Homoeologous Chromosome Pairing and Alien Introgression in Backcrossing Progenies Derived from Hybrids Solanum tuberosum (+) Mexican 2x (1 EBN) B-Genome Potato Species

Abstract

We applied a genomic in situ hybridization (GISH) to analyze the genomic constitution of and meiotic pairing in interspecific somatic hybrids, and in a wide subset of backcrossing derivatives (BC₁–BC₅), from three interspecific combinations involving the cultivated potato, Solanum tuberosum (AAAA genome), and three diploid (1 EBN) wild Mexican potato species (genome BB)—S. tarnii, S. pinnatisectum, and S. bulbocastanum. The theoretically expected genomic composition was detected in the somatic hybrids (AAAABB) and in the BC₁ progeny (AAAAB), whereas in the subsequent BC₂–BC₄ generations, the partial loss of alien chromosomes was observed and almost all the BC₅ genotypes showed a complete chromosome elimination of wild species. GISH revealed a homoeologous pairing between the chromosomes of the A- and the B-genomes in each of the hybrid progenies. Using GISH, we selected introgression lines with a single chromosome of the wild species in a potato genome background, as well as introgression lines with intergenomic recombinant chromosomes. Moreover, via molecular screening, BC hybrids with diagnostic markers for the R-genes conferring resistance to late blight disease and to the quarantine pest of the potato–Columbia root-knot nematode—were selected. The potential application of the results obtained for the planning of introgressive schemes directed to the breeding of advanced lines with multiple disease and pest resistance is discussed.

1. Introduction

The common potato, Solanum tuberosum L. (2n = 4x = 48, 4 EBN, AAAA genome), is the third most important crop and the most important of the non-cereals; however, its cultivation has been continually challenged by numerous diseases and pests. Interspecific hybridization and alien introgression from the wild potato species of section Petota Dumort. of the genus Solanum L. is an important tool in potato breeding for the transfer of important agronomic traits into the cultivated potato gene pool. Wild diploid B-genome potato species (2n = 2x = 24, 1 EBN, genome BB) native to Mexico have been shown to be a valuable source of resistance to many harmful organisms, such as: Phytophthora infestans (Mont.) de Bary, that causes the most destructive potato disease late blight, viruses (PVY and PLRV), root-knot nematodes (Meloidogyne spp.), different aphid species, and the Colorado potato beetle [1]. According to the taxonomic treatment of Hawkes [1], Mexican diploid B-genome species belong to the series Pinnatisecta (Solanum cardiophyllum Lind., Solanum pinnatisectum Dunal, Solanum tarnii, Hawkes and Hjert., S. × michoacanum (Bitter.) Rydb.) and Bulbocastana (Solanum bulbocastanum Dun.). These wild relatives belong to the tertiary gene pool and are sexually incompatible or very difficult to cross with S. tuberosum using double-bridge crosses ((Solanum acaule × S. bulbocastanum) × Solanum phureja) × S. tuberosum) [2,3,4], or through manipulations of EBN and ploidy levels and the use of the S-locus inhibitor gene (Sli) [5,6,7]. Bridge crosses of the diploid Mexican species S. bulbocastanum with South American potato species followed by subsequent backcrosses with potato cultivars took about 50 years to enrich the genetic diversity of the common potato with the alien introgressed Rpi-blb2 gene, conferring broad-spectrum resistance to late blight pathogens and developing two new cultivars, ‘Bionica’ and ‘Toluca’ [8].

To increase the efficiency of interspecific hybridization for broadening the genetic diversity of S. tuberosum (tbr), protoplast fusion techniques have been frequently used to overcome the interspecific incompatibility between the common potato and the diploid B-genome Mexican potato species: S. bulbocastanum (blb) [9,10,11,12,13,14,15,16,17], S. pinnatisectum (pnt) [12,13,18,19,20,21,22,23,24,25,26,27,28], and S. tarnii (trn) [29]; S. cardiophyllum (cph) [23,30,31] and S. x michoacanum (Bitter.) Rydb. (mch) [32,33]. In most cases, the interspecific somatic hybrids between the potato and diploid B-genome Mexican species had a low level of fertility or were sterile, were characterized by poor agronomic characters, and the degree of their disease and/or pest resistance was lower than in the parental clones of wild species [12,15,17,25,32,34,35]. These undesirable characteristics could be due to somaclonal variation induced during protoplast isolation and fusion and the regeneration of hybrid plants [17,36,37]. Thus, cytogenetic instability as mitotic and meiotic irregularities, aneuploidy, and mixoploidy, were detected using cytological methods in the somatic hybrids tbr(+)blb [12,38,39] and tbr(+)pnt [23]. The elimination of parental alleles and markers and genome structural variation were demonstrated in tbr(+)blb, tbr(+)pnt, and tbr(+)mch somatic hybrids using species-specific RAPD [38,40], RFLP [40], ISSR [15,27], and SSR [17] markers; DArT analysis [33]; and genome sequence analysis [28].

In several cases, genetically stable and fertile genotypes have been selected from the numerous interspecific somatic hybrids (SH) and advanced introgression lines that maintain disease and/or pest resistance in their repeated backcross (BC) progenies:

-

SH tbr(+)blb and their BC₁–BC₅ lines with resistance to root-knot nematodes (Meloidogyne chitwoodi Golden and Meloidogyne fallax Karssen), having the R_Mc1(blb) gene introgressed from blb [41,42,43];

-

SH tbr(+)blb and their BC₁–BC₂ lines with late blight resistance, that possess the Rb/Rpi-blb1 gene [10,40,44,45] and the Rpi-blb1 and Rpi-blb3 genes introgressed from blb [16];

-

SH tbr(+)blb and BC₁–BC₂ clones with resistance to viruses PVY and PLRV, and resistance to the aphid species from blb [14];

-

SH tbr(+)trn and BC₁–BC₃ clones with extreme resistance to PVY and resistance to different aphid species from trn [21,29,46];

-

SH tbr(+)pnt and BC₁ hybrids with resistance to PVY and to aphids transferred from pnt [21,46];

-

SH tbr(+)pnt and BC₁ lines with late blight resistance, having the Rpi-blb2 gene from pnt [27,28];

-

SH tbr(+)mch and BC₁ clones with late blight resistance introgressed from mch [47].

To monitor alien introgressions into cultivated potato genomes, molecular markers were applied in the BC progenies of the interspecific somatic hybrids between S. tuberosum and the diploid B-genome Mexican species. The data from a segregation analysis of the species-specific RFLP and RAPD markers in the BC₁–BC₂ populations derived from the tbr(+)blb somatic hybrids indicate that intergenomic recombination may occur in these hybrids [40,41].

Knowledge about genome affinity in parental genomes is important for developing efficient breeding strategies [48]. Data on the affinity between the A-genome of S. tuberosum and the B-genome of the Mexican diploid (1 EBN) species indicate the potential for intergenomic pairing and meiotic recombination in interspecific hybrids. Synteny in the BAC position and orientation and identical oligo-FISH patterns were demonstrated across the homoeological chromosomes of S. tuberosum and S. bulbocastanum [48,49,50,51]. Braz et al. [51] demonstrated that the arm ratio and relative length of individual S. bulbocastanum chromosomes were highly similar to those of the homoeologous potato chromosomes. A majority of the DArT markers derived from S. bulbocastanum and South American potato species were collinear, although some amount of variability between their genomes was detected [52,53]. A genome sequence analysis showed relatedness and gene collinearity between the S. pinnatisectum genome and the A-genome of the potato [28].

