,  C. and  J.P.T.  Valkonen (2001) Organization of genes controlling disease resistance in the potato genome. Annu. Rev. Phytopathol. 39: 79–102.
•  Givry,  S.D.,  M.  Bouchez,  P.  Chabrier,  D.  Milan and  T.  Schiex (2005) Carthagene: multipopulation integrated genetic and radiation hybrid mapping. Bioinformatics 21: 1703–1704.
•  Grattapaglia,  D. and  R.  Sederoff (1994) Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a Pseudo-Testcross: Mapping strategy and RAPD markers. Genetics 137: 1121–1137.
•  Habe,  I. (2018) An in vitro assay method for resistance to bacterial wilt (Ralstonia solanacearum) in potato. Am. J. Potato Res. 95: 311–316.
•  Hämäläinen,  J.H.,  K.N.  Watanabe,  J.P.T.  Valkonen,  A.  Arihara,  R.L.  Plaisted,  E.  Pehu,  L.  Miller and  S.A.  Slack (1997) Mapping and marker-assisted selection for a gene for extreme resistance to potato virus Y. Theor. Appl. Genet. 94: 192–197.
•  Hanemian,  M.,  X.  Barlet,  C.  Sorin,  K.A.  Yadeta,  H.  Keller,  B.  Favery,  R.  Simon,  B.P.H.J.  Thomma,  C.  Hartmann,  M.  Crespi et al. (2016) Arabidopsis CLAVATA1 and CLAVATA2 receptors contribute to Ralstonia solanacearum pathogenicity through a miR169-dependent pathway. New Phytol. 211: 502–515.
•  Hawkes,  J.G. (1990) The potato: Evolution, biodiversity and genetic resources. Belhaven Press, London, p. 259.
•  Hayward,  A.C. (1964) Characteristics of Pseudomonas solanacearum. J. Appl. Bacteriol. 27: 265–277.
•  Hayward,  A.C. (1991) Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu. Rev. Phytopathol. 29: 65–87.
•  Hayward,  A.C. (1994) The hosts of Pseudomonas solanacearum. In:  Hayward,  A.C. and  G.L.  Hartman (eds.) Bacterial wilt: the disease and its causative agent, Pseudomonas solanacearum. CAB International, Wallingford, pp. 9–24.
•  Hendrick,  C.A. and  L.  Sequeira (1984) Lipopolysaccharide-defective mutants of the wilt pathogen Pseudomonas solanacearum. Appl. Environ. Microbiol. 48: 94–101.
•  Horita,  M. and  K.  Tsuchiya (2001) Genetic diversity of Japanese strains of Ralstonia solanacearum. Phytopathology 91: 399–407.
•  Horita,  M.,  Y.  Suga,  A.  Ooshiro and  K.  Tsuchiya (2010) Analysis of genetic and biological characters of Japanese potato strains of Ralstonia solanacearum. J. Gen. Plant Pathol. 76: 196–207.
•  Horita,  M.,  K.  Tsuchiya,  Y.  Suga,  K.  Yano,  T.  Waki,  D.  Kurose and  N.  Furuya (2014) Current classification of Ralstonia solanacearum and genetic diversity of the strains in Japan. J. Gen. Plant Pathol. 80: 455–465.
•  Hosaka,  K. and  R.E.  Hanneman (1998) Genetic of self-compatibility in a self-incompatible wild diploid potato species Solanum chacoense. 1. Detection of an S locus inhibitor (Sli) gene. Euphytica 99: 191–197.
•  Iwata,  H.,  M.F.  Minamikawa,  H.  Kajiya-Kanegae,  M.  Ishimori and  T.  Hayashi (2016) Genomics-assisted breeding in fruit trees. Breed. Sci. 66: 100–115.
•  Jaworski,  C.A.,  R.E.  Webb,  R.W.  Goth and  S.C.  Phatak (1980) Relative resistance of potato cultivars to bacterial wilt. Am. Potato J. 57: 159–165.
•  Katayama,  K. and  S.  Kimura (1984) Prevalence and temperature requirements of biovar II and IV strains of Pseudomonas solanacearum from potatoes. Jpn. J. Phytopathol. 50: 476–482.
•  Katayama,  K. and  S.  Kimura (1986) Ecology and protection of bacterial wilt of potato: 1. Ecology and strains of P. solanacearum. Bull. Nagasaki Pref. Agri. Fore. Exp. Stn. 13: 1–30.
•  Katayama,  K. and  S.  Kimura (1987) Ecology and protection of bacterial wilt of potato: 2. Some control methods and their integration. Bull. Nagasaki Pref. Agri. Fore. Exp. Stn. 14: 29–57.
•  Kelman,  A. (1954) The relationship of pathogenicity of Pseudomonas solanacearum to colony appearance in a tetrazolium medium. Phytopathology 44: 693–695.
•  Kosambi,  D.D. (1944) The estimation of map distance from recombination values. Ann. Eugen. 12: 172–175.
