Distance Only Brings You Closer: Application of ISSR Markers to Analyze Molecular Relationships in Roses (Rosa spp.)- The Symbol of Love

Distance Only Brings You Closer: Application of ISSR Markers to Analyze Molecular Relationships in Roses (Rosa spp.)The Symbol of Love Tanay Joshi 1, , Santosh Kumar , Lalit Arya 3 and Amritbir Riar 1 ---------------------------------------------------------------------------------------------------------Abstract Genetic diversity is inevitable in making any crop improvement program successful. DNA fingerprinting technology to assess the genetic relationship among the selected genotypes for identification and cataloging of different species and cultivars of roses is a promising tool for Rosa genomes. The inter-simple sequence repeats markers (ISSRs) were used to investigate the genetic diversity among twenty-one diverse Rosa genotypes belonging to two different species, Rosa hybrida and R. damascena, and three distinct groups of rose varieties, namely Hybrid Tea, Floribunda, and Damask roses. Twenty-four ISSR primers yielded a total of 280 scorable amplified fragments from 250-1800 bp in length, from which 244 were polymorphic, resulting in an average of 86.4% polymorphism. UPGMA cluster analysis based on Jaccard’s pairwise similarity coefficient values ranged from 0.264 to 0.818, clearly distinguished different species and genotypes, grouping them into three distinct clusters. The results confirmed a high degree of variation in the rose germplasm studied highlighting the potential of improvement in roses for the ornamental and perfume industry.


Introduction
Flowers and gardens for a long have been imperative for three main considerations viz., aesthetic, economic, and social. Floriculture is identified as a fast-emerging industry with potential for domestic and export markets in India [1]. Rose is one of the most important crops in the floriculture industry [2], and is used as cut flowers, potted plants, and garden plants [3]. Roses have also been used in the food, perfumery, and cosmetics industries for many years [4][5][6] Roses belong to the genus Rosa in the Rosaceae family and are one of the most commercially cultivated ornamental plants in the world [7]. Modern rose cultivars are generally triploid or tetraploid hybrids derived from 7-10 wild diploid rose species and a few tetraploid species. The genus Rosa consists of about 200 species and thousands of cultivars that are widely distributed in Europe, Asia, the Middle East, and North America with the greatest diversity of species found in western China [8,9] . More than 150 species have already been catalogued [10,11]. The exact numbers of the rose cultivars are unknown, whereas the number is estimated at more than 20000 rose cultivars in the world [12,13]. Also, only 11 out of 200 Rosa species have contributed to the origin of modern cultivars [14].
The present-day garden roses are complex hybrids involving interspecific hybridization, polyploidy with high female and male sterility [15]. In their study, Raymond et al. [16] reported the rose whole-genome sequencing and assembly and resequencing of major genotypes that contributed to rose domestication. A wide range of variability in flower type and plant growth has been developed in the genus Rosa due to considerable advancement in rose breeding technology for the last 200 years. Unfortunately, just a small portion of this variability has been used in the present breeding.
Traits-based morphological identification and classification of the rose species and cultivars become very difficult when the genetic distance between varieties becomes smaller [17,18]. Potential of Wild rose species help widen the rose gene pool which has not been fully exploited yet. There is always demand and the need for new varieties in floriculture, and the global flower industry prospers on novelty traits such as flower color, form, and scent which are primary novelty markers in consumer choice. For the development of a new variety, the creation of genetic variability is a prerequisite. The assessment of genetic diversity is the most important for a meaningful breeding improvement program because hybrids between genetically diverse parents manifest greater heterosis than those between closely related parents.
Molecular characterization needs to be conducted to clarify the relationships between genotypes as well as for identification and genetic conservation of rose genotypes [19]. In recent years, several molecular marker systems have been developed for fast identification which is accurate and equally effective. ISSRs, a microsatellite-based technique is used for genetic characterization of different plant organisms. Evaluating genetic diversity of reproducible fingerprinting profiles with a sufficient degree of polymorphism can be achieved using ISSR markers [13,20,21].
The present study is the molecular fingerprinting analysis of the locally grown and maintained rose genotypes in North India with a unique primer set to fulfil the gap of genetic relationships among different species and cultivar types including hybrid tea, floribunda and damask roses for breeding novel rose cultivars for better use in ornamental, medicinal and aromatic industry.

