1 Crop Breeding and Applied Biotechnology - 23(3): e45152337 , 2023 New homologues of the Rpi-chc1 gene in wild and cultivated Solanum species

: The Rpi-chc1 gene confers resistance to late blight (LB) in the wild South American species Solanum chacoense. The goal of this study was to enhance our insight into polymorphism of this gene in the genus Solanum, which is the source of valuable donors of resistance to LB. To this end, we analyzed 122 accessions of the working collection, consisting of potato cultivars, complex interspecific hybrids, and representatives of 11 wild Solanum species. We studied the poly - morphism of the region of this gene that encodes the most polymorphic LRR domain. As a result, in the species S. chacoense, S. berthaultii, S. tuberosum, S. microdontum, and S. maglia we found previously unknown variants of the Rpi-chc1 gene, which differ in their amino acid sequence from both the functional and non-functional variant of the Rpi-chc1 gene. Therefore, the function of these homologues cannot be unambiguously predicted, but must be further studied.


INTRODUCTION
Potato (Solanum tuberosum) is the third most important food crop after rice and wheat.Late blight (LB) caused by the oomycete Phytophthora infestans is one of the most devastating potato diseases.The global economic cost of LB is approximately €9.4 billion per year (Haverkort et al. 2016).One strategy to control LB is the introgression of LB resistance genes (R genes) from potato wild relatives.Most of these resistance genes have been introduced into commercial potato varieties from the wild species S. demissum.In particular, in S. demissum the R1, R2, R3a, R3b, R8, and R9a genes were mapped, characterized, and then bred to cultivated potato varieties (Ballvora et al. 2002, Huang et al. 2005, Lokossou et al. 2009, Li et al. 2011, Jo et al. 2015, Vossen et al. 2016).However, the resistance conferred by these genes is being overcome by new virulent strains of P. infestans (Jo et al. 2014).Therefore, it is important to search for new R genes that provide broad-spectrum resistance to a wide range of pathogen races at once.The main sources of these new LB resistance genes (Rpi genes) are wild Solanum species.To date, over 70 Rpi genes have been identified and mapped in 32 Solanum species (Paluchowska et al. 2022).One of these genes is the Rpi-chc1 gene discovered in the wild species S. chacoense (Vossen et al. 2011).
S. chacoense is a diploid South American wild potato species native to Bolivia.The locus associated with resistance to LB has been mapped in S. chacoense on chromosome 10, and the gene conferring this resistance has been found at this locus.This gene was named Rpi-chc1.The Rpi-chc1 gene encodes a protein V Martynov and M Beketova from the NB-LRR family, consisting of 1302 amino acids and containing 29 leucine-rich repeats (LRR) (Vossen et al. 2011).Later it was found that the Rpi-chc1 gene had two allelic variants, Rpi-chc1.1 and Rpi-chc1.2,and it was shown that these alleles recognized different effectors from the PexRD12/31 superfamily of effector proteins of P. infestans.The Rpi-chc1.1 allele recognizes several PexRD12 proteins, and the Rpi-chc1.2allele recognizes several PexRD31 proteins (Monino-Lopez et al. 2021).In addition, homologues of the Rpi-chc1 gene were found in some Solanum species other than S. chacoense, in particular in S. berthaultii, S. tarijense and S. tuberosum, and among these homologues were both functional and non-functional variants (Monino-Lopez et al. 2021).At the same time, the functional variant Rpi-chc1.1 was found to differ from its non-functional homologue from S. tuberosum by 21 amino acid substitutions, of which 19 are in the LRR domain (Monino-Lopez 2021).These data suggest that the Rpi-chc1 gene is a member of a large R gene family that is still poorly understood in Solanaceae.Therefore, the search for new homologues of the Rpi-chc1 gene in S. chacoense and other Solanum species makes sense, since in order to keep up with the rapid evolution of Avr genes, Rpi genes also rapidly evolve with the formation of new variants with different functional activities (Leister 2004, Mcdowell andSimon 2006).Besides, it should be noted that new data on the polymorphism of the primary structure of the Rpi-chc1 gene and its homologues and the possible relationship of this polymorphism with the function will help in selecting targets for genome editing when breeding new LB-resistant potato varieties.
Thus, the goal of this research was to study the polymorphism of the Rpi-chc1 gene in potato varieties and interspecific hybrids cultivated in Russia, as well as in accessions of wild potato species from the collection of the N.I.Vavilov Institute of Plant Genetic Resources (VIR).This collection is one of the largest collections of cultivated potato and wild tuberbearing species in the world.

