SSR polymorphism
Of the total 215 SSR markers employed for polymorphism survey among the hybrids and their parents, 60 markers conveyed a polymorphism percent of 27.9%. The list of polymorphic SSR markers and their details is provided as supplementary table 1. The higher polymorphism percent observed in the present study might be due to inclusion of different species of cotton in the study. In our earlier diversity studies, we observed SSR polymorphism of 16% and 27% in G. arboreum (Santosh et al. 2020) and tetraploid cotton (Santhy et al. 2019), respectively. Earlier, Selvakumar et al. (2010) observed 30% polymorphism during genetic purity analysis of three cotton hybrids using SSR markers. Menka et al. (2016) noted 20% polymorphism while studying hybrid purity in two cotton hybrids. Marker polymorphism depends on many factors such as breeding behaviour of the species, genetic diversity in the study material, sample size, sensitivity of genotyping method and location of primers in the genome used for study. The information conveyed by the polymorphic SSR markers was utilized to assess the molecular divergence among the study material.
Genetic dissimilarity
The genetic dissimilarity among the genotypes under study is presented as Table 2. Maximum genetic dissimilarity of 0.66 was noted between inter-specific H×B hybrid, Phule 388 with G. arboreum line LD 327 (Sel.), and, between G. hirsutum line RHC-006 with G. arboreum line DS 5 (GMS). The dissimilarity of 0.65 was observed between inter-specific H×B hybrid, Phule 388 with intra-arboreum hybrid, CICR2 and its male parent DS 5 (GMS), between G. hirsutum line CSH 43 with G. arboreum line LD 327 (Sel.), between G. hirsutum line RHC-006 and G. arboreum line LD 327 (Sel.). The lesser genetic diversity was observed between the hybrids and its parents. The minimum genetic dissimilarity of 0.07 was observed between intra-hirsutum hybrid, CSHH1862 with its female parent CB 33, followed by 0.11 between intra-arboreum hybrid, CICR2 with its male parent DS 5 (GMS). The genetic similarity of 0.85 was noted between intra-hirsutum hybrid, CSHH243 with its male parent CSH 2013, and between intra-hirsutum hybrid, CSHH1862 with its female parent GMS 16A, and between GMS 16A with CB 33. The SSR markers were highly efficient in capturing both intra-species and inter-species diversity (Abd El-Moghny et al. 2017; Santhy et al. 2019; Santosh et al. 2020) as they revealed higher genetic diversity between different species and lesser diversity within species or between hybrids and their parents.
Table 2
Genetic dissimilarity among the genotypes under study
Dissimilarity co-efficient | LD 327 (Sel.) | DS 5 (GMS) | CICR2 | CSH 8 | CSH 19 | CSHH198 | PIL 8 (Sel.) | SH 2379 9Y | CSHH238 | CSH 43 | CSH 2013 | CSHH243 | CB 33 | GMS 16A | CSHH1862 | RHCb-001 | RHC-006 |
DS 5 (GMS) | 0.25 | | | | | | | | | | | | | | | | |
CICR2 | 0.17 | 0.11 | | | | | | | | | | | | | | | |
CSH 8 | 0.61 | 0.59 | 0.58 | | | | | | | | | | | | | | |
CSH 19 | 0.64 | 0.63 | 0.61 | 0.40 | | | | | | | | | | | | | |
CSHH198 | 0.61 | 0.59 | 0.57 | 0.24 | 0.18 | | | | | | | | | | | | |
PIL 8 (Sel.) | 0.59 | 0.57 | 0.56 | 0.28 | 0.44 | 0.32 | | | | | | | | | | | |
SH 2379 9Y | 0.64 | 0.62 | 0.62 | 0.45 | 0.20 | 0.27 | 0.42 | | | | | | | | | | |
CSHH238 | 0.60 | 0.58 | 0.56 | 0.31 | 0.29 | 0.18 | 0.26 | 0.22 | | | | | | | | | |
CSH 43 | 0.65 | 0.64 | 0.62 | 0.35 | 0.45 | 0.31 | 0.20 | 0.45 | 0.27 | | | | | | | | |
CSH 2013 | 0.62 | 0.61 | 0.59 | 0.29 | 0.29 | 0.26 | 0.33 | 0.29 | 0.25 | 0.33 | | | | | | | |
CSHH243 | 0.61 | 0.58 | 0.57 | 0.26 | 0.36 | 0.21 | 0.24 | 0.34 | 0.18 | 0.18 | 0.15 | | | | | | |
CB 33 | 0.58 | 0.57 | 0.55 | 0.30 | 0.32 | 0.19 | 0.27 | 0.36 | 0.24 | 0.29 | 0.30 | 0.22 | | | | | |
GMS 16A | 0.57 | 0.58 | 0.54 | 0.30 | 0.37 | 0.28 | 0.32 | 0.40 | 0.32 | 0.35 | 0.36 | 0.31 | 0.15 | | | | |
CSHH1862 | 0.55 | 0.53 | 0.51 | 0.31 | 0.34 | 0.23 | 0.28 | 0.38 | 0.22 | 0.31 | 0.32 | 0.26 | 0.07 | 0.15 | | | |
RHCb-001 | 0.63 | 0.63 | 0.63 | 0.54 | 0.55 | 0.52 | 0.55 | 0.57 | 0.55 | 0.59 | 0.55 | 0.54 | 0.55 | 0.55 | 0.55 | | |
RHC-006 | 0.65 | 0.66 | 0.63 | 0.37 | 0.40 | 0.34 | 0.39 | 0.45 | 0.37 | 0.45 | 0.31 | 0.36 | 0.39 | 0.46 | 0.40 | 0.54 | |
Phule 388 | 0.66 | 0.65 | 0.65 | 0.46 | 0.53 | 0.43 | 0.50 | 0.55 | 0.48 | 0.53 | 0.50 | 0.47 | 0.50 | 0.53 | 0.51 | 0.18 | 0.39 |
Clustering and factorial analysis
