MOLECULAR DIVERSITY ANALYSIS OF SOME SELECTED BRRI DEVELOPED RICE VARIETIES USING SSR MARKERS

Molecular characterization of 26 modern rice varieties of Bangladesh was performed using 52 simple sequence repeat markers (SSRs) to estimate the genetic diversity and to reveal genetic relationships among rice cultivars. A total of 156 alleles were detected where the number of alleles per locus generated by each marker varied from 2 to 7, with an average of 3. The band size for a given microsatellite locus ranged from 79 to 278 bp. The polymorphism information content (PIC) for the SSR loci ranged from 0.08 to 0.79, with an average of 0.35. RM566 was the best marker for identification and variability assessment of varieties as revealed by PIC values. A UPGMA dendrogram generated using the NTSYS-pc revealed four clusters with a similarity coefficient of 0.55, whereas phylogenetic cluster analysis of the SSR data based on Nei-genetic distance divided the varieties into three groups. Two-and three-dimensional graphical views of principal coordinate analysis showed the spatial distribution of the varieties and revealed that BRRI dhan56, BRRI dhan51, BRRI dhan52, BRRI dhan61, BRRI dhan62 and BRRI dhan66 were found far away from the centroid of the cluster. The findings of this study are helpful for varietal identification, background selection during backcross breeding and for selecting the suitable genetically diverse parents for crossing programs.


INTRODUCTION
Rice is the first major grain crop and is the main source of energy of Bangladeshi people.It contributes about 46% of the protein and 62% of the calories to the average person's daily diet (HIES, 2010).Additionally, it guarantees the nation's political stability and gives its citizens a sense of food security.Bangladesh Rice Research Institute (BRRI) has released 113 high yielding rice varieties including eight hybrid varieties that have high yield potential, several biotic and abiotic stress tolerances and premium quality for varied rice ecosystems around the country (http://www.brri.gov.bd).These varieties are cultivated in about 80 percent of the total rice areas and contribute almost 91 percent of total rice production of the country (BRRI, 2022).As a result, the gap between technology and farmer has been bridged, allowing Bangladesh to finally become rice self-sufficient.
In Bangladesh, variety identification and DNA fingerprinting of rice through SSR marker have been started few years back but they are very discrete and fragmentary.DNA fingerprinting at the molecular level is crucial to safeguarding rice cultivars against biopiracy.Till 2013, Biotechnology division of BRRI accomplished DNA fingerprinting of BR1 to BRRI dhan-50 rice varieties.In 2016/17, the Genetic Resources and Seed Division (GRSD) conducted DNA fingerprinting ranging from BRRI dhan-51 to BRRI dhan-73, and three hybrid varieties.However, assessment of genetic diversity and molecular characterization among elite rice varieties of BRRI is important for varietal identification.The use of DNA markers has been suggested for precise and reliable characterization and discrimination of rice genotypes (Khalequzzaman et al., 2022).For genetic variability assessment, DNA markers are extensively used, because these are not affected by environmental factors.Microsatellites (SSRs) are the marker of choice because of their advantages over other markers.The SSRs are most suitable for rice because of their reproducibility, multiallelic nature, hypervariability, codominant inheritance, relative abundance, and genome-wide coverage (Powell et al., 1996;Das et al., 2013).The SSR markers are particularly suitable for evaluating genetic diversity and relationships among plant species, populations, or individuals (Choudhury et al., 2013;Roy et al., 2016), studying morpho-molecular divergence of restorer lines for hybrid rice (Islam et al., 2019); marker-assisted selection breeding (Rani and Adilakshmi 2011); cultivar identification; hybrid purity analysis and gene mapping studies (Sarao et al., 2010).
Information regarding genetic variability at molecular level could be used to help, identify and develop genetically unique germplasm that complements existing cultivars (Rahman et al., 2012;Babu et al., 2014).Diversity of modern BRRI rice varieties of Bangladesh has great importance for food security that seeks to assess the genetic diversity among these varieties.The study was, therefore, conducted to evaluate genetic variation among some modern BRRI developed rice varieties at DNA level using SSR markers.The objectives of this research were to (i) assess the genetic variation and diversity of 26 elite BRRI rice varieties, (ii) determine the genetic relationships among these varieties for breeding purposes, and (iii) characterize these rice varieties at molecular level.