LB resistance assessment
LB resistance of varieties and hybrids was evaluated in the laboratory tests by the method of infection of detached leaves.Detached leaves of plants grown in a greenhouse were infected by applying to their surface 0.1 ml of a suspension of zoosporangia (approximately 3000 zoosporangia) of a highly virulent and aggressive isolate of P. infestans N161 from the collection of the Institute of Phytopathology (race 1.2.3.4.5.6.7.8.9.10.11,mating type A1), using the cultivar Santé as a reference control (Kuznetsova et al. 2014).Leaves were placed bottom side out in wet chambers.The lesion was evaluated in five days post infection.The resistance of the sample was scored on a 9-point scale, wherein 9 points correspond to the highest resistance level.The average score for each sample was calculated based on the results of damage of three leaves.

DNA isolation
Total DNA was isolated from young leaves of two-week-old plants using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's protocol.For each accession DNA was isolated from eight plants and then these DNA preparations were combined into one common sample.

Primer design and PCR conditions
PCR primers for specific amplification of the Rpi-chc1 gene were designed based on the sequence of this gene from the International Patent Application WO2011/034433 (Vossen et al. 2011).In this application it was designated as 543-5_C10_C15_C24.We made a multiple alignment of this sequence with the sequences of other closely related homologues of the Rpi-chc1 gene described in this patent application, as well as potato homologues available from the NCBI database.As a result, primers were selected that could discern the Rpi-chc1 gene from its homologues due to the specific 3'-terminal sequence characteristic only of this gene.We designed primers that amplify the region of the Rpi-chc1 gene encoding the LRR domain of the receptor protein, since this domain is responsible for pathogen recognition and it is the most polymorphic.The nucleotide sequences of the designed primers were as follows: 5¢-CTATTTGACTTCCCTCGAATTCT-3¢ for the forward primer and 5¢-CTTCTAACAATGGACAATCACGT-3¢ for the reverse primer.DNA amplification was performed in a thermal cycler GeneAmp PCR System 2700 (Applied Biosystems, Inc., USA) using the following cycling condition: one cycle of 94 °C for 5 min followed by 35 cycles of 94 °C for 35 sec, 60 °C for 35 sec and 72 °C for 1 min 20 sec; and final extension at 72 °C for 7 min.The volume of the reaction mixture was 25 μl.A sample of 50 ng of total DNA was taken per each reaction.PCR products were separated by electrophoresis in 1% agarose gel in 1X TAE buffer and visualized under UV after staining with 0.5 μg/ml ethidium bromide.

Cloning and sequencing
PCR products were cloned using pAL-TA vector (Evrogen, Moscow, Russia) in accordance with the manufacturer's protocol and then sequenced with a nucleic acid analyzer ABI PRIZM 3730 (Applied Biosystems, Inc., USA) using the Big Dye Terminator v.3.1 reagent kit (Applied Biosystems, Inc., USA) according to the manufacturer's instructions.

Bioinformatics analysis
Multiple alignment of nucleotide sequences was performed with the Clustal Omega software (http://www.ebi.ac.uk/Tools/msa/clustalo/), followed by analysis of this alignment with the GeneDoc software.Phylogenetic analysis was performed with the Treecon software (Van de Peer and de Wachter 1994).Deduced amino acid sequences were obtained with the EditSeq software.