Cytogenetic techniques are particularly helpful in monitoring interspecific chromatin transfers and estimating the potential for developing genetically stable alien introgression lines. The B-genome of the Mexican diploid species S. bulbocastanum has been identified in interspecific hybrids through genomic in situ hybridization (GISH). Thus, GISH has been successfully performed for parental genome discrimination in somatic hybrids between S. tuberosum and S. bulbocastanum [16,44], and in sexual hybrids between S. pinnatisectum and S. tuberosum and their progenies [7]. However, there is still little information available about the potential for intergenomic pairing and meiotic recombination between homoeologous chromosomes of the A-genome of S. tuberosum and the B-genome of the diploid (1 EBN) Mexican species, because GISH analysis was previously performed mainly on mitotic preparations of interspecific hybrids [16,44] and because there was only a single systematic study of meiotic chromosome pairing. This includes the recent study of Kikuchi and colleagues [7], who applied GISH to analyze the genomic affinity between the A-genome potato species and S. pinnatisectum in sexual hybrids and their progenies [7], and the work of Ramanna and Hermsen [54], who have extensively studied meiotic pairing behavior in the sexual hybrids between S. bulbocastanum and the South American A-genome potato species, S. acaule and S. phureja, using conventional cytological methods [3,54,55]. Both studies have demonstrated meiotic pairing between homoeologous chromosomes and the potential for alien gene introgression.

We provide here cytogenetic information which was obtained using the GISH method about homoeologous chromosome pairing and alien introgression in the backcrossing progenies derived from the somatic hybrids between S. tuberosum and the diploid Mexican B-genome potato species: S. tarnii, S. pinnatisectum, and S. bulbocastanum. Additional objectives of our research were to select perspective genotypes among the backcrossing derivatives for further pre-breeding programs: introgression lines with alien segment introgressions in recombinant chromosomes, putative monosomic alien addition lines, and hybrid genotypes with diagnostic markers for the R-genes conferring resistance to late blight disease and to quarantine pests of the potato–Columbia root-knot nematode.

The hybrid material analyzed here was characterized in previous research using nSSR and AFLP markers, and was also analyzed for resistance to PVY and aphids, and/or for resistance to late blight [16,21,29,39,46,56,57,58].

2. Materials and Methods

2.1. Plant Materials

The plant material in the present study was represented by 8 parental accessions that were involved in protoplast fusion or backcrossing, 2 selected genotypes of somatic hybrids, and 46 selected backcross derivatives from BC₁–BC₅ progenies from three interspecific combinations (Figure 1):

(a)

(tbr+trn)–somatic hybrids were obtained through protoplast fusion between S. tuberosum cv. Delikat and accession GLKS 2870 of the wild Mexican species S. tarnii [29,56]; two fertile somatic hybrids, 838/2 and 838/7, were selected for the present study. BC₁–BC₅ progenies were produced in sexual crosses between selected hybrid genotypes as female and potato cultivars as a male parent. Somatic hybrids and their backcrossing derivatives have been tested in previous research mainly for resistance to PVY and aphids, as well as to late blight, and have been characterized for agronomic characters [21,29,46,57,58].

(b)

(tbr+pnt)–somatic hybrids were obtained using protoplast fusion between S. tuberosum cv. Rasant and accession GLKS 31607 of the wild Mexican species S. pinnatisectum [21,57]. Resistant to PVY and aphids and/or resistance to late blight genotypes were selected in the subsequent BC generations [21,57,58].

(c)

(tbr+blb)–somatic hybrids were produced through protoplast fusion between potato cultivars and accession GLKS 31741 of the wild Mexican species S. bulbocastanum and backcross progenies were obtained in subsequent crosses of selected hybrid genotypes with different potato cultivars [16,39,57]. Parental clones, somatic hybrids, and their backcross derivatives were characterized for resistance to late blight and agronomic characters in previous studies [16,57].

GISH was performed for the somatic hybrids and for 37 of 46 backcross derivatives from three interspecific combinations. While GISH was applied for the first time for all the hybrid genotypes from combinations (tbr+trn) and (tbr+pnt) (Figure 1), several hybrids from the third combination, (tbr+blb), were analyzed by GISH previously. Rakosy-Tican et al. [16] performed GISH analysis for four hybrid genotypes from the combination—S. tuberosum, cv. Delikat (+) S. bulbocastanum, GLKS 31741—using preparations of somatic chromosomes. One of the four genotypes (BC₂ 95/1/4/59) was used in GISH analysis here to study meiotic chromosome behavior. Here we selected an additional seven backcross genotypes from this combination to continue the study of Rakosy-Tican and colleagues [16], and included in our research five new backcross derivatives from another hybrid family (S. tuberosum, cv. Rasant (+) S. bulbocastanum, GLKS 31741) that was not analyzed by GISH before.

Molecular screening was performed for the whole subset: 8 parental accessions, 2 somatic hybrids, and 46 backcross derivatives.

2.2. In Situ Hybridization

The plant material for the genomic in situ hybridization (GISH) assays was grown in a greenhouse. Root tips and flower buds were used for detecting the genome composition of the hybrids. After pretreatment in water with ice for 24 h, the root tips were fixed in Carnoy’s solution (an ethanol: glacial acetic acid = 3:1) and further stored at −20 °C for chromosome counts. The flower buds were fixed directly in Carnoy’s solution and then stored at −20 °C for meiotic analysis in pollen mother cells (PMC). Chromosome slides were prepared after enzymatic treatment of the anthers and root meristems. The enzyme solution contained 4% cellulase (1.14 U/mg) and 1% pectolyase (0.94 U/mg); the time of incubation was 65–120 min.

Genomic DNA was isolated from the young leaves according to the protocol of Bernatzky and Tanksley [59]. For GISH, DNA was isolated from the young leaves of the parental accessions of the cultivated potato S. tuberosum (AAAA genome), cv. Delikat, and from the accessions of the B-genome diploid species: S. tarnii (GLKS 2870) and S. bulbocastanum (PI 498223). The DNA of the A- and the B-genome species were labeled using Nick-Translation Digoxigenin-NT Labeling Kit or BIO-NT Labeling Kit (Jena Bioscience, Jena, Germany, PP-310-DIGX and PP-310-BIO16). The differentially labeled DNA from the A- and B-genomic species were used in the hybridization mix. GISH was performed according to the standard techniques [60] with slight modifications [61].

After GISH, chromosome preparations of some selected introgression lines were re-probed in FISH experiments using an rDNA probe. Selected slides were washed and re-probed using 18S/25S-labeled rDNA in the FISH experiments to identify satellite chromosomes. The 18S/25S rDNA plasmid sequence in the VER17 probe [62] was labeled using DIG-Nick Translation Mix (Roche Diagnostics, Monneheim, Germany 11745816910).

