Assessment of molecular diversity among Indian mustard [Brassica juncea (L.) Czern and coss] genotypes using SSR marker

Introduction

Indian mustard [Brassica juncea (L.) Czern and Coss], commonly known as rai or raya, belongs to the family Brassicaceae and genus Brassica. Indian mustard is a natural amphidiploid (2n = 36) derived from interspecific cross of B. campestris (2n = 20) and B. nigra (2n = 16). It is largely a self-pollinated crop (85-90%). However, owing to insects, especially honeybees, the extent of cross-pollination varies from 4.0 to 16.6 per cent (Rambhajan et al., 1991) [13] . India occupies first position in area and second in production after China. Genetic diversity of the germplasm is the major interest for the plant breeder. So, they are producing high yielding varieties which can tolerate environmental fluctuations for increasing the production and productivity of pulses. Genetic diversity can be assessed through molecular markers at the DNA level because of their abundance as compared to morphological characters and independence of environmental influences. It can give information about genetic diversity and structure of germplasms from various geographical origins (Akkale et al., 2010) [2] . In addition, they allow more accurate characterization of the genetic variations and genetic relationships. Thus, molecular markers can successfully provide an alternative tool for genetic analysis. Variations of the DNA sequences (base-pair additions, substitutions, deletions or sequence patterns) are the bases for molecular marker to detect polymorphisms. List of structural markers is long viz., RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism), Cleaved Amplified Polymorphic Sequence (CAPS), Microsatellite or Simple Sequence Repeat (SSR), Inter-SSR (ISSR), etc. SSRs are short tandem repeats of 1 to 6 bases that occur randomly and frequently in the eukaryotic genome (Tautz and Renz, 1984) [17] . SSR markers are abundant, locus specific, reproducible, hyper variable, codominant in nature and uniformly present in the nuclear and organelle genome. The SSRs markers are more advantageous compared to other marker systems. They are reproducible, co-dominant in nature, abundant, highly polymorphic and easily scorable representing multi-allelic variation (Adhikari et al., 2017) [1] . Numerous SSRs markers have been used in different Brassica species such as in B. juncea (Vinu et al., 2013; Pratap et al., 2014) [18,10] , B. napus (Li et al., 2013) [7] and B. rapa (Suwabe et al., 2002) [16] . Hence, we used SSR markers for diversity analysis.

Materials and Methods

Total genomic DNA Isolation procedure For the isolation of genomic DNA, twenty-two diverse mustard genotypes were grown in Agronomy Instructional Farm, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar. All the procedure start from the DNA isolation to PCR amplification was conducted at biotechnology laboratory of Department of Genetics and Plant Breeding, Chimanbhai Patel College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar. The isolation of genomic DNA of Indian mustard genotypes was carried out as per CTAB method of Doyle and Doyle (1987) [4] . Various stock solutions and buffers were prepared and used during isolation of genomic DNA and during gel electrophoresis. The primers used for present study (Table 1) was taken from Pratap et al. (2014) [10] , Prajapati et al. (2014) [9] , Sharma et al. (2018) [15] , Rajpoot et al. (2020) [12] , Kumar et al. (2020) [6] , Panigrahi et al. (2018) [8] .

Genomic DNA Isolation, Purification and Quantification

The fresh leaves were taken from 20-25 days old seedlings and were made to grind to a fine powder using liquid nitrogen in a pestle and mortar and were filled into 2 ml centrifuge tube. 1 ml of CTAB extraction buffer and 10μl βmercaptoethanol was added into the tubes and kept in a water bath for 45 minutes to 1 hour, where tubes are given a jerk for every 10-15 minutes interval. After incubation the tubes were centrifuged at 12000 rpm for 12 minutes at 24 0 C. The supernatant was carefully transferred to a fresh tube and an equal volume of chloroform: isoamyl alcohol (24:1) was added and again centrifuged for 12 minutes at 12000 rpm. The supernatant was transferred to a fresh tube and given a RNase A treatment and kept in water bath at 37 0 C for 45 minutes. Equal volume of chloroform: isoamyl alcohol (24:1) was added after the RNase A treatment and centrifuged at 12000rpm for 12 minutes. The supernatant was taken and equal volume of chloroform: isoamyl alcohol (24:1) was added and again centrifuged at 12000 rpm for 12 minutes. After centrifugation the supernatant was transferred to 1.5 ml centrifuge tube and 0.6 volume of chilled isopropanol was added and stored overnight at-20 0 C for better precipitation of DNA. Next day morning, the fine pellets of DNA will be obtained after it has been given a centrifugation at 4 0 C for 10 minutes at 10000 rpm. The pellets obtained was washed with 70% ethanol, twice and kept in the oven for the evaporation of ethanol. Later, the pellets were made to dissolve in 100 μl of 1X TE buffer by tapping on the tubes and stored at-20 0 C. The quantification of nucleic acid was performed using UV spectrophotometer. The good quality DNA will have the O.D. in the range of 1.8-2.0. The O.D. below 1.8 or 1.7 indicates protein contamination and O.D. above 2.0 will be indicating contamination of RNA. The 0.8% agarose gel run will show bands with less shearing indicates good quality and amount of DNA.

