Genomic Markers Linked to Meloidogyne Chitwoodi Resistance Introgressed From Solanum Bulbocastanum to Cultivated Potato and Their Utilility in Marker-assisted Selection

Meloidogyne chitwoodi is a major threat to potato production in the Pacic Northwest region of United States. Infected tubers are rendered unmarketable, hence growers’ protability is adversely affected. Breeding for nematode resistance is a long-term process and phenotyping the segregating populations for nematode resistance is the most time-consuming and laborious part of the process. Using DNA-based markers closely linked to the nematode resistance trait for marker-assisted selection (MAS) could enhance breeding eciency and accuracy. In the present study, a pool of phenotyped progenies segregating for nematode resistance and susceptibility were ngerprinted using a 21K single nucleotide polymorphism (SNP) array. Eight candidate SNPs located on potato Chromosome 11, segregating with the nematode resistance trait were identied and used as landmarks for discovery of other marker types such as, simple sequence repeat (SSR) and insertion-deletion (INDEL) markers. Subsequently, a total of eight SNPs, 30 SSRs and four INDELS located on scaffold 11 of Solanum. bulbocastanum were used to design primers; markers were validated on a panel of resistant and susceptible clones. Two SNPs (SB_MC1Chr11-PotVar0066518 and SB_MC1Chr11-PotVar0064140), ve SSRs (SB_MC1Chr11-SSR04, SB_MC1Chr11-SSR08, SB_MC1Chr11-SSR10, SB_MC1Chr11-SSR13 and SB_MC1Chr11-SSR20) and one INDEL (SB_MC1Chr11-INDEL4) markers differentated between the resistant and susceptible clones in the test panel as well as other segregating progenies using simple PCR technique and high resolution melting curve analysis. These markers are robust, highly reproducible and easy to use for MAS of nematode resistant potato clones to enhance the breeding program. greenhouse phenotyping for nematode resistance screening to enhance breeding efforts. Markers developed present show for our breeding program for MAS and development of nematode resistant potato varieties for Pacic


Introduction
The Paci c Northwest contributes approximately 65% of the total potato production in the United States, valued at $2.21 Billion for 2019 (NASS-USDA 2020). Meloidogyne chitwoodi Golden et al. (Columbia root-knot nematode; CRKN) is a major pest of potato in this region (Santo et al. 1980). M. chitwoodi infects potato roots as well as tubers, causing both external and internal defects and reducing the market value of the crop. Preplant soil fumigation with nematicides and/or fumigants is the most effective approach to control CRKN populations. Soil fumigants and nematicides are not only expensive but also pose a threat to the environment and human health. Hence, growing potato varieties with natural resistance to most prevalent nematode populations would be an effective strategy, but at present none of the commercial potato varieties are resistant to CRKN.
Conventional breeding is the backbone of crop domestication and improvement. Although it is a time-consuming process, its bene ts to modern agriculture supercede its drawbacks. In potato, natural resistance to CRKN was rst identi ed in the diploid, wild species, Solanum bulbocastanum accession 22 (aka, SB22) (Austin et al. 1993). This race-speci c resistance was mapped onto Chromosme 11 (Chr 11) using RFLP markers and the resistance is controlled by a single dominant locus R MC1(blb) (Brown et al. 1996). This locus from S. bulbocastanum was introgressed into tetraploid potato using protoplast fusion and subsequent crossings with elite tetraploid cultivars Mojtadehi et al. 1995). The result of this introgression was an advanced tetraploid breeding selection resistant to CRKN, PA99N82-4. Recent study using differential transcriptome analysis during nematode infection in PA99N82-4 suggests that the introgressed resistance is in the form of hypersensitive response and cell death, which is a characteristic response displayed by a single-dominant resistance gene (Bali et al. 2019).
PA99N82-4 is the main source of race-speci c nematode resistance used in potato breeding program at Oregon State University. Phenotyping the breeding clones developed through controlled crosses is among the most time consuming and tedious parts of nematode resistance breeding. Phenotyping is typically performed in greenhouses or small trial plots. True potato seeds (resulting from controlled crosses) are germinated and tubers are harvested after 9-12 weeks. Those seedling tubers are grown for up to 2-weeks in one gallon pots and young plants are inoculated with freshly extracted nematode eggs, which hatch out, infect the roots, and multiply for 8-12 weeks. Subsequently, infected plants are uprooted, roots are washed thoroughly, eggs are extracted and counted to calulate the multiplication/reproduction factor. These phenotype screening assays present the most challenging step in nematode resistance breeding thus resulting in selection and inclusion of a very limited number of plants for eld trial assessment the following season. Molecular markers linked to resistance loci are promising alternative to phenotyping assays for nematode resistance screening; markers support the more e cient marker-assisted selection (MAS) and breeding processes.
The development of modern molecular biology techniques such as DNA markers have contributed immensely to the precision, accuracy and faster development of crop improvement tools and related processes (Dayteg and Tuvesson 2010). DNA ngerprinting is now integral to the plant breeding programs of major crops cultivated across the globe. It has contributed to the development of crop genetic resources while maintaining genetic diversity in germplasm resources (Nybom et al. 1991). There has been tremendous improvement to the genomic resources in potato breeding. Since the rst potato genome published in 2011 by the international Potato Genome Sequencing Consortium (PGSC, 2011), potato breeders have had ready access to genomic information for its usage in enhancing their breeding programs. SpudDB (http://solanaceae.plantbiology.msu.edu/cgi-bin/gbrowse/potato/), a potato genomic resources database maintained by Michigan State University, houses genome browsers for the PGSC double monoploid S. tuberosum group Phureja DM v4.03 pseudomolecules, as well as the updated version of pseudomolecules (v6. In the present study, we used a combined phenotyping-genotyping methodology on a pool of segregating nematode resistant and susceptible clones using high-throughput SNP markers to locate the markers linked with resistance locus R MC1(blb) on S. bulbocastanum Chr11. We rst phenotyped several advanced selections for nematode resistance in the greenhouse and selected ve each of resistant and susceptible clones for high throughput SNP-genotyping using 21K SNPs. The primary source of nematode resistance, SB22 and the immediate introgressed resistant parent PA99N82-4 were also genotyped with the same SNP array. All the markers clearly segregating with the trait and located on Chr11 were further analyzed. These validated DNA-markers can be effectively used for MAS in potato to select the nematode resistant lines for their inclusion in the early development of the program. This will dramatically reduce the time required to phenotype the individual clones to identify the promising nematode resistant clones, thus enhancing potato breeding outcomes.