•  Kreike,  C.M.,  A.A.  Kok-Westeneng,  J.H.  Vinke and  W.J.  Stiekema (1996) Mapping of QTLs involved in nematode resistance, tuber yield and root development in Solanum sp. Theor. Appl. Genet. 92: 463–470.
•  Lan,  T.,  S.  Zheng,  L.  Yang,  S.  Wu,  B.  Wang,  S.  Zhang,  Z.  Tong,  Y.  Chen,  S.  Chen,  Y.  Duan et al. (2014) Mapping of quantitative trait loci conferring resistance to bacterial wilt in tobacco (Nicotiana tabacum L.). Plant Breed. 133: 672–677.
•  Lebeau,  A.,  M.C.  Daunay,  A.  Frary,  A.  Palloix,  J.F.  Wang,  J.  Dintinger,  F.  Chiroleu,  E.  Wicker and  P.  Prior (2011) Bacterial wilt resistance in tomato, pepper, and eggplant: genetic resources respond to diverse strains in the Ralstonia solanacearum species complex. Phytopathology 101: 154–165.
•  Lebeau,  A.,  M.  Gouy,  M.C.  Daunay,  E.  Wicker,  F.  Chiroleu,  P.  Prior,  A.  Frary and  J.  Dintinger (2013) Genetic mapping of a major dominant gene for resistance to Ralstonia solanacearum in eggplant. Theor. Appl. Genet. 126: 143–158.
•  Mori,  K.,  N.  Mukojima,  T.  Nakao,  S.  Tamiya,  Y.  Sakamoto,  N.  Sohbaru,  K.  Hayashi,  H.  Watanuki,  K.  Nara,  K.  Yamazaki et al. (2012) Germplasm release: Saikai 35, a male and female fertile breeding line carrying Solanum phureja-derived cytoplasm and potato cyst nematode resistance (H1) and potato virus Y resistance (Ry_chc) genes. Am. J. Potato Res. 89: 63–72.
•  Murashige,  T. and  F.  Skoog (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.
•  Obidiegwu,  J.E.,  R.  Sanetomo,  K.  Flath,  E.  Tacke,  H.R.  Hofferbert,  A.  Hofmann,  B.  Walkemeier and  C.  Gebhardt (2015) Genomic architecture of potato resistance to Synchytrium endobioticum disentangled using SSR markers and the 8.3k SolCAP SNP genotyping array. BMC Genet. 16: 38.
•  Ortiz,  R. and  S.J.  Peloquin (1994) Use of 24-chromosome potatoes (diploids and hihaploids) for genetical analysis. In:  Bradshaw,  J.E. and  G.R.  Mackay (eds.) Potato genetics. CAB international, Wallingford, pp. 133–154.
•  Patil,  V.U.,  J.  Gopal and  B.P.  Singh (2012) Improvement for bacterial wilt resistance in potato by conventional and biotechnological approaches. Agric. Res. 1: 299–316.
•  Peloquin,  S.J.,  R.W.  Hougas and  G.L.  Yerk (1990) The use of potato haploids in genetics and breeding. In:  Gupta,  P.K. and  T.  Tsuchiya (eds.) Chromosome engineering in plants: genetics, breeding, evolution, part B. Elsevier, Amsterdam, pp. 79–92.
•  Qian,  Y.L.,  X.S.  Wang,  D.Z.  Wang,  L.N.  Zhang,  C.L.  Zu,  Z.L.  Gao,  H.J.  Zhang,  Z.Y.  Wang,  X.Y.  Sun and  D.N.  Yao (2013) The detection of QTLs controlling bacterial wilt resistance in tobacco (N. tabacum L.). Euphytica 192: 259–266.
•  Rahman,  M.A.,  H.  Abdullah and  M.  Vanhaecke (1999) Histopathology of susceptible and resistant Capsicum annuum cultivars infected with Ralstonia solanacearum. J. Phytopathol. 147: 129–140.
• R Core Team (2017) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.
•  Rowe,  P.R. and  L.  Sequeira (1970) Inheritance of resistance to Pseudomonas solanacearum in Solanum phureja. Phytopathology 60: 1499–1501.
•  Sakamoto,  Y.,  K.  Mori,  Y.  Matsuo,  N.  Mukojima,  W.  Watanabe,  N.  Sobaru,  S.  Tamiya,  T.  Nakao,  K.  Hayashi,  H.  Watanuki et al. (2017) Breeding of a new potato variety ‘Nagasaki Kogane’ with high eating quality, high carotenoid content, and resistance to diseases and pests. Breed. Sci. 67: 320–326.
•  Sequeira,  L. and  P.R.  Rowe (1969) Selection of utilization of Solanum phureja clones with high resistance to different strains of Pseudmonas solanacearum. Am. Potato J. 46: 451–462.