FITS anti-DIG conjugate (FAB fragments) (Roche, Monneheim, Germany 11207741910) and Rhodamine conjugated avidin Red TM-X (Thermo Fisher Scientific, Waltham, MA, USA, S6366) were used in a two-color GISH; FITS anti-DIG conjugate (FAB fragments) was used in FISH.

An AxioImager M2 epifluorescence microscope with an AxioCam, Carl Zeiss, Germany, MRm camera and AxioVision Rel 4.8 software was used to analyze the slides, and create and process the images. In addition, Adobe Photoshop 6.0 was used for image processing.

2.3. Marker-Assisted Selection

Genomic DNA was isolated from the leaf material of the greenhouse-grown plants following the CTAB-extraction method with some modifications [63]. The quality and quantity of the isolated DNA samples were checked using agarose gel electrophoresis (0.8% gel) and a spectrophotometer (Implen NanoPhotometer N60 Touch).

Molecular screening was performed for the whole subset that included 56 genotypes: 8 parental accessions, 2 somatic hybrids, and 46 backcrossing derivatives from three interspecific combinations. The seven markers of the four R-genes conferring resistance to root-knot nematode, R_Mc1(blb) [43], and resistance to late blight, R8 [64], Rpi-blb1 [65,66], and Rpi-blb3 [67,68], used in this study are shown in Supplementary Table S1. PCR reactions were carried out in a total volume of 20 µL, containing 10 ng DNA template, 1 × PCR reaction buffer (‘Dialat’ [13]), 2 mM MgCl₂, 0.4 mM of each dNTP (‘Dialat’ [69]), 0.5 µM of forward/reverse primer (synthesized by ‘Evrogen’ [70]), and 1U Taq polymerase (‘Dialat’). For marker 193I9 the MgCl₂ concentration was reduced to 1.5 mM. PCR was carried out according to the standard procedures. The annealing temperatures and cycling conditions corresponded to those indicated by the primer-developers (see references in Supplementary Table S1). All reactions were conducted with at least three replicates.

For CAPS-markers, enzyme RsaI (‘SibEnzyme’ LLC, Novosibirsk, Russia [71]) was used. The restriction was performed in a total volume of 30 µL overnight according to the manufacturer’s protocol.

The PCR products were separated by electrophoresis in 2.0% agarose gels in a TBE buffer, followed by ethidium bromide staining, and visualization in UV light using a GelDoc XR gel system (BIO-RAD, Hercules, CA, USA). The sizes of the amplification products corresponded to those published by the authors (Supplementary Table S1). Diagnostic bands of molecular markers were registered for the hybrids and introgression lines involved in our research.

3. Results

3.1. GISH Analysis of the Genomic Composition and Alien Chromosome Introgression in the Somatic Hybrids and their Backcross Progenies

The genomic composition of the 39 hybrid genotypes from three interspecific combinations of tbr(+)trn, tbr(+)pnt, and tbr(+)blb, were determined using GISH both in mitotic and meiotic cells.

Table 1. Genomic composition of somatic hybrids and backcrossing derivatives from three interspecific combinations between S. tuberosum and the wild Mexican diploid B-genome potato species.

Hybrid Genotype	2n	Number of Chromosomes of the
		A-Genome	B-Genome
(a) Fusion combination: S. tarnii, GLKS 2870 (+) S. tuberosum, cv. Delikat
SH 838/2	72	48	24
SH 838/7	72	48	24
BC1 838/2/25	60	48	12
BC1 838/2/80	60	48	12
BC1 838/7/18	60	48	12
BC1 838/7/53	60	48	12
BC2 838/2/25/1	50	48	2
BC2 838/2/80/5	52	48	4
BC2 838/7/18/3	53	48	5
BC2 838/7/53/3	56	48	8
SBC3 838/2/25/1/3/13 (self-pollinated BC3)	48	48	0
SBC3 838/2/25/1/3/15 (self-pollinated BC3)	48	48	0
BC4 838/7/53/3/23/6	51	48 one of them with the B-genome fragment	3
BC4 838/7/53/3/23/7	49	48	1
BC4 838/7/53/3/23/9	50	48	2 one of them with the A-genome fragment
BC4 838/7/53/3/23/4	54	48	6
BC5 838/7/53/3/23/4/15	48	48	0
BC5 838/7/53/3/23/4/7	48	47	1
BC5 838/7/53/3/23/1/18	48	48	0
BC5 838/7/53/3/23/1/3	48	48	0
BC5 838/7/53/3/23/19/14	48	48	0
BC5 838/7/53/3/23/19/5	48	48	0
BC5 838/7/53/3/23/19/6	48	48	0
(b) Fusion combination: S. pinnatisectum, GLKS 31607 (+) S. tuberosum, cv. Rasant
BC3 2045/2/7/17/7	49	48	1
BC4 2045/2/7/17/7/1	49	48	1
BC4 2045/2/7/17/7/26	49	48	1
(c′) Fusion combination: S. bulbocastanum, GLKS 31741 (+) S. tuberosum, cv. Delikat
BC1: 82/4/43	60	48	12 one of them with terminal A-genome fragment
BC1: 83/9/11	60	48	12 one of them with terminal A-genome fragment
BC1: 83/9/35	60	48	12
BC1: 83/9/44	58	48	10
BC2: 82/4/46/14	53	48	5
BC2 82/4/68/2	52	48	4
BC2 82/4/68/7	52	48	4
BC2: 95/1/4/59	53	48 two of them with the B-genome fragments	5
(c″) Fusion combination: S. bulbocastanum, GLKS 31741 (+) S. tuberosum, cv. Rasant
BC2 08/5/1/10	53	48	5
BC2 08/5/1/13	51	48	3
BC2 08/5/1/20	50	48	2
BC2 08/5/1/24	52	48	4
BC3 82/4/68/32/27	48	48	0

and Figure 2, Figure 3 and Figure 4 present the content of the B-genomes of the wild diploid Mexican species S. tarnii, S. pinnatisectum, and S. bulbocastanum in the somatic hybrids and backcrossing derivatives.

3.1.1. Genomic Constitution of the Somatic Hybrids, S. tuberosum (+) S. tarnii, and Their Introgression Lines from Backcross Progenies BC₁–BC₅

In our study, GISH was used for the first time to differentiate the parental chromosomes in the interspecific hybrids between S. tuberosum and S. tarnii. Two somatic hybrids, 838/2 and 838/7, derived from the protoplast fusion of S. tuberosum (2n = 4x = 48, AAAA) and the wild species S. tarnii (2n = 2x = 24, BB), were hexaploid (2n = 6x = 72) and had the theoretically expected AAAABB genomic composition, with 48 chromosomes of potato and 24 chromosomes of S. tarnii (Table 1a, Figure 2a,b). Their BC progenies from two hybrid families were further analyzed using GISH. Correspondingly, the BC₁ lines, derived from crosses between the hexaploid somatic hybrids (AAAABB) and the tetraploid parental cultivar Delikat (AAAA), had a 5x (2n = 60) ploidy level and an AAAAB genome constitution, with 48 chromosomes of the A-genome of S. tuberosum and 12 chromosomes of the B-genome of S. tarnii (Table 1a, Figure 2c).