Plant material
The clones used for genotyping and bulk segregation were developed and phenotyped at USDA-ARS, Prosser, WA. The population used for marker validation was developed and phenotyped at Oregon State University. Tubers were planted in the green house and fresh leaf material was collected for DNA isolation.
Nematode resistance phenotyping: All clones used in the study were rst phenotyped for nematode resistance in the greenhouse. True potato seeds from the crosses with PA99N82-4 as pistillate parent were germinated on root-initiation media; multiple cuttings were transferred to individual tissue culture tubes containing propagation media. Four-week-old tissue culture seedlings were transferred to a pasteurized mix of sand and virgin sandy loam soil in one gallon clay pots. Each plant grew for two-weeks, and was then inoculated with 5000 M. chitwoodi eggs. The pots received regular watering for 8 weeks (~ 55 days) under greenhouse conditions. Subsequently, plants were uprooted, roots were washed thoroughly under tap water and eggs were extracted by the NaOCl (hypochlorite) method (Hussey 1973). Extracted eggs were counted using 1ml counting slide to calculate a reproduction factor (R): R = nal egg density ÷ initial egg density (Mojtahedi et al. 1988) to determine whether the clone was resistant or susceptible to M. chitwoodi. Clones with lower egg densities were considered resistant.Five resistant and ve susceptible clones were used for high-throughput SNP ngerprinting along with SB22 (diploid resistance source) and PA99N82-4 (tetraploid resistant parent) ( Table 1). DNA isolation DNA isolation was performed using a slightly modi ed Dellaporta protocol described by Presting et al. 1995. Brie y, 500 mg of fresh leaf tissue was placed in an Agdia grinding bag (Agdia Inc., Elkhart, IN) with 1.5 ml of freshly prepared extraction buffer (100 mM Tris, 50 mM EDTA, 500 mM NaCl and 10 mM 2mercaptoethanol). Tissue was ground completely using a marble pestle; the slurry was collected over a mesh lter to avoid tissue debris, and placed into a 2 ml centrifuge tube to which 100 µl of 10% SDS was added. After thorough mixing, the tube contents were incubated at 65°C for 30 minutes. Subsequently, 200 µl of 5 M potassium acetate was added to the slurry and incubated on ice for 15 minutes, followed by centrifugation at 12,000 rpm for 5-6 minutes to separate the tissue debris. The clear supernatant was transferred to a fresh tube. DNA was precipitated by adding 300 µl of cold isopropanol and holding the tube on ice for up to 30 minutes. The DNA pellet was washed with 70% ethanol, dried completely and dissolved in 100 µl DNase-free ultrapure water. DNA quality and quantity were evaluated with Nanodrop (Thermo Fisher Scienti c, Waltham, MA) and stored at -20°C until use.