Occurrence of homologues of the Rpi-chc1 gene in the working collection
We amplified the total DNA samples isolated from 122 accessions of the working collection, consisting of potato varieties, interspecific hybrids and wild Solanum species, with primers specific to the Rpi-chc1 gene.As a result, the expected PCR product was obtained only in 30 samples.However, we probably failed to amplify the specific PCR product in some other samples due to the inherent disadvantage of the PCR method, which is that if mutations occur in the DNA at the primer binding sites, then PCR is not feasible, resulting in false negative results.PCR results for these 30 samples are shown in Figure 1.In any of the five studied North American species S. bulbocastanum, S. cardiophillum, S. pinnatisectum, S. stoloniferum and S. verrucosum the specific PCR product was not detected.This finding may support the assumption that the ancestral form of the Rpi-chc1 gene arose after the separation of the North American and South American Solanum species, and the presence of functional alleles of Rpi-chc1 gene in the latter may be the result of a recent cross of the species (Monino-Lopez et al. 2021).Among South American species we found the specific PCR product not only in S. chacoense, S. berthaultii and S. tuberosum, but also in two species, S. microdontum and S. maglia.In particular, in the case of S. chacoense, out of 19 analyzed samples, the expected PCR product was present in six, and out of eight analyzed samples of S. microdontum it was found in two samples.In recent decades, the species S. microdontum has already been used in programs for breeding LB resistant potato varieties (Sandbrink et al. 2000), and one LB resistance gene Rpi-mcd1 was discovered in this species (Tan et al. 2008).In our study, for the first time, we found in S. microdontum a homologue of the Rpi-chc1 gene, which was the most identical to the prototype gene (see Table 1 and discussion below).This finding makes S. microdontum an even more valuable donor of LB resistance and should serve as a starting point in an in-depth study of the structural and functional features of the homologue of the Rpi-chc1 gene in this species.Also, the expected PCR product was found in S. bertaultii (in four samples out of five) and in both analyzed samples of S. maglia.For the species S. maglia, the homologue of the Rpi-V Martynov and M Beketova chc1 gene obtained in this study is the first reported homologue of the LB resistance gene.In varieties of cultivated potatoes, the specific PCR product was found in 12 of the 17 analyzed samples, and it was found in only four of ten screened hybrids.

