Genetic Resources and Crop Evolution

, Volume 63, Issue 7, pp 1113–1126 | Cite as

Genetic diversity analysis of Pakistan rice (Oryza sativa) germplasm using multiplexed single nucleotide polymorphism markers

Research Article


A broad base of genetic diversity is crucial to provide variation to withstand biotic and abiotic stresses and to continue to make yield improvements. Pakistan has a wide range of climates and rice is ranked second as a staple food; however, very little is known in terms of rice genetic diversity in Pakistan with respect to global germplasm. This research was performed to investigate the genetic diversity and structure of the most commonly grown rice varieties in Pakistan, including the aromatic Basmati varieties, along with accessions from other countries. One hundred and five rice accessions were analyzed, including 21 Pakistan accessions, 44 IRRI lines, 6 wild rices, and 34 global accessions representing all five groups of rice as a reference in this study. These accessions were genotyped with 373 multiplexed SNP markers using the Illumina GoldenGate assay RiceOPA2.1 run at the Genotyping Services Lab at IRRI. The overall call percentage of the 373 SNPs across 105 accessions was 98.7 %. Structure analysis, principal coordinate analysis, and clustering with Neighbor Joining and UPGMA separated the genotypes in five major groups: indica, japonica, wild, aromatic and aus, while the Structure analysis at K = 7 further divided the indica cluster into 4 indica sub-groups. The Pakistan varieties were found in both the indica and aromatic clusters, but with an overall lower genetic diversity than seen in germplasm from other countries. Information on the diversity of rice germplasm can help guide future initiatives to widen the genetic base of rice cultivation in Pakistan.


Basmati Multiplexed SNP marker genotyping Oryza sativa Pakistan Rice diversity 



International Rice Research Institute


Minor allele frequency




Oligo pool assay


Single nucleotide polymorphism



We gratefully acknowledge the technical assistance of Maria Ymber Reveche, Christine Jade Dilla-Ermita, Dennis Nicuh Lozada, Erwin Tandayu, Pauline Capistrano, and Geraldine Ann M. Layaoen in the Genotyping Services Lab at IRRI. Also special thanks to Yoo Jin Lee for provision of wild rice from her research work. This study was funded in part by the Higher Education Commission (HEC) of Pakistan, the Global Rice Science Partnership and the International Rice Research Institute, Philippines. This research forms part of the Ph.D. thesis study of S.M.S.

Author contribution

S.M.S. analyzed data and wrote the manuscript. K.A. and G.S. assisted with data analysis and provided insight during manuscript preparation. M.A. provided rice germplasm, conceived of the study and provided guidance for manuscript writing. M.J.T. provided guidance for data analysis and helped with editing the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest. This research does not involve human participants or animals.

Supplementary material

10722_2015_304_MOESM1_ESM.docx (167 kb)
Supplementary material 1 (DOCX 166 kb)