Four BC₂ hybrid genotypes selected from the crosses between the pentaploid BC₁ hybrids (AAAAB) and the tetraploid cultivar Sonate (AAAA) were aneuploids, indicating the loss of the B-genome chromosomes in the backcrossing. According to the GISH results, these BC₂ hybrids have the 48 chromosomes of the A-genome and from two to eight alien chromosomes of the B-genome of S. tarnii (Table 1a).

Introgression lines with 1–3 and 6 alien chromosomes of S. tarnii were detected in the subsequent backcross generations (Table 1a, Figure 2d–f); the genotypes that lost all alien chromosomes of the B-genome were also identified.

Almost all the analyzed BC₅ hybrids lost all the chromosomes of the B-genome. Out of seven BC₅ hybrids, six had 48 chromosomes of S. tuberosum, and one BC₅ genotype (838/7/53/3/23/4/7) had chromosome counts of 2n = 48, with 47 chromosomes of the A-genome of potato and one chromosome of the B-genome from S. tarnii (Table 1a, Figure 2f). A FISH analysis revealed only three hybridization sites of 18S/25S rDNA genes in this genotype, and all three signals were located on three chromosomes of the A-genome of S. tuberosum, indicating the absence of one of four satellite chromosomes of the potato (Figure 2f,g). Hence, the single chromosome of the B-genome in the BC₅ genotype, 838/7/53/3/23/4/7 (2n = 48), did not replace one of the missing satellite chromosomes of the potato; it is an alien-addition monosome chromosome transferred from S. tarnii into the genome of S. tuberosum.

3.1.2. Genomic Constitution of the Introgression Lines from Backcross Progenies BC₃–BC₄ of the Interspecific Somatic Hybrids S. tuberosum (+) S. pinnatisectum

One selected BC₃ hybrid had the chromosome count 2n = 49, with 48 chromosomes of the A-genome of S. tuberosum and one extra alien chromosome of the B-genome from S. pinnatisectum (Figure 3). This alien chromosome was transferred into its two BC₄ derivatives analyzed here (Table 1b, Figure 3a,b). This monosomic alien chromosome from S. pinnatisectum was not a satellite, as confirmed using FISH, with the 18S/25S rDNA (Figure 3c,d).

3.1.3. Genomic constitution of the Introgression Lines from Backcross Progenies BC₁–BC₃ of Interspecific Somatic Hybrids S. tuberosum (+) S. bulbocastanum

Three of four BC₁ hybrids from the combination tbr(+)blb, which were analyzed using GISH, had an AAAAB genomic composition, with 48 chromosomes of the A-genome and 12 chromosomes of the B-genome of S. bulbocastanum. The other BC₁ hybrid (83/9/44) was an aneuploid missing two alien chromosomes (2n = 5x = 58; 48 chromosomes of the A-genome and 10 chromosomes of the B-genome) (Table 1c′, Figure 4a,b).

The white arrows indicate homoeologous pairing between chromosomes of the A- and the B-genomes. The blue arrows indicate recombinant chromosomes. The scale bar = 5 µ.

All eight analyzed BC₂ hybrids from the combination blb(+)tbr were aneuploids having 48 chromosomes of the A-genome and from 2 to 5 chromosomes of the B-genome of S. bulbocastanum (Table 1c′,c″; Figure 4c,d). One BC₃ genotype (82/4/68/32/27) analyzed here lost all alien chromosomes according to the GISH data (Table 1c′).

3.2. Detection of Alien Chromatin Introgression in Recombinant Chromosomes of Selected BC Genotypes

GISH clearly distinguished between the chromatin of the A- and B-genomes of the parental species, both on the mitotic and meiotic preparations of the somatic hybrids and backcrossing derivatives (Figure 2, Figure 3 and Figure 4). In GISH, the cross-hybridization with genomic DNA probes of both parental species was observed only at the nucleolus organizer region (NOR) that is localized at the short arm of chromosome II in the potato species. Alien introgressed fragments detected with GISH are not always easy to distinguish from cross-hybridization sites, especially at late diakinesis and MI, because the chromosomes of potato species are small and have a similar shape (see review [72]). To exclude misinterpretation of the cross-hybridization sites as introgressed segments in the recombinant chromosomes, we washed the slides after performing GISH and re-probed them in FISH experiments using DIG-labeled 18S/25S rDNA as the probe. It was shown that the cross-hybridization sites observed in GISH were always co-localized with the rDNA hybridization sites detected in FISH, but not with the recombinant chromosomes (Figure 2f,g and Figure 4d–g). Thus, GISH/FISH re-probing was helpful in precisely identifying the introgression of alien chromatin in the recombinant chromosomes of five backcrossing derivatives (Table 1). They included: two BC₄ hybrids from the combination trn(+)tbr (Table 1a, Figure 2e) and two BC₁ hybrids from the combination blb(+)tbr, with one recombinant chromosome each, and one BC₂ hybrid (95/1/4/59) that possessed two chromosomes of the A-genome with introgressions from the B-genome (Table 1c′, Figure 4d,e).

One of the 37 hybrids analyzed here was involved in a previous GISH analysis [16]; this is BC₂ 95/1/4/59 (2n = 53) from the blb(+)tbr combination. According to our results, it has 48 chromosomes of potato, including 2 recombinant chromosomes and 5 alien chromosomes of S. bulbocastanum. We did not reveal any instability in the chromosome number or in the genomic composition of the microsporocytes of this introgression line. Our results for this genotype do not coincide with those of Rakosy-Tican et al. [16] (2n = 48, 50—mixoploidy; the hybrid had four alien chromosomes and recombinations; chromosome counts were performed in somatic cells). It can be assumed that in this case, during the development of sporogenous tissue in this mixoploid genotype, the genetically stable PMCs were selected.

3.3. Homoeologous Chromosome Pairing Behavior in Hybrid Genotypes from Three Interspecific Combinations Analyzed using In Situ Hybridization

GISH differentiation among the chromatin of the A- and B-genomes allowed identification of the homoeologous genomes involved in each meiotic pairing configuration in the somatic hybrids and introgression lines. Figure 2b,f,g, Figure 3 and Figure 4 present the chromosome behavior during diakinesis and MI for selected hybrid genotypes. Meiotic chromosome pairing was analyzed in 11 hybrid genotypes from three interspecific combinations, tbr(+)trn, tbr(+)pnt, tbr(+)blb, that are representatives of all hybrid generations: somatic hybrids and BC₁–BC_5.

Table 2. Chromosome pairing in hybrids derived from three interspecific combinations: S. tuberosum (+) S. tarnii, S. tuberosum (+) S. pinnatisectum, and S. tuberosum (+) S. bulbocastanum.