Rpi-chc1 gene
In order to determine the nucleotide sequence of the PCR products obtained in all five abovementioned South American species, we cloned the amplified fragment from seven samples.These were samples of the wild species S. chacoense K19264, S. microdontum CGN 20640, S. berthaultii K19961 and S. maglia K240604 from the VIR collection and samples of cultivated potato S. tuberosum, represented by varieties "Sarpo Mira" and "Bintje" and a complex interspecific hybrid 2372-60.The cloned fragment corresponded to the region of the LRR domain in the C-terminal part of the Rpi-chc1 protein.
Five clones were sequenced for each sample.Hence, 35 nucleotide sequences of structural homologues of the Rpi-chc1 gene were obtained in total.For each sample, the sequences of five clones were substantially similar to each other.A few single-nucleotide polymorphisms (from four to six) can be considered as artifactual, since each of them occurs only in one clone out of five.An exception was the "Sarpo Mira" sample.In this sample, four clones were almost identical (99.8% of sequence identity), and the fifth clone was significantly different from them (91.2% of sequence identity).
Thus, it suggests that the cultivar "Sarpo Mira" is polymorphic in the Rpi-chc1 gene and has at least two variants of this gene in its genome.The highly resistant cultivar "Sarpo Mira" was known to have not only homologues of the R3a, R3b and R4 genes from S. demissum, but also own resistance genes Rpi-Smira1 and Rpi-Smira2 (Rietman et al. 2012).The most homologous sequences are in bold.In our study, we showed for the first time that, in addition to the known Rpi genes, the "Sarpo Mira" genome contains at least two variants of the Rpi-chc1 gene homologues.This discovery sheds light on the pedigree of the cultivar "Sarpo Mira" and suggests that the genetic material of S. chacoense was used when this cultivar was being bred.The size of the cloned fragment for all samples was 575 nucleotides, except for the S. microdontum sample, for which the size of the resulting product was 572 nucleotides.Due to the high identity, we deposited one of the obtained sequences for each sample at the NCBI GenBank under the following accession numbers: OQ411253 for the S. chacoense K19264, OQ411254 for the S. microdontum 20640, OQ411256 for the S. berthaultii 19961, OQ411257 for the S. maglia K240604, OQ411252 for the S. tuberosum cultivar "Bintje" and OQ411255 for the S. tuberosum hybrid 2372-60.The exception was the "Sarpo Mira" sample, for which we deposited two sequences found in this sample.The accession numbers of these sequences are as follows: OQ414957 and OQ414958.
Thus, further we analyze and discuss these eight deposited sequences.The sequences from S. chacoense, S. berthaultii, S. maglia, the cultivar "Bintje" and the hybrid 2372-60, as well as the sequence OQ414958 from the cultivar "Sarpo Mira", are translated in silico, suggesting that they are the expressed genes.In contrast, the translation of the sequence from S. microdontum and the second sequence OQ414957 from the cultivar "Sarpo Mira" terminates at an early stop-codon, indicating that most probably these sequences are pseudogenes, or they are coding a truncated protein.
We compared the obtained sequences with the sequences of two allelic variants of the prototype gene Rpi-chc1.1 and Rpi-chc1.2.The results are shown in Table 1.As can be seen from this table, sequences from S. microdontum, the cultivar "Sarpo Mira" (the sequence OQ414958) and S. berthaultii are the most similar to the prototype, and sequences from S. chacoense, S. maglia, the hybrid 2372-60 and the cultivar "Bintje", as well as from the cultivar "Sarpo Mira" (the sequence OQ414957) are significantly less similar to the prototype.At the same time, the level of homology of all obtained sequences with both Rpi-chc1.1 and Rpi-chc1.2 is approximately the same -on average 91% for less homologous sequences and 96% for more homologous sequences, while the homology between the variants Rpi-chc1.1 and Rpi-chc1.2 is 98.57%, and the homology between variants Rpi-chc1.1/Rpi-ber1.1/Rpi-tar1.1 is 99%.Therefore, none of the obtained homologues is the known variant Rpi-chc1.1 or Rpi-chc1.2.Then, we compared the obtained nucleotide sequences with known homologues of the Rpi-chc1 gene in S. berthaultii, S. tarijense and S. tuberosum described in Monino-Lopez et al. (2021).
The results of the comparison are presented as a phylogenetic tree in Figure 2. On this dendrogram the sequences of known variants of the Rpi-chc1 gene from S. chacoense, S. berthaultii, S. tarijense and S. tuberosum form a large common cluster (Cluster I), and within this cluster, homologues of the Rpi-chc1 gene are not grouped according to species origin, but according to belonging to the variant of this gene.The sequences obtained in this study are clustered separately from the previously known sequences of the Rpi-chc1 gene and its homologues and form their own distinct cluster (Cluster IV).The exceptions are the sequence from S. berthaultii, which clusters together with the sequence of the known homologue Rpi-ber1.4(Cluster III), and the sequences from "Sarpo Mira" OQ414958 and S. microdontum, which form their own separate cluster (Cluster II).It is worth mentioning that the new obtained    V Martynov and M Beketova homologue of the Rpi-chc1 gene from S. chacoense is closer to the homologues of this gene from potato than to the homologues from S. chacoense, and together with the former forms a common cluster.
The functional variant Rpi-chc1.1 in S. chacoense was shown to differ from its non-functional homologue Rpi-tub1.3 in S. tuberosum by 21 amino acid substitutions (Monino-Lopez 2021).Seven of these substitutions are located in the region of the LRR domain that we amplified (Figure 3), and we compared the amino acids at these positions in the obtained homologues and the functional and non-functional variants of the Rpi-chc1 gene.The comparison results are shown in Table 2.As can be seen from this table, none of the obtained homologues in terms of its amino acid composition at these positions corresponds to both the functional and non-functional variant of the Rpi-chc1 gene.Homologues from the cultivar "Sarpo Mira" (OQ414958) and S. microdontum are represented by a combination of amino acids characteristic of both functional and non-functional variants.The homologue from S. microdontum has five amino acids characteristic of the functional variant, while the homologue from the cultivar "Sarpo Mira" contains four such amino acids.The homologue from S. berthaultii has three amino acids characteristic of the functional variant.The other obtained homologues have amino acid residues at positions 1035, 1057, 1161 and 1188 that have not been described for the functional and non-functional variants of the Rpi-chc1 gene.
Summarizing the obtained results, we can conclude that the homologues obtained in this study cannot be unambiguously classified as any of the previously known variants of the Rpi-chc1 gene, with the exception of the homologue from S. berthaultii, which most likely is the Rpi-ber1.4,since it is clustered with the Rpi-ber1.4on the dendrogram.All other Bold letters -amino acids of the functional variant Rpi-chc1.1;italic letters -amino acids of the non-functional variant Rpi-tub1.3.

Figure 3 .
Figure 3. LRR domain of the Rpi-chc1 protein.Arrows indicate the location of this domain (the numbers of the initial and final amino acids) relative to the full-length protein.Numbers above the vertical lines indicate the positions of the functionally important amino acids that are discussed in this article.Bold numbers indicate the positions in which previously unknown amino acids were found.Italicized numbers indicate the positions where the amino acids correspond to known variants of the Rpi-chc1 protein.Numbers on the LRR domain itself indicate the LRR numbers in which the abovementioned substitutions are found.

Table 1 .
Homology of the obtained sequences (%) with

Table 3 .
Comparison of the results of molecular analysis with the data of laboratory resistance to LB of the studied cultivars and hybrids «+» -presence of the marker, «-» -absence of the marker