PCR Amplification

For the PCR based amplification total thirty number of SSR primers were used for the validation. PCR reactions were performed in the reaction volume of 15 μl containing 1.5μl of Taq buffer (10X) with MgCl2, 0.5 μl dNTP mix (10 mM), 1.0 μl of each primer (5 p mol), 0.1 μl of Taq DNA polymerase and 1.0 μl of genomic template DNA (50ng/ml).

Results and Discussion

Total 30 SSR primers were used for screening of twentytwo genotypes for the assessment of molecular diversity. Out of 30 SSR primers, 10 primer pairs were found to be polymorphic, 12 primers found monomorphic while rest of the primers not amplified properly among the mustard genotypes. The results obtained using 10 polymorphic primers and 12 monomorphic primers out of total 30 have been presented in Table 2. The SSR amplification profiles obtained in the present study with primers BR-A01, BR-A03, BR-A05, BRMS-08, BRMS-37, BRMS-240, SR-9222, SR-94102, BG-41 and BG-48 were found polymorphic. The results obtained using 10 polymorphic primers have been presented in Table 2 and result is shown in Plate-1 To 10. Number of alleles per locus was found to be 2 alleles. Previous studies by Rajpoot et al. (2020) [12] found an average of 3.86 alleles per locus which was higher than present study. The band size of primers BG-41 and BG-48 used in present genetic diversity study in mustard was in accordance with molecular size of 160-260 bp as reported by Neeraj et al. (2020) [6] , the band size of BRMS-37 was in accordance with 137-176 bp as reported by Prajapati et al.

(2014) [9] whereas, the band size of BRMS-017 was in accordance with 200-350 bp as reported by Prajapati et al.

(2014) [9] . Based on the simple sequence repeat data, cluster analysis was performed by using genetic similarity values and a dendrogram (Fig. 1) was generated showing the genetic relationships among twenty-two genotypes of mustard. The dendrogram was constructing based on UPGMA (un weighted pair-group method with arithmetic mean) by using Jaccard's similarity coefficient through NTSYSpc (version 2.0) software. Jaccards's similarity coefficient values for 22 genotypes ranged from 0.60 to 1.00 with average of 0.88 indicating moderate level of genetic variation existing among the mustard genotypes used in this study. Kumar et al. (2020) [6] also reported Similarity coefficient average of 0.72 among 30 cultivars. Clustering pattern of dendrogram generated by pooled SSR data of twenty-two genotypes by using 22 primer pairs constructed using UPGMA showed two major clusters A and B formed at a similarity coefficient of 0.93 (Fig: 1). The similarity coefficient was ranged from 0.86-1.00 which is an indicative of presence of moderate variation among genotypes. Relationships between the mustard cultivars were graphically represented in the form of dendrogram. These results indicated that this genotypes GMMo2122A, GMMo2130A, GMMo2128A, GMMo2102A were found to be the most diverse type that the genetically divergent parents could be chosen which indicate adequate polymorphism. The knowledge of genetic relationships among the genotypes provides useful information to mark breeding programs and germplasm resource management (Roldan-Ruiz et al., 2001) [14] . The present investigation suggested that SSR markers are suitable to evaluate genetic diversity in mustard and suitable to individually distinguish all the twenty-two genotypes of mustard. Molecular data must be useful for exposing DNA patterns especial for a given genotype or set of genotypes. Overall, it was concluded that the genetic diversity derived in this investigation might be useful in future mustard improvement programmes and selection in mustard crop should be based on the phenotypic characters as well as based on the molecular markers analysis. Therefore, studies on morphological and molecular markers are completely useful in analysis the genetic diversity in mustard. Normally, it can be observed that the combination of microsatellites and morphological data can be highly informative so that they could be effective and accurate tools for recognizing genetic diversity in mustard germplasm. The assessment of genetic divergence and relatedness among breeding materials has significant implications for the improvement of crop plants (Chandra et al., 2013) [3] . GMMo2103A, GMMo2105A, GMMo2106A, GMMo2108A, GMMo2109A, GMMo2111A, A A2 GMMo2113A, GMMo2114A, GMMo2116A, GMMo2119A, GMMo2121A, GMMo2123A, GMMo2124A, GMMo2125A, GMMo2127A, 16 GMMo2131A A2:2 GMMo2118A, GMMo2129A 02 B GMMo2122A, GMMo2130A

GM1A-GMMo2102A, GM2A-GMMo2103A, GM3A-GMMo2105A, SKM109A-GMMo2106A, SKM 125A-GMMo2108A, SKM 139A-GMMo2109A, SKM 149A-GMMo2111A, SKM 201A-GMMo2113A, SKM 214A-GMMo2114A, SKM 301A-GMMo2116A, SKM 303A-GMMo2118A, SKM 319A-GMMo2119A, SKM 9928A-GMMo2121A, RSK 88A-GMMo2122A, RSK 89A-GMMo2123A, RSK 90A-GMMo2124A, RSK 91A-GMMo2125A, RSK 92A-GMMo2127A, RSK 93A-GMMo2128A, RSK 94A-GMMo2129A, RSK 95A-GMMo2130A, RSK 96A-GMMo2131A

Acknowledgement

AcknowledgementSpecial thanks to Oilseeds Research, Sardarkrushinagar for giving material and C. P. College of agriculture along with Directorate of research, S.D.A.U. for giving research facility.

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