Plant materials and genomic DNA extraction
The experiment was conducted in the Molecular Laboratory of Genetic Resources and Seed Division (GRSD) of BRRI during 2016-2017.Twenty-six modern rice varieties developed by BRRI were used in this investigation as shown in Table 1.Seeds were germinated in germination chamber at 30°C temperature.Three days after germination, germinated seeds were sown in earthen pots.The pots were then kept in a net house.Genomic DNA was extracted from young leaves of 20-day-old seedlings following the Mini scale method described by Zheng et al. (1995).

SSR Markers and PCR amplification
From the prior rice research (McCouch et al., 2002;Islam et al., 2018a), sixty pairs of primers were used, with some of them being chosen at random.Detailed information of the primers is obtained from the website (www.gramene.org/markers/microsat).
Markers that showed monomorphic banding patterns was excluded.Finally, 52 polymorphic SSR markers were selected for diversity analysis.The total PCR reaction was performed using the procedure established by Islam et al. (2018b).

Electrophoresis and visualization of amplified products
The 10 μL of each PCR product with 2 μL of a loading dye was analysed using 8% polyacrylamide gel electrophoresis in 1 × TBE buffer at 75 V for about 2.0-2.5 h depending upon the allele size.The gels were stained with ethidium bromide solution (0.5 mg/mL) for 25 min and exposed to UV light using the molecular Imager gel documentation unit (XR System, Uvitec Cambridge, France) for visualization.

Data analysis
The size of the band for each marker was calculated by AlphaEaseFC-4.0 software.The summary statistics, including the number of alleles, major allele size and frequency, gene diversity and polymorphism information content (PIC) value were determined using Power Marker version 3.25 (Liu and Muse, 2005).Binary format (allele presence= "1" and allele absence = "0") for allele frequency was prepared using Power Marker software and used for dendrogram construction by NTSYS-pc version 2.2 (Rohlf, 2002).A similarity matrix was calculated with the SimQual subprogram using the Dice coefficient, followed by cluster analysis with the SAHN subprogram using the UPGMA (unweighted pair group method using arithmetic mean) clustering method as implemented in NTSYS-pc.The similarity matrix was also used for principal coordinate analysis (PCoA) with the DCenter, Eigen, Output, and MXPlot subprograms in NTSYS-pc.Again, software MEGA 5.1 was applied to make the neighbor joining tree (Tamura et al., 2011;Hall, 2013).

SSR markers and allelic diversity
In this study, 60 SSR markers distributed 12 chromosomes of rice were genotyped in 26 BRRI developed rice varieties.Among the 60 SSR markers, 52 (86.67%) were polymorphic while 8 markers revealed mono-morphic patterns and excluded from the study.A total of 156 alleles were detected for the 52 polymorphic primers (Table 2).The number of alleles per locus generated by each marker ranged from 2 to 7, with an average number of alleles identified per locus was 3.00.The gene diversity varied from 0.09 to 0.82, with an average of 0.39, whereas the size of alleles ranged from 79 to 278 bp.The polymorphism information content (PIC) for the SSR loci ranged from 0.08 (RM5, RM487, RM178, RM214, RM542, RM500) to 0.79 (RM566), with an average of 0.35.RM566 had the highest PIC value (0.79) and the highest number of alleles ( 7) revealed the highest level of polymorphism.PIC value revealed that RM566 was supposed to be the best marker for characterizing the BRRI developed 26 rice varieties.Again, the second highest PIC value (0.73) was obtained for RM1337, followed by RM591, RM5473, RM21 and RM304.Total 14 highly informative markers (PIC>0.50),19 informative markers (0.50>PIC>0.25)and 19 slightly informative markers (PIC<0.25) were obtained which might be effectively used for genetic diversity and relationships study of rice.The variability in allele number per locus, the frequency of the most common allele at each locus, ranged from 22.23% (RM566) to 96.15% (RM487, RM214).On average, 70.25% of the 26 rice varieties shared a common major allele at any given locus (Table 2).The DNA profile of 26 rice varieties with SSR marker (RM224) was depicted in Fig. 1.
Table 3. Genetic dissimilarity between pair of BRRI varieties obtained from SSR data analysis Figure 3.An UPGMA cluster dendrogram showing the genetic relationships between 26 rice varieties of Bangladesh based on the alleles detected by 52 SSR markers.

