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SSR based genetic diversity analysis in a diverse germplasm of groundnut (Arachis hypogaea L.) from Pakistan [Australian Journal of Crop Science](Australian Journal of Crop Science Via Acquire Media NewsEdge) Abstract The current study was aimed to explore the genetic diversity among seventy Pakistani accessions of Arachis hypogaea. In Pakistan their morphological and biochemical variations have already documented but still so far, molecular variations need to be studied for this valuable crop. For molecular diversity study DNA was extracted from all seventy accessions of Arachis hypogaea. The extracted DNA was primed with thirty SSR primers and amplified through PCR. Fifteen out of thirty primers generated polymorphic bands among the selected accessions. In total, forty different polymorphic loci were determined across the selected accessions. The range of number of polymorphic loci detected was ranged from 2 to 4 for each primer, with an average of 2.6 loci per primer. Polymorphic Index Content (PIC) value was calculated for each marker. The dendrogram was constructed on the basis of allelic data from fifteen SSR markers across the selected accessions. All the accessions were divided into six clusters at 0.67 coefficients of similarity. This study of variations at molecular level of Pakistani groundnut accessions will be helpful for conservation and breeding purposes of groundnut and other legumes. Keywords: Simple Sequence Repeat, Polymorphic Index Content, Groundnut, Loci. Abbreviations: PCR Polymerase Chain Reaction, SSR Simple Sequences Repeat, PIC_Polymorphic Index Content. Introduction Groundnut (Arachis hypogaea L.) is grown throughout the world as a source of oil and protein. The Arachis hypogaea is an allotetraploid (AABB, 2n = 4x = 40 chromosomes), resulting from duplication of AA and BB wild type species (Leal-Bertioli et al., 2009). A hypogaea belong to genus Arachis of Fabaceae. Two main classes of A hypogaea are hypogaea and fàstigiata. A hypogaea have flowers on the main axis while fàstigiata lack flowers on the main axis (Krepovickas and Gregory, 1994). The classification of groundnut only on the basis of their morphological characteristics is not sufficient therefore the assessment of variation at gene level during germplasm collections and pedigree construction is necessary. The use of molecular markers will be helpful for the collection of advanced and novel genotypes of groundnut. DNA based markers provide accurate knowledge at gene level which was not possible with phenotypic markers (Altinkut et al., 2003). Molecular markers have been used for tagging of important traits of groundnut in inter specific introgression populations. Many markers have been identified which were resistant to late leaf spot (Mace et al., 2006). Despite significant physiological, agronomic and morphological variation the peanut exhibits variations at low level by RAPD (Mondai et al., 2005), SSR (Raina et al., 2001) and AFLP (Herselman, 2003). The use of microsatellites to track advantageous traits in plant breeding and as helpful point in gene cloning program explores their importance (Brown et al., 1996). Microsatellites were mostly used in genetic diversity studies, gene flow mating system and paternity studies (Rosseto et al., 1999). Being with highest PIC, due to their high mutation rate, SSRs are routinely used in finger-printing Chen and Du, (2006), molecular mapping (Zhao et al., 2005) phylogenetic and genetic relationship studies (Yang et al., 2005) and markerassisted breeding (Sun et al., 2006). PCR amplified the SSR loci using primers created from distinctive contiguous nucleotides. Polymorphism is due to the differences in the amount of repeats. Point mutations and polymerase slippage are the main reason of variation in number of repeats (Kruglyak et al., 1998). Microsatellites, (SSRs) have been used to study genetic variation and to construct molecular maps in numerous crops (Lee et al., 2004). ATT and CTT are the most common tri nucleotide sequences in plant genomes (Ferguson et al., 2004). Similarly AT is proved to be the most abundant dinucleotide repeat sequences followed by AG/CT and GT/CA in plant genomes (Cue et al., 2008). The aim of the current study was to explore the genetic diversity at molecular level among accessions of Arachis hypogea from