Hybrid Genotype (Interspecific Combination)	No. of PMCs	No. of Chromosomes of the Genome				Chromosomal Associations on Average per Cell										A-B Associations on Average Per cell X
				I *		II **			III ***			IV ****			V *****
		A	B	A	B	AA	BB	A-B	AAA	AA-B	A-BB	AAAA	AAA-B	AA-BB	AAAA-B
SH 838/7 trn(+)tbr, Delikat	20	48	24	6.15 (3–11)	4.90 (2–9)	17.80 (13–21)	7.55 (5–10)	3.45 (0–6)	0.3 (0–2)	0.15 (0; 1)	0.05 (0; 1)	0.2 (0; 1)	0.15 (0; 1)	0.05 (0; 1)	0.05 (0; 1)	4.75 (0–6; 8; 10; 14)
BC1 82/4/43 blb(+)tbr, Delikat	59	48	12	5.77 (2–11)	8.71 (6–12)	18.79 (15–21)	0	3.02 (0–5)	0.14 (0–2)	0.17 (0–2)	0	0.12 (0; 1)	0.12 (0; 1)	0	0	3.63 (0–8)
BC1 83/9/11 blb(+)tbr, Delikat	27	48	12	5.18 (2–12)	9.41 (7–12)	19.34 (16–22)	0	2.48 (1–5)	0.22 (0; 1)	0.11 (0; 1)	0	0.19 (0; 1)	0	0	0	3.05 (1–6)
BC1 83/9/35 blb(+)tbr, Delikat	16	48	12	5.44 (2–9)	8.38 (3–12)	18.94 (12–22)	0	3.19 (0–6)	0	0.44 (0–2)	0	0.13 (0; 1)	0	0	0	3.56 (0–8)
BC1 83/9/44 blb(+)tbr, Delikat	30	48	10	6.33 (2–10; 14)	7.30 (5–8; 10)	18.73 (16–22)	0	2.57 (0–4)	0.1 (0; 1)	0.07 (0; 1)	0	0.23 (0; 1)	0.1 (0; 1)	0	0	3.20 (0–6)
BC2 82/4/46/14 blb(+)tbr, Delikat	31	48	5	3.71 (0–7)	2.65 (1–5)	20.77 (18–23)	0	2.09 (0–4)	0.03 (0; 1)	0.26 (0–3)	0	0	0	0	0	2.38 (0–4)
BC2 95/1/4/59 blb(+)tbr, Delikat	25	48	5	5.48 (3–9)	3.08 (1–5)	18.80 (15–21)	0	1.85 (0–4)	0.71 (0–2)	0.13 (0; 1)	0	0.21 (0; 1)	0.08 (0; 1)	0	0	2.16 (0–5)
BC3 2045/2/7/17/7 pnt(+)tbr, Rasant	60	48	1	3.13 (0–8; 11)	0.38 (0; 1)	21.33 (17–24)	0	0.50 (0; 1)	0.10 (0; 1)	0.05 (0; 1)	0	0.29 (0; 1)	0.05 (0; 1)	0	0	0.65 (0–2)
BC4 2045/2/7/17/7/1 pnt(+)tbr, Rasant	5	48	1	4 (3; 5; 6)	0.20 (0; 1)	21.00 (19; 21; 22)	0	0.80 (0; 1)	0.40 (0; 1)	0	0	0	0	0	0	0.80 (0; 1)
BC4 2045/2/7/17/7/26 pnt(+)tbr, Rasant	29	48	1	4.48 (0; 2–8)	0.55 (0; 1)	20.55 (17–23)	0	0.31 (0; 1)	0.24 (0; 1)	0.07 (0; 1)	0	0.24 (0; 1)	0.07 (0; 1)	0	0	0.55 (0–2)
BC5 838/7/53/3/23/4/7 trn(+)tbr, Delikat	5	47	1	9.00 (8; 9; 11)	0.80 (0; 1)	18.40 (18; 19)	0	0.20 (0; 1)	0.20 (0; 1)	0	0	0	0	0	0	0.20 (0; 1)

* I—univalents; ** II—bivalents; *** III—trivalents; **** IV—quadrivalents; ***** V—pentavalents. Number of chromosomal arms per PMC which are involved in homological or homoeological chromosomal associations is indicated in brackets. ^X Frequency of A-B chromosomal associations on average per cell, including homoeological II, III, IV, and V; the number of chromosomal arms per PMC which are involved in homoeological chromosomal associations is indicated in brackets.

summarizes the following meiotic chromosome pairing characters: average frequency of homologous and homoeologous chromosomal associations per cell, type of meiotic configurations, and the number of chromosomal arms involved in homoeologous A/B pairing.

SH (2n = 6x = 72, AAAABB).

According to the GISH results, the most frequent chromosome configurations in hexaploid somatic hybrid 838/7 (2n = 6x = 72, AAAABB genome), were homologous bivalents; on average, 17.8 per cell involving chromosomes of the A-genome and 7.55 involving the B-genome of S. tarnii (Table 2, Figure 2b). Nevertheless, the average frequencies of homologous bivalents were less than the theoretically expected preferential bivalent pairing (24 II AA + 12 II BB) for the distant hybrid with genome constitution AAAABB, because of the appearance homoeologous chromosomal associations. The average frequency of intergenomic chromosomal association in the somatic hybrid was recorded as 4.75 per cell, including 3.45 II A-B + 0.15 III AA-B + 0.05 III A-BB + 0.15 IV AAA-B + 0.05 IV AA-BB + 0.05 V AAAA-B.

Note that among the homoeologous bivalents (II A-B), we detected not only rod, but also ring (closed-type) bivalents with two chiasmata, as well as closed-type associations among the homoeologous quadrivalents (IV AAA-B). Therefore, when summing up all the chromosomal pairs involved in allosyndetic associations, the obtained value of 3.90 on average per cell increased up to 4.75 (Table 2).

The average chromosome configuration also included univalents—6.80 per cell of S. tuberosum and 4.90 of S. tarnii (Table 2).

BC₁ hybrids (2n = 5x = 60, AAAAB).

None of the chromosomes of the B-genome formed homologous B-B bivalents in the four analyzed BC₁ hybrids; alien chromosomes of the B-genome were present as univalents in all PMCs, which means that these plants possess 12 individual alien chromosomes of the wild species S. bulbocastanum in a univalent state (basic chromosome number x = 12, as in all potato species) (Table 2, Figure 4a,b). At the same time, homoeologous chromosomal associations were detected in each of the four analyzed BC₁ hybrids that included A-B bivalents, and occasionally AA-B trivalents and AAA-B quadrivalents. The average frequency of the homoeologous associations varied from 3.05 to 3.63 per cell (Table 2, Figure 4a,b).

BC₂ hybrids.

Two selected BC₂ hybrids each possessed five alien chromosomes of the wild species S. bulbocastanum and recombinant chromosomes—one in BC₂ 82/4/46/14 and two in BC₂ 95/1/4/59 (Table 1c′). At meiosis for these hybrids, the average frequency of homoeologous chromosomal associations per cell was recorded as 2.38 and 2.16 per cell, respectively (Table 2, Figure 4c,d). GISH results showed that the recombinant chromosomes were paired with chromosomes of the A-genome of potato (Figure 4a,d,f).

BC₃–BC₅ hybrids.