Principal coordinate analysis
The two dimensional graphical views of principal coordinate analysis showed the spatial distribution of the BRRI rice varieties.The rice varieties namely BRRI dhan56, BRRI dhan51, BRRI dhan52, BRRI dhan61, BRRI dhan62 and BRRI dhan66 were to be found far away from the centroid of the cluster and the rest of the varieties were close to the centroid (Fig. 4).Genotypes close to the centroid suggest similar characteristics, while genotypes distant from the centroid suggest different traits (Islam et al., 2018c).Molecular characterization has been earlier documented in rice genotypes (Salgotra et al., 2015;Ahmed et al., 2016;Siddique et al., 2016a, b, c;Islam et al., 2021;Vabna et al., 2021).In the present study, 156 alleles were detected among 26 studied rice varieties with an average number of 3 alleles per locus and an average PIC of 0.35.The genetic diversity observed in the present study is similar to earlier studies (Shah et al., 2013), they detected 2.75 alleles per locus and an average PIC value of 0.38 among 40 rice accessions of Pakistan.Similarly, SSR diversity was also observed in a study with 36 polymorphic SSRs in which they detected 2.22 alleles per locus and an average PIC value of 0.25 in 375 Indian rice varieties collected from different regions of India (Singh et al., 2013).The result differs with the result of Siddique et al. (2016a and2016b) where average PIC value of 0.90 and 0.81 for 96 T. Aman and 20 GI rice cultivars, respectively.
The UPGMA dendogram formed four clusters which is similar to other studies.Khalequzzaman et al. (2022) studied diversity and population structure of 48 Boro rice landraces showed four major groups.Pachauri et al. (2013) made the UPGMA dendogram based on molecular marker analysis and clustered the 41 genotypes into four major clusters.Similarly, Das et al. (2013) found four groups among a set of 26 rice cultivars.
Though the genetic divergence was not very high, but these studied varieties showed considerable genetic diversity.Narrow genetic base of modern rice varieties are available from different countries like Latin America (Herrera et al., 2008;Rabelo et al., 2015), Japan (Wanga et al., 2014), USA (Xu et al., 2004) and Korea (Song et al., 2002).This might be due to the varietal selection pressure and adaptation capability of the specific variety in the specific climatic conditions.

CONCLUSION
This study showed that the tested samples possessed a considerable level of genetic variation.The PIC values for the microsatellite loci varied from 0.08 to 0.79 with a mean of 0.35.Comparatively higher genetic distance was observed between BRRI dhan56 and BRRI Hybrid dhan4 genotypes pair followed by BRRI dhan60 and BRRI dhan64 genotypes pair than the other combinations.The identified informative (0.50>PIC>0.25)and highly informative (PIC>0.50)SSR markers could be effectively used in background selections during backcross breeding.

Table 1 .
List of BRRI developed rice varieties with special characters used for the study.

Table 2 .
Number of alleles, allele size range, highest frequency allele, gene diversity and polymorphism information content (PIC) found among 26 varieties for 52 microsatellite markers Figure 1.DNA Profile of 26 BRRI Rice varieties with RM224