The information on genetic dissimilarity among the genotypes was utilized for clustering and factorial analysis. Both clustering based on unweighted Neighbour Joining (Fig. 1) and factorial analysis (Fig. 2) depicted pattern of genetic diversity and the grouping of genotypes was in congruence with the ploidy of the species. The diploid species (G. arboreum) hybrid, CICR2 along with their parents [DS 5 (GMS) and LD 327 (Sel.)] were clustered separately and distinctly from the rest of the genotypes. All the hirsutum genotypes (Hybrids CSHH198, CSHH238, CSHH243, CSHH1862 and their respective parents were also found closely clustered. Similarly, the inter-specific hybrid, Phule 388 along with its G. hirsutum (RHC-006) and G. barbadense (RHCb-001) parent formed a distinct group. Factorial coordinate analysis provides overall representation of diversity while, clustering tends to faithfully represent the individual relations (Santosh et al. 2017). Clustering based on molecular makers revealed that particular hybrid and its parents were grouped together as cluster and hybrid was positioned in a near midway between its two parents (Rana et al. 2006; Chauhan et al. 2016). A similar pattern of distinct grouping was observed for the hybrids Phule 388, CSHH1862 and CICR2.
Confirmation of genetic purity
Out of the 215 SSR markets surveyed, 60 were observed as polymorphic among the material included in the present study. Polymorphic markers which clearly differentiated the male and female parent of each of the hybrid were identified from the 60 polymorphic markers. The genetic purity was confirmed in each of the hybrid using identified markers that differentiated male and female parents of each hybrid by clear, scorable and unambiguous amplified fragments. The markers producing multiple bands with heterozygosity were excluded for genetic purity analysis. Microsatellite markers in cotton are known to reveal multiple banding patterns per locus (Rudmann-Maurer et al. 2007; Rana et al. 2006; Selvakumar et al. 2010; Chauhan et al. 2016), which may be the result of polyploidy or amplification of repetitive sequences or due to pollen contamination.
The markers GH486, BNL1421, BNL3594 and JESPR151 differentiated the parents (CSH19 and CSH8) of G. hirsutum hybrid, CSHH198 and confirmed the genetic purity of the hybrid by producing alleles from both the parents (Fig. 3). The SSR markers viz., GH486, BNL2449, JESPR151 and TMB0436 produced parent-specific alleles in the SH2379-9Y and PIL8 Sel. and hybridity was confirmed in G. hirsutum hybrid, CSHH238 by producing both the parental alleles (Fig. 4). The parents (CSH2013 and CSH43) of G. hirsutum hybrid, CSHH243 produced genotype specific alleles for the markers BNL2449, JESPR151 and JESPR152 (Fig. 5). These markers produced heterozygous bands specific to male and female parents of the hybrid, thus confirming the hybrid purity. The parents of inter-specific hybrid, Phule 388 were found to be homozygous for different alleles of GH527, BNL3812, TMB1484, TMB1645, NAU1190 and BNL3816 (Fig. 6). The hybrid produced both G. hirsutum and G. barbadense parent specific alleles for each of these markers, thus confirming genetic purity of the hybrid. Markers distinctly differentiating the parents of intra-arboreum hybrid, CICR2 and intra-hirsutum hybrid CSHH1862 and also unambiguously confirming the genetic purity of these hybrids were not observed in the study. SSR markers are known for their efficiency in genetic purity analysis and were utilized for genetic purity testing of different cotton hybrids (Rana et al. 2006; Selvakumar et al. 2010; Rao et al. 2015; Chauhan et al. 2016; Menka et al. 2016).
Phenotyping based on morphological traits is very important as they represent the expressed part of the genome. Since, most of these morphological traits are quantitative in inheritance and environmentally influenced, more often, there exists a risk of categorising genetically different cultivars as similar or vice-versa owing to subjective assessment (Santhy and Meshram, 2015). The SSR markers can be used in efficient analysis of hybrid seed purity since this technique is simple to use, more accurate and not affected by environment when compared with GOT. Moreover, SSR based clustering is known to have better correlation with the pedigree than the dendrogram from morphological data (Giancola et al. 2002). Pattanaik et al. (2018) carried out the comparison of traditional grow-out test and DNA-based PCR assay to estimate F1 hybrid purity in cauliflower and proposed that molecular marker-based hybrid purity assessment may serve as an effective substitute to traditional GOT. A combination of SSR markers and morphological descriptors are proposed for comprehensive and unambiguous cultivar identification and differentiation (Santhy et al. 2019; Santosh et al. 2020). The present study has identified polymorphic SSR markers which can be used in hybrid purity testing. The information generated in the study about genetic diversity and genetic purity testing will greatly facilitate seed production of these cotton hybrids. The polymorphic SSR markers identified in the study will facilitate their robust identification and thus, their licensing and commercialization.