Pakistan. Morphological and physiological variations have been already documented but molecular variation has been not properly studied for this valuable crops. The analysis of variations at molecular level will be helpful for conservation and breeding purposes of groundnut. Results SSR fragment size range and polymorphism in accessions From the initial assessment of thirty SSR markers, fifteen were selected for analysis of polymorphism among seventy groundnut accessions. A total of forty polymorphic loci were identified across seventy accessions (Table 3). Amplification with SSR primers resulted in fragment size from 75-320 base pair. Maximum fragment length (320bp) was observed in local genotypes while Australian genotype NCFLA-14 showed minimum fragment size. The number of polymorphic loci detected ranged from 2 to 5 (against each primer), with an average of 2.9 loci per primer. Among the polymorphic markers, two primers amplified five alleles each. The maximum genetic distance of 0.97 was observed between PG-1074 and USA-7 presenting them as the most divergent members. Similarly the most related members among these seventy accessions were ALKAT and ICGS-12 with 0.162 genetic distances. PIC was calculated for each marker ranged from 0.255 to 0.662 with an average of 0.548. Marker Ah-08 explored maximum diversity (PIC=0.662) while marker Ah21 showed minimum diversity (PIC=0.255) among these 70 genotypes. Fig. 1 illustrates the result of one of the fifteen SSR primers. The individual primers gave different banding pattem. In this study, not all markers clearly showed definite amplification fragments. Some markers showed no amplification band within several genotypes while showing clear and bright amplification bands in other genotypes. For example the primer Ah-26 and Ah-08 showed clear amplification products in all genotypes except Banki, Chakori and Bari-89 genotypes suggesting the deletion of primer binding site or complete lack of the primer locus in these genotypes. Few markers were noted to be varietyspecific producing different alleles in different genotypes. Primer Ah-27 amplified five alleles in Icgs-38, three in Yh11 and two in the other genotypes. Ouster analysis The dendrogram was constructed on the basis of allelic data from fifteen SSR markers across the selected accessions as shown in Fig. 2. At 0.67 coefficients of similarity all the accessions were grouped into six clusters. The selected accessions were grouped mainly according to their breeding /research stations. These clusters were further divided into sub clusters. The cluster A contains 24 genotypes evolved at BARI. These varieties showed relatively high similarity with one other. Genotypes ICGS-493 and ICGS-38 showed maximum similarity and the genotype ICGS-14 is the most divergent genotype of this group. Similarly the cluster B contains 13 genotypes evolved at AARI. The genotypes of this group showed maximum similarity with each other when compared with the rest of genotypes. Genotypes ICGS-12 and ALKAT showed maximum similarity while PG-864 is the most divergent genotype of this group. Ouster C consists of 20 genotypes developed at NARC. PG-1142 is the most diverse member of this group. In this group the two genotypes PG-953 and PG-1070 are the most similar members. Cluster D contains only 4 genotypes. All the genotypes are of Chinese origin. The genotypes of this group showed minimum similarity with each other when compared with the rest of genotypes. YH-14 is the most divergent member of the group. The cluster E contains 10 genotypes of Australian origin while the cluster F has only two genotypes of USA origin. Eight genotypes of Sub-group E showed relatively less similarity with each other as compared to sub group F. Pakistani origin accessions group together at 0.59 co-efficient and are much diverse from each other while Chinese and US origin accessions were group together at 0.75 co-efficient. Discussion The identification of agronomically valuable and diverse cultivare at molecular level will be very useful for linkage mapping and genetic improvement of specific traits in groundnut. Microsatellites have widely used as molecular markers in recent years. Among the DNA markers, SSR markers are more preferable as it is highly abundant, genetically co-dominant, analytically simple and are highly reproducible. The first report of microsatellites in plants