Identification and Characterization of Simple Sequence Repeats (SSR) Markers Associated with Downy Mildew Resistance Locus “ RPV1 ” in Grapes

The cultivation of grapes is severely impacted by the emergence of downy mildew (DM) disease which negatively affects quality and yield possibly re-sulting in heavy losses. Due to certain shortcomings in the usage of fungicides and the development of new cultivars by plant breeding, marker assisted selection (MAS) will be an efficient alternative method to introduce desired genes into the cultivated varieties in a short time period. The Simple sequence repeats (SSR) markers seem to be the most popular genetic marker of choice for MAS. In the present study, we identified 14 new SSR markers in RPV1 locus that are associated with downy mildew resistance in grapes. The characterization of the identified markers was carried out on the basis of various parameters such as types of repeat motifs, number of repeats, different classes and structure of microsatellites. Additionally, SSR genotyping in 56 different grape accessions was done to determine the susceptibility or resistance of these accessions to DM.


Introduction
Grapes are one of the world's most widely cultivated horticultural crops, providing enormous nutritious products such as drinks, jellies and jams. Many fun-How to cite this paper: Goyal, N., Nag, A., Samarth, R.R., Upadhyay, A. and Singh, K. The air quality decreases in grape growing areas, persistence of chemical residues may occur in grape products, and the development of fungicides resistant fungal strains can take place [4]. During the 19th century, the management of fungal diseases by genetic resistance was the paramount goal of the scientific community in grape breeding. Though the V. vinifera cultivars were found to be susceptible to P. viticola, many North American and Asian Vitis species such as Muscandinia rotundifolia, V. riparia, V. cinerea, V. labrusca, V. rupestris, V. berlandieri, V. lincecumii, show variable resistance levels to this pathogen [5]- [12].
The genetic basis of resistance to downy mildew was therefore examined and mapped in various genetic backgrounds.
However, the breeding of new grape varieties is quite resource-intensive and time-consuming; therefore, an alternate method of Marker Assisted Selection (MAS) may also be used efficiently to integrate desirable genes into cultivated varieties. In grapes, considerable advances have been made in the production of MAS tools in recent times. A huge number of DNA sequence based markers such as amplified fragment length polymorphism (AFLP), diversity arrays technology markers (DArT), restriction fragment length polymorphism (RFLP), single nucleotide polymorphism (SNP), and simple sequence repeats (SSRs) associated with the gene of interest were created previously. Such markers can be used to generate genetic linkage maps to identify genomic regions that are linked  [20].
Amongst several, the most widely used marker type is simple sequence repeats (SSR) markers, also called microsatellites, short tandem repeats (STR), and simple sequence length polymorphisms (SSLP) [21] [22]. These are the short, repetitive nucleotide motifs of 1 to 6 base pairs dispersed across most eukaryotic species' genomes [23]. Litt and Luty first provided the term "microsatellite" in 1989 to describe the simple sequence stretches amplified by PCR [24]. They can be used for multiple purposes, such as genetic map construction, genetic variation study, phylogenetic studies to create evolutionary trees, and MAS [25].

Marker Identification and Primer Designing
To identify SSR markers linked to RPV1 locus, the "FASTA" file of the above sequence was submitted to "WebSat" online program (https://bioinfo.inf.ufg.br/websat/) [28]. For analysis, the criteria for marker motif length was di to hexa nucleotide repeat motif; minimum number of 4 motif repetitions was chosen for di-, tri-, and tetra-nucleotide repeats; minimum of 2 motif repetitions was chosen for penta-and hexa-nucleotide repeats; mononucleotides were excluded from the analysis. Its means that for di-nucleotide repeat motifs, the minimum repeat number was kept as 4 and maximum was 12.
Likewise, the same criteria was followed for other types of repeats. Next, the SSR finder program processed the sequence and highlighted several regions which contained SSR markers in an output. Next, the BLASTX of the regions having SSR markers as obtained from "Websat" was performed to find blast homology with TIR-NB-LRR type resistant proteins so as to make it easier for further screening on the basis of R proteins. Consequently, such sequences were identified that showed the highest identity with TIR-NB-LRR type resistant proteins.
Next, the BLASTX of the regions with SSR markers as obtained from "Websat" was performed to find blast homology with TIR-NB-LRR type resistant proteins.

SSR Analysis
The characterization of SSRs was done to test the specificity of SSRs in grapes.
Various SSR characteristics analyzed were types of SSR motifs, number of repeats, different classes and structure of microsatellites.