•  Sharma,  S.K.,  D.  Bolser,  J.  de Boer,  M.  Sønderkær,  W.  Amoros,  M.F.  Carboni,  J.M.  D’Ambrosio,  G.  de la Cruz,  A.  Di Genova,  D.S.  Douches et al. (2013) Construction of reference chromosome-scale pseudomolecules for potato: integrating the potato genome with genetic and physical maps. G3 (Bethesda) 3: 2031–2047.
•  Siri,  M.I.,  G.A.  Galván,  L.  Quirici,  E.  Silvera,  P.  Villanueva,  F.  Ferreira,  L.  Franco Fraguas and  M.J.  Pianzzola (2009) Molecular marker diversity and bacterial wilt resistance in wild Solanum commersonii accessions from Uruguay. Euphytica 165: 371–382.
•  Stahl,  Y.,  S.  Grabowski,  A.  Bleckmann,  R.  Kühnemuth,  S.  Weidtkamp-Peters,  K.G.  Pinto,  G.K.  Kirschner,  J.B.  Schmid,  R.H.  Wink,  A.  Hülsewede et al. (2013) Moderation of Arabidopsis root stemness by CLAVATA1 and ARABIDOPSIS CRINKLY4 receptor kinase complexes. Curr. Biol. 23: 362–371.
•  Suga,  Y.,  M.  Horita,  M.  Umekita,  N.  Furuya and  K.  Tsuchiya (2013) Pathogenic characters of Japanease potato strains of Ralstonia solanacearum. J. Gen. Plant Pathol. 79: 110–114.
•  Sul,  I.W. and  S.S.  Korban (1996) A highly efficient method for isolating genomic DNA from plant tissues. Plant Tiss. Cult. Biotech. 2: 113–116.
•  Tan,  M.Y.A.,  T.H.  Park,  R.  Alles,  R.C.B.  Hutten,  R.G.F.  Visser and  H.J.  van Eck (2009) GpaXI^l_tar originating from Solanum tarijense is a major resistance locus to Globodera pallida and is localised on chromosome 11 of poato. Theor. Appl. Genet. 119: 1477–1487.
•  Tanksley,  S.D.,  M.W.  Ganal,  J.P.  Prince,  M.C.  de Vicente,  M.W.  Bonierbale,  P.  Broun,  T.M.  Fulton,  J.J.  Giovannoni,  S.  Grandillo,  G.B.  Martin et al. (1992) High density molecular linkage maps of the tomato and potato genomes. Genetics 132: 1141–1160.
•  Thurston,  H.D. and  J.C.  Lozano (1968) Resistance to bactrial wilt of potatoes in Colombian clones of Solanum phureja. Am. Potato J. 45: 51–55.
•  Tung,  P.X.,  E.T.  Rasco Jr.,  P.  Vander Zaag and  P.  Schmiediche (1990a) Resistance to Pseudomonas solanacearum in the potato: I. Effects of sources of resistance and adaptation. Euphytica 45: 203–210.
•  Tung,  P.X.,  E.T.  Rasco Jr.,  P.  Vander Zaag and  P.  Schmiediche (1990b) Resistance to Pseudomonas solanacearum in the potato: II. Aspects of host-pathogen-environment interaction. Euphytica 45: 211–215.
•  Voorrips,  R.E. (2002) MapChart: software for the graphical presentation of linkage maps and QTLs. J. Hered. 93: 77–78.
•  Wang,  J.F.,  J.  Olivier,  P.  Thoquet,  B.  Mangin,  L.  Sauviac and  N.H.  Grimsley (2000) Resistance of tomato line Hawaii7996 to Ralstonia solanacearum Pss4 in Taiwan is controlled mainly by a major strain-specific locus. Mol. Plant Microbe. Interact. 13: 6–13.
•  Wang,  J.F.,  F.I.  Ho,  H.T.  Hong Truong,  S.M.  Huang,  C.H.  Balatero,  V.  Dittapongpitch and  N.  Hidayati (2013) Identification of major QTLs associated with stable resistance of tomato cultivar ‘Hawaii 7996’ to Ralstonia solanacearum. Euphytica 190: 241–252.
•  Wang,  S.,  C.J.  Basten,  P.  Graffney and  Z.B.  Zeng (2005) Windows QTL Cartographer 2.5 user manual. Bioinformatics Research Center, North Carolina State University, Raleigh.
•  Watanabe,  K.,  H.M.  El-Nashaar and  M.  Iwanaga (1992) Transmission of bacterial wilt resistance by First Division Restitution (FDR) 2n pollen via 4x × 2x crosses in potatoes. Euphytica 60: 21–26.
•  Watanabe,  J.A.,  M.  Orrillo and  K.N.  Watanabe (1999) Frequency of potato genotypes with multiple quantitative pest resistance traits in 4x × 2x crosses. Breed. Sci. 49: 53–61.
•  Yanping,  Z.,  L.  Hui,  Z.  Hairui and  G.  Gao (2013) Identification and utility of SRAP markers linked to bacterial wilt resistance gene in potato. Vegetos 26: 131–138.