was reported by Condit and Hubbel, 1991, who reported that SSR markers are abundant in plants. The report SSR polymorphisms on soybean by Akkaya et al., 1992 have opened up a novel source of PCR-based DNA markers for other crops. The high polymorphism and stability of SSR markers made them usefiil and superior in molecular genetic studies and varietal identification (Varshney et al., 2005).The present study demonstrates the presence of significant polymorphic SSR markers in A hypogaea. Fifty percent polymorphism was identified among the tested genotypes in the current research work. The results revealed the presence of significant level of polymorphism in cultivated peanut. Several other studies have also reported a significant level of polymorphism in cultivated peanut (Mace et al., 2007) observed a high level of polymorphism within germplasm of peanut which are resistant to bacterial wilt. He reported that 99.4% of the genotypes showed polymorphism when screened with thirty two SSR primers. Similarly 76.5% polymorphism was described by Mondai S and Badigannavar AM (2010) when they studied the connection of simple sequence repeat (SSR) with late leaf spot and rust resistance in groundnut. The study of (Cue et al., 2008) in which 44% polymorphism was observed, were similar to our results In contrast to our work various previous works have resulted in a low level of polymorphism among cultivated peanut genotypes (Halward et al., 1991; He and Prakash, 1997; Hopkins et al., 1999; Herselman, 2003; Moretzsohn et al., 2004; Mace et al., 2006). Insignificant molecular diversity among wild and cultivated peanut genotypes was detected by using SSR markers (Hopkins et al., 1999, Krishna et al., 2004, He et al., 2005). Hie insignificant molecular diversity among the parental lines might be the reason of low level of polymorphism in cultivated peanut. The origination of cultivated groundnut from a single polyploidization practice is the main reason of low level of genetic polymorphism (Young et al., 1996). In the current study the PIC value was calculated for each marker ranged from 0.255 to 0.662 with an average of 0.548. The study of (Cue et al., 2008) in which an average of 0.46 PIC was calculated, are in support of our findings. Similarly (Geleta et al., 2006) observe an average of 0.64 PIC value for SSR markers among 45 accessions of groundnut. The number of alleles per marker detected in this study, ranged from 2 to 4 with an average of 2.9 alleles per locus matched with the earlier studies. (Cue et al., 2008) showed similar results of 2 to 5 alleles with an average of 2.9 alleles per locus in 32 genotypes of groundnut. Our results are in pipeline with the finding of (Tang et al., 2007) in which 2.0-5.5 alleles per locus for were observed. The work of (Hopkins et al., 1999) also favor multiloci concept of SSR markers. The main reasons behind the contrary results of molecular markers might be due to the different genetic sources of peanut germplasm and primer variation in quality and quantity. In short it is summarized that molecular level genetic diversity is superior as compared to morphological and biochemical polymorphism. Furthermore the assessment of molecular level diversity is more important for preservation of genetic assets, recognition of best germplasm resources and the collection of superior cultivare for hybridization purposes (Dwived et al., 2001). The results from the genetic similarity-based analysis of seventy groundnut accessions with fifteen polymorphic microsatellite markers prominently differentiate the local varieties into their breeding/research station. The clustering of the same institutions accessions into one major group is consistent with conventional taxonomical classification of cultivated peanut (Tang et al., 2008). One possible reason for this close association could be the involvement of common parents/lines in the development of these varieties. Thus the enhancement of diverse populations by using diverse germplasm resources is needed in the future for various purposes such as population genetic structure, germplasm analysis, identification of cultivare, selection of parents and phylogenetic relationships. Materials and methods Plant materials A total of seventy accessions of groundnut were collected from different research institute such as NARC, BARI and AARI for the current study (Table 