Plant Material and DNA Extraction
The leaf tissues of different grape accessions were collected from National Research Centre for Grapes (NRCG), Pune (Table S1). Total genomic DNA was isolated from the leaf samples obtained and the protocol given by Dellaporta et al., 1983 was followed [29]. The quality and integrity of the extracted DNA was checked on 0.8% agarose gel. The quantification and purity of DNA were assessed on a nano-spectrophotometer (Eppendorf, USA). The purity of DNA sample was determined by measuring the absorbance at 260 nm and 280 nm.

Primer Validation
For the validation of effective SSR primers associated with downy mildew resistance, the PCR amplifications were done using a single V. vinifera accession ter. The forward and reverse primers that were added to master-mix are given in Table S2. All components were mixed well and PCR was performed (Bio-Rad T100 TM Thermal Cycler) with PCR conditions mentioned in Table S2 for RPV1 associated markers. PCR products were analysed on 2.5% agarose gel.

Identification of SSR Markers Associated with Downy Mildew Resistance
Simple sequence repeats (SSRs) are 1 -6 bp long, tandem-repeated genomic segments that prevail in both coding and non-coding regions [30]. They are extensively used in molecular genetics and plant breeding areas due to their abundance, multi-allelism, co-dominance, ubiquity and variation etc. [31]. As SSR markers and QTLs are known to be associated with each other, the RPV1 locus of grape genome was examined to identify highly polymorphic SSR markers.

Sequence Retrieval
The RPV1 locus resides on chromosome 12 of grape genome. As identified in literature, the total region of RPV1 locus detected is upto 7 Mb from 13. 10 -20.37 Mb between the markers VMC4f3-1 and VMC8g9 in Muscandinia rotundifolia [27]. The region between 16.752623 and 16.755849 Mb on chromosome 12 is found to be coding for RGA8; a full length TIR-NBS-LRR gene (also called RPV1 gene) [27]. We have analyzed the genomic sequence of this region for the identification of SSR markers. The locus ID for this sequence is JQ904634 and the total length of sequence analyzed is up to 14,677 bp.

Marker Identification and Primer Designing
For analysis, the genomic sequence was retrieved from NCBI and analyzed at "Websat" to identify various SSR markers. Initially, a large number of sequences with a repeat number of 2 to 6 for penta-and hexa-nucleotide motifs; repeat number of 4 to 6 for di-, tri-and tetra-nucleotide motifs were generated. Then, after screening for the presence of TIR, NBS and LRR domains in SSR marker containing sequences, a total of 14 such sequences were identified that shared blast homology with TIR-NBS-LRR domains and consisted of SSR markers.
Thus, in total, 14 SSR markers associated with DM resistance were identified.
Also, as the SSRs were identified in gene coding region, they are fewer in number. The primers were designed by using the same software. The primer name, marker category and repeat sequence are given in Table 1. Because of their wide use in multiple applications such as genotyping, mapping and breeding, the development of SSR markers in grapes was also done previously [32] [33] [34]. In the present study, we have focused on the identification of SSR markers associated with downy mildew resistant locus "RPV1" in grapes.