Four selected introgression lines from the BC₃–BC₅ generations had a tetraploid ploidy level. Each of them possessed one additional alien chromosome of the B-genome; three of them were from the tbr(+)pnt combination and one was from tbr(+)trn combination (Table 2). As expected, the average chromosome configurations were mostly A-A bivalents between homologous S. tuberosum chromosomes, with frequencies of 18.40–21.33. Moreover, t I, III, and IV involving A-genome chromosomes of potato were observed in these introgression lines (Table 2, Figure 2f,g and Figure 3).

The frequency of intergenomic associations between the chromosomes of the A-genome and an additional monosomic chromosome of the B-genome were detected in each of the four introgression lines. The single alien chromosome of S. tarnii in the BC₅ hybrid (838/7/53/3/23/4/7) took part in homoeologous pairing, with average frequency of 0.2 per cell (Table 2, Figure 2f,g).

The BC₃ and BC₄ hybrids from the combination tbr(+)pnt represent the same family. These three putative monosomic alien addition lines bring the same single alien chromosome from S. pinnatisectum (Table 1b, Figure 3) that was not lost during backcrossing, because it took part in homoeologous pairing in the BC₃ hybrid 2045/2/7/17/7, with an average frequency of 0.65 per cell (Table 2). Our data demonstrate that this alien chromosome was involved in at least the second cycle of homoeologous pairing and recombination in the BC₃ and in the BC₄ hybrids.

3.4. Marker-Assisted Selection of the Somatic Hybrids and Introgression Lines

The whole subset of 56 genotypes (backcrossing derivatives, somatic hybrids, and parental accessions) was tested with seven DNA markers of four resistance genes to select the hybrid genotypes with introgressed alien genes.

3.4.1. Molecular Screening with Markers of the R_Mc1(blb) Gene Conferring Resistance to M. chitwoodi

Two DNA markers, 193I9 and 39E18, closely linked to the R_Mc1(blb) gene [43] conferring resistance to Columbia root-knot nematode, were used in the molecular screening (Supplementary Tables S1 and S2). Both markers were detected in parental accessions of the wild species S. tarnii, GLKS 2870 and S. bulbocastanum, GLKS 31741. Only one of these markers (193I9) was detected in accession GLKS 31607 of S. pinnatisectum. Both markers 193I9 and 39E18 were absent in the potato cultivars analyzed here that were involved in protoplast fusion and backcrossing (Supplementary Table S2).

Two somatic hybrids, SH 838/2 and SH 838/7 (2n = 6x = 72, AAAABB) from the combination trn(+)tbr, possessed both of these markers, whereas their four BC₁ derivatives (2n = 5x = 60, AAAAB) lost them. This might be explained by the heterozygosity of the locus R_Mc1(blb) in the parental accession of S. tarnii that was segregated in the BC₁ progeny. In contrast, in the combination blb(+)tbr, the markers of the R_Mc1(blb) gene were detected in all 10 BC₁ hybrids and in 5 BC₂ hybrids (Supplementary Table S2, Supplementary Figure S1).

3.4.2. Molecular Screening with Markers of the Genes Conferring Resistance to Late Blight Diseases

The marker 184-81-RsaI, specific for the R8 gene conferring resistance to late blight, was detected in all three parental accessions of the wild species: S. tarnii, GLKS 2870, S. bulbocastanum, GLKS 31741, and S. pinnatisectum, GLKS 31607. At the same time, it was absent in the potato cultivars analyzed here that were involved in protoplast fusion and backcrossing. Both somatic hybrids SH 838/2 and SH 838/7 (2n = 6x = 72, AAAABB) from the combination trn(+)tbr, and three out of four of their BC₁ hybrids (2n = 5x = 60, AAAAB), had this marker, whereas one BC₁ genotype (BC₁ 03.838/2/25) lost it. This might be explained by the heterozygous condition of the R8 gene in the parental accession of S. tarnii. Two of the four BC₂ derivatives analyzed here from the combination trn(+)tbr had the R8 gene. In the combination blb(+)tbr, the marker 184-81-RsaI of the R8 gene was detected in all the BC₁ hybrids and in three BC₂ genotypes. The backcross derivatives from combination tbr(+)pnt lost it (Supplementary Table S2, Supplementary Figure S2).

The parental accession GLKS 31741 of S. bulbocastanum possess four markers of the two Rpi genes conferring broad-spectrum late blight resistance—two markers, 1/1′ and blb1 F/R, specific for the Rpi-blb1 gene, and two markers, Rpi-blb3 and blb3 F/R, specific for the Rpi-blb3 gene (Supplementary Table S2, Supplementary Figures S3–S6). These markers were not detected in the parental accessions of S. tarnii, GLKS 2870, or S. pinnatisectum, GLKS 31607.

All 10 BC₁ hybrids from the combination blb(+)tbr had markers of the Rpi-blb1 and Rpi-blb3 genes. Moreover, we detected six BC₂ hybrids with Rpi-blb1, two with Rpi-blb3, and one BC₃ genotype, 82/4/68/32/27, with the marker Rpi-blb3 (Supplementary Table S2, Supplementary Figures S3–S6).

Thus, 12 genotypes (10 BC₁ and 2 BC₂) from the combination blb(+)tbr with four resistance genes from S. bulbocastanum, GLKS 31741, were selected using MAS for a further breeding program.

4. Discussion

4.1. Genomic Composition of Interspecific Hybrids and Transmission of Alien B-Genome Chromosomes through Backcrossing

The desirable alien genes of wild species could be introgressed into the recipient genome of crop plants through alien chromosome additions or substitutions, intergenomic translocations, homoeologous pairing, and meiotic recombination. The application of GISH for the offspring of distant hybrids is an efficient approach for the monitoring of alien chromatin introgression into the genome of crop species [73,74], especially for species with small chromosomes and low levels of karyotype differentiation, such as potato.

Previous research has showed that GISH has been applied effectively for the distinction of parental genomes in hybrids of potato and non-tuber-bearing species of the genus Solanum, belonging to the sections Lycopersicon and Etuberosum (see reviews [48,72,75]). Within the section Petota Dumort, which includes numerous tuber-bearing species (potatoes) of the genus Solanum, GISH has been successfully used to differentiate the A-genome of S. tuberosum and the B-genome of Mexican diploids in interspecific somatic and in sexual hybrids and their progenies [7,16,44]. It has also been used to distinguish the B-subgenome in natural Mexican allopolyploid species [61,76] or chromosomes of the B-subgenome in sexual hybrids between the wild Mexican allotetraploid species and the common potato [77].

In the present study, we detected the genomic composition of 39 hybrid genotypes of different generations from three interspecific combinations between the common potato, S. tuberosum (genome AAAA), and the wild diploid (1 EBN) Mexican species: S. tarnii, S. pinnatisectum, and S. bulbocastanum (BB genome). The genomic composition of the hexaploid somatic hybrids from the combinations trn(+)tbr corresponded to what was theoretically expected—AAAABB, as well as the genomic composition of the BC₁ hybrids—seven BC₁ hybrids from the combinations trn(+)tbr and blb(+)tbr were pentaploids having an AAAAB genomic constitution. As expected, these BC₁ hybrids produced aneuploid progeny, indicating the loss of the B-genome chromosomes in the subsequent BC₃–BC₅ backcrossing. From six to one alien chromosomes were detected in the BC₄ introgression lines; six of the seven BC₅ genotypes lost chromosomes of the B-genome.