DNA Fingerprinting by ISSR Markers
A total of 33 primers with different repeat motifs were used for initial screening with 3 genotypes, one each from Hybrid Tea, Floribunda, and Oil Content. Out of these, 9 primers gave no amplification at all, while only 24 primers were found to give clear and polymorphic patterns. In similar study Matsumoto et al. [22], also reported that out of 13 primers tested 7 primers generated clear and consistent banding patterns with clonal plants of Marina and rootstock. These twenty-four primers were subsequently employed to analyze the entire set of 21 rose genotypes, as given in Table 1. The amplification products of the 24 primers screened on 21 genotypes yielded a total of 280 scorable bands among these 244 were polymorphic with an average of 11.66 bands per primer in the current study (Figure 1). The size of clearly detectable amplified ISSR-PCR fragments ranged from 250 bp to 1800 bp and the number of bands generated by each primer varied. The UBC 825 primer produced the highest number of polymorphic bands (17), followed by P-21 (15 bands) and primer (P-02, P-03 & UBC 826) 14 bands each, while the lowest number of polymorphic bands (7) were obtained with the P-18 primer ( Table 2).   [25,26]. The reason for high percentage of polymorphism can be attributed to the heterozygous nature of the polyploidy genome structure of rose species. A positive correlation in percent polymorphism was observed between ISSR Primers with CA and AC repeat motif when compared to the primers with AG motifs that showed low percentages of polymorphism. These findings were contrary to the result reported in the case of Gerbera by Bhatia et al. [27] and by Namita et al. [28], in marigold where CA motifs showed low polymorphism.
Two approaches were adopted for the efficient utilization of ISSR markers in the identification of rose cultivars. In one method, an effort was made to identify the most efficient primer(s) that could individually or in combination distinguish all cultivars. For this purpose, two parameters were calculated, Resolving Power (Rp) and Marker Index (MI). These Markers showed variation in the percentage of polymorphism, band informativeness (Ib), which is a measure of closeness of a band to be present in 50% of the genotypes under assessment and resolving power (Rp) which is the sum of Ib values of all the bands amplified by a primer [29].
The average Ib ranged from 0.15 to 0.60 while Rp ranged from 2.19 to 9.52. The highest average value Ib (0.60) was shown by P-02 while P-03 revealed the highest value of Rp (9.523) as shown in Table 2. The primers with high Rp were examined for their ability to uniquely distinguish all 21 genotypes. Primer P-02 has the highest discrimination power i.e., the genotype index (GI) of    [30].

Genetic variation among rose genotypes
The binary data matrix generated by the amplified fragments of the 21 rose individuals in the ISSR-PCR analyses was used for computing Jaccard's similarity indices. From Jaccard's similarity analysis it was deduced that the values observed in the similarity matrix ranged from 0.264 between Noorjahan and Laher to 0.818 between Chaiti Gulab and Jawala. The minimum similarity was observed between genotype Noorjahan and Laher (0.264) followed by Noorjahan and Happiness (0.269) as shown reported in Figure 2. Thus, these genotypes are said to be distantly related and the cross between them may prove beneficial maybe because both of the genotypes belong to different species i.e., hybrida and damascena as well as the different type of varieties i.e., Hybrid Tea, Floribunda and Oil containing cultivars (Damask Roses).
The clustering of genotypes may be due to various reasons, first and foremost is the genotype and their parentage, the second reason may be the phenotypical difference (or) similarity between the cultivars including inflorescence type, leaf size, and type of flower color. In Cluster-I cultivar Gruss n Tepliz which belongs to species Rosa damascena has branched off from its main cluster (Cluster-III) and is grouped with cultivar Happiness. This might be because all the other oil content varieties that grouped belong to species Rosa damascene and are of Indian (desi) origin The clustering of genotypes may be due to various reasons, first and foremost is the genotype and their parentage, the second reason may be the phenotypical difference (or) similarity between the cultivars including inflorescence type, leaf size, and type of flower color. In Cluster-I cultivar Gruss n Tepliz which belongs to species Rosa damascena has branched off from its main cluster (Cluster-III) and is grouped with cultivar Happiness. This might be because all the other oil content varieties that grouped belong to species Rosa damascene and are of Indian (desi) origin whereas, Gruss n Tepliz is an exotic cultivar. All the 14 varieties that have pooled in Cluster-II  are grouped possibly because all the genotypes in this cluster belong to species Rosa hybrida, as well as all of them, are Hybrid Tea varieties and Floribunda varieties. As Abhisarika is the mutant of Kiss of fire so it has paired into a separate sub-cluster in Cluster-II as even discussed by Sasikumar et al. [31] Markedly, all the genotypes that belong to Damask roses (R. damascena) have grouped in Cluster-III as they represent the same species and have distinctly formed a separate cluster with an exception of cultivar Gruss n Tepliz which have out grouped with cultivar Happiness in Cluster-I [5]. The above results and discussion showed ISSR profiling is an effective tool for evaluating genetic variation and thus would be useful for differentiation of elite breeding lines and varieties [28]. It surfaces as an effective and reliable alternative to the conventional methods which are based on morphological markers.

Materials and Methods
For the present investigation, the experimental material comprised of 21 rose varieties (  [36], were also calculated. Computation for multivariate analysis was done using NTSYS-pc Version 2.02 software [37] and a similarity matrix was subjected to the cluster analysis of the Unweighted Paired Group Method using Arithmetic Averages (UPGMA) and a dendrogram was constructed.

Conclusion
ISSR primer P-01, P-02, P-14, P-25, UBC-824, and UBC-825 showed the highest values of percentage of polymorphism so these markers may be used for diversity analysis. Primer P-03 revealed the highest value of Rp (9.523) proves to be the best to uniquely distinguish all 21 genotypes. The minimum similarity was observed between genotype Noorjahan and Laher. These genotypes are said to be distantly related and the cross between them may prove beneficial.
Cultivar Happiness may be used to develop SCAR (Sequenced characterized amplified region) markers for this particular genotype as more than one unique band could be detected in primers P-17 & P-30. Conversion of specific ISSR segments into SCAR markers can be of help in improving the value of these markers for the identification of rose genotypes. Genetic variability observed among these genotypes would be useful for selecting parents in the crop improvement program, it will also aid in the development of promising varieties that meet the demand of ornamental, medicinal and aromatic industry.