Characterization of SSRs
The region of RPV1 locus analyzed was genomic sequence consisting of both introns and exons. However, the SSR markers were developed after confirming through BLASTX for the presence of TIR-NBS-LRR domains, therefore we can say that the markers are developed from the exonic regions, thus the markers are EST-SSRs (Expressed sequenced tagged microsatellites) or Genic. Such types of markers (EST-SSRs) have also been detected previously in several plants, such as  [38]. The primers were also designed from the coding regions, hence the primers designed were exonic and were designed to amplify the coding region of the locus.
The motif characterization was done on the basis of repeat motif type and number of repeats. The repeat motif for microsatellites can be mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide repeats [39]. Through analysis, a total of 1 di-; 2 tri-; 1 tetra-; 5 penta-and 5 hexa-nucleotide repeat motifs were identified in 14 SSR markers. In plants, the most commonly found microsatellites are di-nucleotide motifs consisting of (GT) n and (AT) n repeats [40] [41]. However, the di-nucleotide repeat motif obtained in our study is (TC) n repeats. The occurrence of tri-nucleotides and tetra-nucleotides in plant genomes is also reported, the commonly observed are (AAG) n and (AAT) n [30]. We also got one (AAT) n tri-nucleotide motif in our analysis. The frequency of occurrence of different types of repeat motifs is penta-/hexa-> tri-> di-/tetra-nucleotide repeat motifs (Figure 1). Penta-and hexa-nucleotide repeat motifs were present in maximum number, whereas di-and tetra-nucleotide repeat motifs were found in minimum number (Figure 1). The advantage of getting higher number of penta-/hexa nucleotide repeat motifs is that there are lesser chances of getting mutations due to longer repeat length, thus the gene expression will not be compromised. As the resistant locus analyzed in this study is known for having resistance (R) genes that provide resistance, therefore it is necessary that the gene frame should not be altered because  that might disrupt the gene function. Therefore, it is advantageous that we got higher number of penta-/hexa nucleotide repeat motifs as compared to di-, triand tetra-nucleotide repeat motifs. There are lesser chances of getting mutations due to longer repeat length, thus the gene expression would not be compromised. Next, the repeat number for SSR markers was analyzed and it was observed that the repeat number varied from 2 to 6 ( Figure 2). The maximum repeat number of 6 was found in one SSR marker consisting of di-nucleotide repeat motif; the repeat number of 5 was also found in one SSR marker consisting of tri-nucleotide repeat motif; the repeat number of 4 was found in two SSR markers consisting of tri-and tetra-nucleotide repeat motifs and the repeat number of 2 was found in ten SSR markers consisting of penta-and hexa-nucleotide repeat motifs ( Figure 2). The reason for the differences in number of repeats of a particular motif at a locus in different individuals is recombination errors, DNA strand slippage during replication, mismatches, and retro-transposition [42]. Next, the SSR markers were grouped into different classes depending upon the length of repeat motifs. Generally, if the length of SSR repeat is 20 bp or more, they are considered Class 1 repeats; between 12 -20 bps-Class 2 repeats; and if the length is less than 12-Class 3 repeats. In our analysis, we found 7 SSR markers to be between 12 -20 bps; hence they are considered as Class 2 repeats. Likewise, in class 3 repeats category also, we got 7 SSR markers. In class 1 category, no SSR marker was detected.  (dC-dA) n (dG-dT) n [19]. The structure of all the SSR markers designed in our study consisted of only 1 type of repetitive unit, hence they are called simple repeats.

SSR Genotyping
Microsatellites are the widely used genetic markers in plant breeding programs due to their properties such as co-dominant markers, wide distribution, high polymorphism levels, and high abundance in genome [21] [43]. Polymorphism detection is done after the amplification of SSR markers through PCR accompanied by interpretation and evaluation of band patterns observed after agarose gel electrophoresis [21] [44].

DNA Isolation
Total genomic DNA was isolated from leaf tissues of 56 different V. vinifera accessions collected from NRCG, Pune by using the method described by Dellaporta et al., 1983 [29]. The absorbance ratio of 260/280 indicated that the DNA was relatively free of proteins and contaminants. The reason for choosing these varieties is that the identification of RPV loci has previously been done in American and Central Asian varieties only, we checked their presence in Indian cultivars [5]- [12]. Presently, there is no commercial table grape variety in India that is resistant to downy mildew, therefore, it is necessary to screen as much grape germplasm as possible to identify the resistant traits in different grape varieties.

Screening of SSR Primers for Downy Mildew Resistance
For the screening of SSR primers associated with downy mildew resistance, the PCR amplification of SSR markers was performed in "Perlette" variety of V. vinifera and analysed on 2.5% agarose gel. This variety was chosen because of its easy availability in our region. "Perlette" was primarily obtained in California in  Table 2). The maximum band size obtained was 390 bp for 3 markers, and minimum band size was 200 bp for 1 marker (Table   2).

Amplification of SSR Markers Associated with RPV1 Locus in Different Genotypes
After validation of SSR markers amplification at the specific annealing temperature, amplifications were done in DNA isolated from 56 different V. vinifera accessions. The amplification of these polymorphic markers at a particular band size indicates whether that variety is susceptible or resistant to downy mildew.
Out of 12, 6 SSR markers namely ATT, TCT, GTAAT, AGGAG, TTCTTT, AGAGGG showed amplifications. The genomic DNA of Muscandinia rotundifolia was also used in the PCRs as a positive control depicting the standard marker allele size associated with resistance. This species of "Vitis" has co-evolved with "Plasmopara viticola" in North America; therefore, it tolerates/resists the infection caused by DM [45].

Data Analysis
All SSR fragments were screened manually and converted to "1" and "0" binary digits for the presence of band and absence of band ( Table 3 Table 3. Chart showing the scoring of RPV1 locus associated SSR markers in binary digit format ("1" for presence of band and "0" for absence of band). "0" indicated the opposite trend of presence/absence of SSR markers with respect to positive control having value "1".

Conclusion
In the present study, we identified and characterized various SSR markers in RPV1 locus that are known to be associated with downy mildew resistance in grapes. Their genotyping was done to detect polymorphisms in grape germplasm using Muscandinia rotundifolia (