Our results indicate the potential for the selection of monosomic alien addition lines of the B-genome Mexican species in a S. tuberosum genome background. Five putative monosomic addition lines with single alien chromosomes were detected among the BC₃-BC₅ derivatives analyzed here—two in the combination trn(+)tbr and three in the combination pnt(+)tbr. The single alien chromosome from S. pinnatisectum was not lost during BC₃ backcrossing and was transferred to the BC₄ hybrids because it took part in intergenomic pairing. Homoeologous chromosomal associations involving a single alien chromosome of S. tarnii were also observed in the BC₅ 838/7/53/3/23/4/7 (2n = 48) introgression line, indicating the possibility of their transfer during subsequent backcrossing.

Similarly, using GISH, Kikuchi et al. [7] selected three introgression lines with a single chromosome of S. pinnatisectum in the potato genome background among the BC₄ progeny of sexual interspecific hybrids. In potato, a set of alien addition and/or substitution lines was established for more distantly related non-tuber-bearing species of the genus Solanum, belonging to the sections Lycopersicon [78,79] and Etuberosum [80], using BC progenies derived from somatic hybrids.

4.2. Meiotic Pairing of A/B Homoeologous Chromosomes, Detection of Recombinant Chromosomes

The results of introgressive breeding in potatoes depend largely on the potential of homoeologous pairing and meiotic recombination, because it predicts the success of desirable alien gene introgression into the S. tuberosum genome through subsequent backcrossing, and helps to develop introgressive hybridization schemes [3,7,48,54,77,81,82,83]. Therefore, information about meiotic chromosome behavior in interspecific hybrids and about the peculiarities of the interaction of the A-genome of S. tuberosum and the B-genome of Mexican diploid species is important for potato introgressive breeding.

To assess the potential of homoeologous meiotic A/B pairing and recombination, a GISH analysis was performed here for the PMCs of 11 hybrid genotypes from three interspecific combinations. Homoeologous meiotic chromosome pairing between chromosomes of the A- and the B-genomes was detected in each of the 11 analyzed introgression lines, even in the somatic hybrid trn(+)tbr (2n = 72, AAAABB), for which we expected to detect preferential bivalent pairing (24 II A + 12 II B). The average frequency of intergenomic chromosomal association in the somatic hybrid was recorded as 4.75 per cell; in the BC₁ hybrids it varied from 3.05 to 3.63 per PMC, including II A-B, III AA-B, and IV AAA-B. An analysis of the homoeological chromosome pairing associations indicates that the intergenomic A-B bivalents prevailed in all the 11 analyzed hybrid genotypes, among which associations of the open type (rod bivalents) predominated; closed (ring) A-B bivalents were less common, but they were observed in all the hybrids, except for one line—BC₄ 2045/2/17/07/1.

Our results are in agreement with a few previous studies of meiotic chromosome behavior in interspecific hybrids, which indicated that chromosomes of the B-genome of wild Mexican potato species can recombine with chromosomes of the A-genome. Thus, intergenomic chromosome pairing at meiosis was demonstrated using GISH in sexual hexaploid interspecific hybrids between S. pinnatisectum and S. tuberosum having four genomes from wild species and two genomes from potato; about 20% of the S. pinnatisectum chromosomes were involved in intergenomic associations, with an average frequency of 6.7 per cell [7]. Regular meiosis was reported in the sexual hybrids between S. bulbocastanum (BB genome) and the wild Mexican species S. verrucosum (AA genome) [81]. A quadrivalent formation was detected in the interspecific sexual hybrid ((Solanum acaule × S. bulbocastanum) × Solanum phureja), that was analyzed using the conventional cytological method [3,54]. A homoeologous pairing was detected using GISH in the sexual hybrids between chromosomes of the B-subgenome of the wild allotetraploid Mexican species Solanum stoloniferum Schltdl. (AABB genome) and chromosomes of the A-genome. The average frequency of intergenomic associations was 0.82 per cell in the hexaploid hybrid (mitotic doubling, genome composition AAAABB), and up to 2.50 in the BC₁ hybrid (genome composition AAAAB) [77].

GISH/FISH re-probing was helpful for precisely identifying the introgression of alien chromatin in recombinant chromosomes. Five introgression lines (two BC₁, one BC_2, and two BC₄) having one–two chromosomes with alien segment introgression were identified among the backcrossing derivatives from the trn(+)tbr and blb(+)tbr combinations. These recombinant chromosomes took part in homoeological pairing and, therefore, have a potential to be transferred to the subsequent offspring, where the size of the alien introgressed segments may change as a result of homoeological pairing and crossing-over. Taking into account the number of homoeological chromosomal associations detected in our study, we can assume that the observed number of recombinant chromosomes recognizable using GISH in the five BC genotypes is significantly less than expected. This can possibly be explained by the fact that the homoeological chromosomes were paired by terminal chiasmata and some of the introgressed fragments were out of range of the GISH resolution.

We could suppose a different origin for some of the recombinant chromosomes that arose, not only from homoeological pairing and meiotic recombination, but also from chromosomal rearrangements due to somaclonal variation. Thus, the findings of Rakosy-Tican et al. [16], who detected a single recombinant chromosome at a very early stage of introgression breeding, still in the somatic hybrid blb(+)tbr, which obviously could be the result of chromosomal rearrangements during protoplast isolation and fusion.

In previous GISH studies of backcrossing progenies of interspecific somatic hybrids between the common potato, S. tuberosum, and the non-tuber-bearing species, S. etuberosum, recombinant chromosomes were not detected [84,85], whereas in the closer combination between S. tuberosum and S. pinnatisectum, intergenomic translocations were often observed in the BC₂–BC₄ progenies of sexual interspecific hybrids [7].

4.3. Marker–Assisted Selection (MAS) of the Somatic Hybrids and the Backcrossing Derivatives

Molecular screening data confirmed the introgression of alien genetic material into the potato genome in the somatic hybrids and their backcrossing derivatives.

MAS forR_Mc1(blb)geneconferring resistance to Columbia root-knot nematode.

We detected the R_Mc1(blb) gene encoding resistance to M. chitwoodi, using two DNA markers closely linked to it, in 17 out of the 48 hybrid genotypes, including 10 BC₁ and 5 BC₂ hybrids from the combination blb(+)tbr and in 2 from SH trn(+)tbr. The utility of these markers for the MAS of resistance to Columbia root-knot nematode genotypes was demonstrated earlier in the nematode resistance tests [43]. A successful program for the introgression of the R_Mc1(blb) gene from S. bulbocastanum into the cultivated potato gene pool has already been carried out at Washington State University, where advanced breeding clones with root-knot resistance were developed from blb(+)tbr hybrids with subsequent backcross cycles [42].

MAS for genes encoding resistance to late blight.