SNP ngerprinting
Approximately 400 ng of high quality DNA of ve nematode resistant and ve susceptible clones, resistant parent PA99N82-4 and source of nematode resistance (S. bulbocastanum accession 22 or SB22) were sent to GeneSeek (Lincoln, NE) for SNP ngerprinting using 21K SNP array. SNP data analysis was performed as described in Bali et al. 2017. Brie y, raw SNP data was analyzed by the Genome Studio-Tetraploid version and allelic data was exported as an Excel le. All the SNPs checked for quality manually in order to eliminate the monomorphic SNPs and SNPs with ≥ 10% no call rate ( Table 2).  Table 4 for primer speci c annealing temperature) for 15 seconds and 72°C for 15 seconds, with a nal extension at 72°C for 5 minutes. The PCR product was fractionated using 1.2% agarose gels run at 95V for 5 hours, stained with an ethidium bromide solution, and visualized with a BIO-RAD Universal Hood II Gel Doc system (Bio-Rad laboratories, Hercules, CA). All 49 markers were rst tested using PCR followed by agarose gel electrophoresis (PCR-AGE).

Marker assisted selection in breeding
To further validate potential markers, we used a segregating progeny (OR09007) of 96 individual clones resulting from the cross PA99N82-4 X CO98067-7RU developed by Oregon state University. A subset of 24 of these clones had been phenotyped in the green house for nematode resistance as described above. The progenies were screened using all promising SNP, SSR and INDEL markers.