1) which consists of four Chinese, two USA, seven Australian and fifty seven of Pakistani origin accessions. The research work was carried out at National Institute for Genomics and Advanced Biotechnology (NIGAB), NARC, Islamabad in 2012. DNA extraction DNA was extracted from the leaves of seventy accessions of groundnut through a modified CTAB-based procedure (Mace et al., 2003). The quality of DNA was measured by running DNA on 1% agarose gel and quantified with spectrophotometer at wavelength of 260 nm. PCR analysis Thirty different SSR markers were used in the current study (Table 2). PCR was carried out in Veriti 96-well thermal cycler (Applied Biosystems, CA) with Taq polymerase (MBI Fermentas). DNA samples were diluted to 20ng/pl for PCR. PCR reaction were performed in 20 pi volumes PCR mixture, containing 30 ng/pl genomic DNA, IX PCR buffer (MBI Fermentas) 1.6 mM MgC12, 0.5 mM dNTPs, 0.5 U DNA polymerase (MBI Fermentas) and 10 p mol of each primer. The initial dénaturation temperature of 94°C for 04 min was adjusted followed by the 40 cycle of Denaturation (94 for 45sec), annealing (58°C for 60 sec) and Extension (72°C for 90 sec). The quality and concentration of extracted DNA was assessed by visualizing it on 2% agarose gel under UV light. Data analysis The presence/absence of each fragment (product length variant) and banding pattem in each accession was recorded against each primer. The size of the amplified product was calculated on the basis of its mobility relative to molecular mass of marker (lOObp, MBI, Fermentas). Pair-wise comparisons of the cultivare based on the ratio of exceptional to common alleles were used to calculate the genetic similarity by Dice coefficients using SIMQUAL sub-program in similarity routine of software NTSYS-pc version 2.2 (Exeter Software, Setauket, NY, U.S.A.) software package (Rohlf, 2005). Estimates of genetic similarity were considered among all pairs of the genotypes according to Nei and Li (1979). Dendrogram was constructed using UPGMA to calculate genetic associations among peanut accessions by the protocol earlier described by Rohlf, 2005. The PIC for each SSR was determined according to Anderson et al. (1993). Conclusion The results showed that fifteen out of thirty markers produced a total of forty loci across Pakistani accessions of groundnut. The number of polymorphic loci detected ranged from 2 to 4 (against each primer, with an average of 2.6 loci per primer. Marker Ah-20 showed maximum diversity (PIC=.663) while marker Ah-21 showed minimum diversity (PIC=.255) among these 70 genotypes. At 0.59 coefficient of similarity all the seventy genotypes were divided into four major groups according to their breeding/research stations. The current study highlights the need of isolation and characterization of more DNA markers in groundnut and their uses in advanced studies such as gene discovery, marker assisted selection and gene mapping. SSR markers used in this study can also be used in other species as well. It will reduce the cost of the study, since microsatellite markers improvement is still costly and sustained. Acknowledgments We are thankful to National Institute for Genomics and Advanced Biotechnology (NIGAB), NARC, Islamabad, Pakistan for providing of all kinds of technical and financial support. References Akkaya MS, Bhagwat AA, Gregan PB (1992) Length polymorphism of simple sequence repeats DNA in soybean. Genetics 132: 1131-1139. 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Fabaceae), In: Paterson A.H.(ed.), Genome Mapping in Plants, Landes Co., Austin, USA 211-227 Zhao XQ, Liang GH and Zhou JS (2005) Molecular mapping of two semidwarf genes in an indica rice variety Aitaiyin 3 (Oryza sativa L). Acta Genet Sin 32: 189-196 Sohaib Roomi1*, Bibi Sabiha2, Arshad Iqbal4, Muhammad Suleman3, Izhar Muhammad 4, Muhammad Amir Zia4, Muhammad Zulfiqar Ahmad4, Farooq Rashid4, Abdul Ghafoor4 and Nabila Tabbasam4 1Department of Biosciences COMSATS Institute of Information Technology, Islamabad, Pakistan 2Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology, Islamabad, Pakistan 3Institute of Biotechnology and Microbiology, University of Swat, Khyber Pakhtunkhwa, Pakistan 4National Institute for Genomics and Advanced Biotechnology, National Agricultural Research Centre, Islamabad, Pakistan *Corresponding author: [email protected] (c) 2014 Southern Cross Publisher |