Previous studies have shown that the parental accessions of three wild diploid Mexican species, trn (GLKS 2870), pnt (GLKS 31607), and blb (GLKS 31741), are involved in somatic hybridization and are highly resistant to late blight, and/or to PVY, and aphids; these resistance traits are transferred to the somatic hybrids and BC progenies [16,21,29,39,56,57,58].

Lokossou et al. [67] reported the presence of the well-studied Rpi genes conferring broad-spectrum late blight resistance, in natural populations of Mexican potato species, including S. bulbocastanum (Rpi-blb1, Rpi-blb2, and Rpi-blb3 genes). The diagnostic fragments of the specific markers of the Rpi-blb1 and Rpi-blb3 genes—BLB1F/R, Blb3F/R [16] and 1/1′, and Rpi-blb3 (the present study)—were detected in the late-blight-resistant parental accession GLKS 31741 of S. bulbocastanum. The primers specific for the Rpi-blb2 gene were not amplified in this accession. Rakosy-Tican and colleagues [16] confirmed the functionality of the detected alleles of the Rpi-blb1 and Rpi-blb3 genes in the parental accession GLKS 31741 of S. bulbocastanum, and in selected genotypes of SH and BC₁-BC₂, using agro-infiltration with the effectors of the corresponding Avr genes. A similar approach was used to detect the functional alleles of the R3a and R3b late-blight-resistance genes in the same genotypes [16]. Molecular screening and phenotyping of the BC₁ and BC₂ hybrids from the combination blb, GLKS 31741(+)tbr, cv. Delikat, with specific primers for the Rpi-blb1, Rpi-blb3, R3a, and R3b genes allowed the identification of plants carrying all four genes that were resistant to foliage blight in greenhouse and field trials [16]. Twelve hybrid genotypes from this previous study were included in our subset, which was expanded with additional BC₁, BC₂, and BC₃ hybrids, and was screened with additional gene-specific primers for the Rpi-blb1, Rpi-blb3, and R8 genes (see Supplementary Tables S1 and S2). A comparison of the data from the literature [16] with our results shows that 11 of 12 late-blight-resistant genotypes have a stacking of 5 genes with distinct resistance specificities to late blight that are located on four chromosomes: Rpi-blb1 (VIII), Rpi-blb3 (IV), R3a (XI), R3b (XI), and R8 (IX). In addition, the markers of the R_Mc1(blb) (XI) gene encoding resistance to Columbia root-knot nematode were detected in each of these 11 genotypes (9 BC₁ hybrids having a complete chromosomal set of alien chromosomes and three BC₂ genotypes, two of them with 4 and 5 alien chromosomes, according to the GISH data).

Three genes from S. bulbocastanum—R_Mc1(blb), R3a, and R3b—are located on the same chromosome XI [41,86,87]. They all were transferred from parental accession GLKS 31741 to 11 BC genotypes (9 BC₁ and 2 BC₂, see Supplementary Table S2). One introgression line, BC₂ 95/1/4/59 (2n = 53, 48 chromosomes of potato, including 2 recombinant chromosomes and 5 alien chromosomes), had the R3a and R3b genes, but lost the markers tightly linked to the R_Mc1(blb) gene, which might be explained by recombination.

Particularly interesting is the BC₃ 82/4/68/32/27 genotype that possesses gene-specific markers of the Rpi-blb3 gene introgressed from blb, GLKS 31741, although it lost all alien chromosomes, according to the GISH results. Based on the data obtained in the present study, it can be assumed that the alien segment with the Rpi-blb3 gene, which is located on chromosome IV of S. bulbocastanum, was introgressed into the A-genome of S. tuberosum by meiotic recombination, and that the size of this alien fragment was too small to be detected with GISH.

MAS of trn(+)tbr and pnt(+)tbr combinations.

The primers specific for the genes Rpi-blb1–Rpi-blb3 were not amplified in the late-blight-resistant parental accession GLKS 2870 of S. tarnii. This parental accession, two SH and five BC hybrids, had the diagnostic fragments of the gene-specific primers for the late-blight- resistant R8 gene.

Lokossou et al. [67] also reported the presence of the Rpi-blb3 gene conferring broad-spectrum late blight resistance, in natural populations of S. pinnatisectum. However, the primers specific for the Rpi-blb3 and Rpi-blb1 genes (the present study) and for Rpi-blb2 were not amplified in the late-blight-resistant parental accession GLKS 31607 of S. pinnatisectum. The novel, major late-blight-resistance locus derived from S. pinnatisectum (GLKS 31607) was mapped onto chromosome VII using DArT markers [88]. However, DNA markers associated with this novel locus and that are well suited for MAS have not yet been developed. Additional studies are required to identify chromosome VII of S. pinnatisectum, which was transferred to the late-blight-resistant hybrid genotypes.

4.4. Strategy for Introgressive Breeding

Knowledge about the level of meiotic chromosome pairing affinity in hybrids is important for developing efficient breeding strategies. In this case, the most significant is the meiosis of the pentaploid (2n = 5x) BC₁ hybrids having the AAAAB genomic composition (48 chromosomes of the A-genome and 12 chromosomes of the B-genome), which possesses a complete haploid set of alien chromosomes of a wild species, because no homologous B-B bivalents were found in any PMCs. At the same time, from 3.05 to 3.63 intergenomic A/B associations per PMC were observed in these BC₁ hybrids. It is important that breeding programs directed to developing advanced lines with resistance to multiple diseases do not lose recombination potential during subsequent backcrosses, in which alien chromosomes are rapidly lost. Therefore, it can be assumed that several cycles of self-pollination of the BC₁ hybrids may accumulate their A/B recombination potential. The subsequent backcrossing of the self-pollinated BC₁ hybrids will promote the cytological stability of the introgression lines, having potato chromosomes with alien segment introgressions.

5. Conclusions

Using the GISH and MAS methods, we provided information about the potential of homoeologous chromosome pairing and alien gene introgression in a wide subset of backcrossing derivatives, from three interspecific combinations between S. tuberosum and the diploid Mexican B-genome potato species: S. tarnii, S. pinnatisectum, and S. bulbocastanum.

Hybrids from BC progenies contain different amounts of chromatin from wild species, including monosomic alien addition chromosomes, alien segments that were introgressed through homoeologous pairing and recombination, as well as hybrid genotypes with stacking up to six genes (Rpi-blb1, Rpi-blb3, R3a, R3b, R8, and R_Mc1(blb)) from S. bulbocastanum.

Selected hybrid genotypes with alien introgressions might be used in potato pre-breeding programs for their resistance to multiple diseases and pests, such as late blight, the quarantine pest of potato, M. chitwoodi, as well as PVY and aphids.

In the present work we demonstrated the efficiency of applying two methods—GISH and MAS—for monitoring the alien introgression of the B-genome diploid (1 EBN) wild Mexican species into the A-genome of potato, S. tuberosum. This is consistent with the recent results of Kikuchi et al. [7] and Rakosy-Tican et al. [16]. At the same time, additional studies will be required to identify the individual alien chromosomes passed on to backcross derivatives from wild Mexican B-genome species.