Results And Discussion
Since nematode resistant potato varieties with acceptable agronomic traits are still unavailable and considering the environmental hazards associated with the use of harmful soil fumigants, there is an urgent need to expedite the breeding process. Conventional breeding for nematode resistance is a lengthy process and phenotyping of offspring for nematode resistance is the most time consuming and tedious part. Conventional breeding further limits the number of progenies tested and selected for further agronomic trials. In the past, DNA-based markers linked to nematode resistance loci have been successfully used in MAS and breeding. , these markers were not only di cult to reproduce across populations but also resulted in false ngerprints during our intial screening for nematode resistance. It may be that these markers are not tightly linked with the R MC1(blb) locus and are lost during the recombination events in advanced selections. We are seeking more advanced and robust co-dominant SNP, SSR and INDEL markers in potato, for their potential contribution to our breeding program. In the present study, we utilized high-throughput SNP genotyping of segregating phenotyped clones generated by crossing nematode resistant and susceptible parents to develop robust markers for MAS. An advanced selection, PA99N82-4, was used as the pestilate parent and is source of resistant in all crosses. Susceptible parents included popular Russet varieties such as GemStar, Alturas, Western Russet and Russet bulk (Table 1). These selections had been phenotyped for nematode resistance multiple times; those with > 5000 egg counts (more than initial inoculumn) were considered susceptible. The selections with lower egg counts were phenotyped at least four more times to con rm resistance. Five susceptible selections with high (> 10,000) egg counts and ve resistant selections with close to zero egg counts in majority of the replicates were selected and SolCAP In nium SNP array with 21K SNP markers were used to genotyping (Supplementary File 1). Affordable high-throughput SNP ngerprinting services allow us to take advantage of these resources.. All the ngerprinted samples showed high call rate (≥ 0.98) except SB22 (0.87 call rate; Table 2). The low SNP call rate in SB22 could be due to the fact that SNP markers were developed from the cultivated potato; S. bulbocastanum. being a diploid wild relative of cultivated potato is expected to posses genomic variations resulting in low SNP call rate. Of an average 20K SNPs called, 15 SNPs showed clear segregation with the nematode resistance phenotype (Table 3, Supplementary File 1). Looking further into the locations of the potential SNPs on the potato genome, four were located on Chr10, eight on Chr11 and three on Chr 01 (Table 3). We were interested in SNPs on Chr11 because the locus of R MC1(blb) is mapped on Chr11 (Brown et al. 1996). A total of eight SNPs located on Chr11 were shortlisted for further analysis. We then looked for the corresponding SNPs in S. bulbocastanum genome. This genome was sequenced by Oregon State University (Sathuvalli et. al. unpublished) and is publicly available at http://solanum.cgrb.oregonstate.edu/cgi-bin/gb2/gbrowse/solanum/. All SNPs could be located on the S. bulbocastanum scaffold 11 which corresponds to S. tuberosum Chr11 (Table 3). A ~ 6Mb region was marked with the borderline corresponding SNPs; the sequence was manually processed to identify other potential marker types including SSRs and INDELS. There are ~ 1259 SSRs and 18 INDELS in the region. We selected 15 SNP's, 30 SSR's and four INDELS for marker development and validation using a panel of 12 phenotyped nematode resistant and seven susceptible potato clones, including 10 clones that were initially genotyped for marker discovery. Most of the markers developed are highly reproducible and easy to scan using PCR-AGE and a highly advanced and faster technology such as HRM curve analysis. HRM is a relatively new technique that uses melting or dissociation of the PCR product to detect variations in DNA sequences. The differences are recorded by quanti cation of the dsDNA binding dyes during the dissociation step. HRM circumvents the requirement for gel electrophoresis in order to check the marker segregation patterns. We were especially interested in developing HRM assays for potential markers because this technique is much faster and less tedious and could be mutilplexed to scan larger number of progenies (1-96-samples in one reaction) in just 1.5 h for nematode resistance or susceptibilty. One marker, SB_MC1Chr11-PotVar0066518, can be used as a dual marker. It ampli es a ~ 120 bp product in the resistant clones and does not amplify in the susceptible clones (it requires a longer electrophoresis run). SB_MC1Chr11-PotVar0066518 clearly distinguishes the clones in melting curve analysis as two variants (resistant and susceptible; Fig. 1). In addition to SNP markers, SSRs are the markers of choice for MAS and breeding due to their high polymorphic information content, co-dominant nature and abundance in plant genomes (Gupta and Varshney 2000). Five SSR markers, SB_MC1Chr11-SSR04, SB_MC1Chr11-SSR08, SB_MC1Chr11-SSR10, SB_MC1Chr11-SSR13 and SB_MC1Chr11-SSR20 differentiated the panel using simple PCR-AGE. Three of the SSR markers, SB_MC1Chr11-SSR04, SB_MC1Chr11-SSR08, SB_MC1Chr11-SSR10 contain trinucleotide repeat motifs of (GAA) 7 , (TGT) 11 , and (TGC) 7 , respectively. One of the markers, SB_MC1Chr11-SSR13, is a dinucleotide repeat (GA) 14 whereas SB_MC1Chr11-SSR20 is a pentanucleotide repeat (GATAG) 5 . The only INDEL marker (SB_MC1Chr11-INDEL4) that could successfully differentiate the selected panel contained a 24 bp deletion. Based on their power to differentiate and ease of scoring, we recommend using a set of at least three markers SB_MC1Chr11-SSR10, SB_MC1Chr11-INDEL4 and SB_MC1Chr11-PotVar0066518 to generate reliable passport data for the progeny selection (Fig. 2). SB_MC1Chr11-SSR10 and SB_MC1Chr11-INDEL4 can be used as PCR-AGE and SB_MC1Chr11-PotVar0066518 can be used as PCR-AGE as well as HRM curve analysis (refer to Table 4 for summary of eight markers). In order to test the utility of these markers for MAS, we screened segregating progeny (OR09007) of 96 individual clones developed by crossing PA99N82-4 X CO098067-7RU. A subset of these clones (24) were phenotyped to validate the accuracy of marker segregation, which was 100% accurate with SB_MC1Chr11-SSR10, SB_MC1Chr11-INDEL4 and SB_MC1Chr11-PotVar0066518 thus validating the potential of these markers to be used for MAS (Fig. 2, Supplementary Fig. 1).

SNP1
Using modi ed resistant-susceptible genotype segregrant analysis, SNP genotyping and genome sequence mining we developed eight breeder-friendly markers linked to resistance locus for M. chitwoodi resistance in potato. Of these eight markers, a set of three markers are robust and have the potential for use in marker-assisted breeding.

Declarations
Ethics approval and consent to participate (Human Ethics, Animal Ethics or Plant Ethics): Not Applicable Consent for publication: All authors (except CB and HM, who are now deceased) have approved the manuscript for publication.
Availability of data and material: All data generated or analyzed in this study are included in the published article and supplementary les.
Con icts of interest/Competing interests: Authors declare no con ict of interest.
Funding: Funding for this work is provided by Northwest Potato Research Consortium and the USDA-ARS state partnership award.
Authors' contributions: SB and VS designed the research; SB performed all the experiments including genotyping, data analysis, marker validation, progeny screening and manuscript preparation; VS conceptualized the project, secured the funding, supervised and reviewed the manuscript; SY, LC, RQ, CB, HM, RI helped in breeding and phenotyping of the progenies. Figure 1 The potential of SB_MC1Chr11-PotVar0066518 as a dual marker. This marker can be used for PCR-AGE (longer run) and HRM (High Resolution Melt) curve analysis.