  1. Akram M (2009) Aromatic rices of Pakistan—a review. Pak J Agric Res 22:154–160Google Scholar
  2. Annoymous (2013) Rice newsletter. Rice Research Institute, Kala Shah Kaku, vol 2, pp 3–5Google Scholar
  3. Ashfaq M, Khan AS (2012) Genetic diversity in Basmati rice (Oryza sativa L.) germplasm as revealed by microsatellite (SSR) markers. Russ J Genet 48:53–62CrossRefGoogle Scholar
  4. Bernardo R (2008) Molecular markers and selection for complex traits in plants: learning from the last 20 years. Crop Sci 48:1649–1664CrossRefGoogle Scholar
  5. Caliskan M (2012) Genetic diversity in microorganisms. In: Abdel-Mawgood AL (eds) DNA based techniques for studying genetic diversity. ISBN:978-953-51-0064-5Google Scholar
  6. Cavalli-Sforza LL, Edwards AWF (1967) Phylogenetic analysis. Models and estimation procedures. Am J Hum Genet 19:233–257PubMedPubMedCentralGoogle Scholar
  7. Chakhonkaen S, Pitnjam K, Saisuk W, Ukoskit K, Muangprom A (2012) Genetic structure of Thai rice and rice accessions obtained from the International Rice Research Institute. Rice 5:1–13CrossRefGoogle Scholar
  8. Deulvot C, Charrel H, Marty A, Jacquin F, Donnadieu C, Lejeune-Hénaut I, Burstin J, Aubert G (2010) Highly-multiplexed SNP genotyping for genetic mapping and germplasm diversity studies in pea. BMC Genom 11:468CrossRefGoogle Scholar
  9. Earl DA, vonHoldt BM (2012) STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour 4:359–361CrossRefGoogle Scholar
  10. Ebana K, Yonemaru J-I, Fukuoka S, Iwata H, Kanamori H, Namiki N, Nagasaki H, Yano M (2010) Genetic structure revealed by a whole-genome single-nucleotide polymorphism survey of diverse accessions of cultivated Asian rice (Oryza sativa L.). Breed Sci 60:390–397CrossRefGoogle Scholar
  11. Falush D, Stephens M, Pritchard JK (2003) Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164:1567–1587PubMedPubMedCentralGoogle Scholar
  12. Garris AJ, Tai TH, Coburn J, Kresovich S, McCouch SR (2005) Genetic structure and diversity in Oryza sativa L. Genetics 169:1631–1638CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gupta PK, Roy JK, Prasad M (2001) Single nucleotide polymorphisms: a new paradigm for molecular marker technology and DNA polymorphism detection with emphasis on their use in plants. Curr Sci India 80:524–535Google Scholar
  14. Hamblin MT, Close TJ, Bhat PR, Chao S, Kling JG, Abraham KJ, Blake T, Brooks WS, Cooper B, Griffey CA (2010) Population structure and linkage disequilibrium in US barley germplasm: implications for association mapping. Crop Sci 50:556–566CrossRefGoogle Scholar
  15. Hyten DL, Song Q, Choi IY, Yoon MS, Specht JE, Matukumalli LK, Nelson RL, Shoemaker RC, Young ND, Cregan PB (2008) High-throughput genotyping with the GoldenGate assay in the complex genome of soybean. Theor Appl Genet 116:945–952CrossRefPubMedGoogle Scholar
  16. Kovach MJ, Calingacion MN, Fitzgerald MA, McCouch SR (2009) The origin and evolution of fragrance in rice (Oryza sativa L.). Proc Natl Acad Sci USA 106:14444–14449CrossRefPubMedPubMedCentralGoogle Scholar
  17. Li YH, Li W, Zhang C, Yang L, Chang RZ, Gaut BS, Qiu LJ (2010) Genetic diversity in domesticated soybean (Glycine max) and its wild progenitor (Glycine soja) for simple sequence repeat and single-nucleotide polymorphism loci. New Phytol 188:242–253CrossRefPubMedGoogle Scholar
  18. Lorenz AJ, Chao S, Asoro FG, Heffner EL, Hayashi T, Iwata H, Smith KP, Sorrells ME, Jannink JL (2011) Genomic selection in plant breeding: knowledge and prospects. Adv Agron 110:77CrossRefGoogle Scholar
  19. Loridon K, Burgarella C, Chantret N, Martins F, Gouzy J, Prospéri J-M, Ronfort J (2013) Single-nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula. Mol Ecol Resour 13:84–95CrossRefPubMedGoogle Scholar
  20. McNally KL, Childs KL, Bohnert R, Davidson RM, Zhao K, Ulat VJ, Zeller G, Clark RM, Hoen DR, Bureau TE (2009) Genomewide SNP variation reveals relationships among landraces and modern varieties of rice. Proc Natl Acad Sci USA 106:12273–12278CrossRefPubMedPubMedCentralGoogle Scholar
  21. Moose SP, Mumm RH (2008) Molecular plant breeding as the foundation for 21st century crop improvement. Plant Physiol 147:969–977CrossRefPubMedPubMedCentralGoogle Scholar
  22. Pervaiz ZH, Rabbani MA, Shinwari ZK, Masood MS, Malik SA (2010) Assessment of genetic variability in rice (Oryza sativa L.) germplasm from Pakistan using RAPD markers. Pak J Bot 42:3369–3376Google Scholar
  23. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedPubMedCentralGoogle Scholar
  24. Rabbani MA, Pervaiz ZH, Masood MS (2008) Genetic diversity analysis of traditional and improved cultivars of Pakistani rice (Oryza sativa L.) using RAPD markers. Electron J Biotechnol 11:52–61CrossRefGoogle Scholar
  25. Rabbani MA, Masood MS, Shinwari ZK, Shinozaki K (2010) Genetic analysis of Basmati and non-Basmati Pakistani rice (Oryza sativa L.) cultivars using microsatellite markers. Pak J Bot 42:2551–2564Google Scholar
  26. Rohlf FJ (2000) Multivariate analysis system, version 2.1. Exeter Software, New YorkGoogle Scholar
  27. Rostoks N, Ramsay L, MacKenzie K, Cardle L, Bhat PR, Roose ML, Svensson JT, Stein N, Varshney RK, Marshall DF (2006) Recent history of artificial outcrossing facilitates whole-genome association mapping in elite inbred crop varieties. Proc Natl Acad Sci USA 103:18656–18661CrossRefPubMedPubMedCentralGoogle Scholar
  28. Sasaki T, Burr B (2000) International Rice Genome Sequencing Project: the effort to completely sequence the rice genome. Curr Opin Plant Biol 3:138–142CrossRefPubMedGoogle Scholar
  29. Shah SM, Naveed SA, Arif M (2013) Genetic diversity in Basmati and non-Basmati rice varieties based on microsatellite markers. Pak J Bot 45:423–431Google Scholar
  30. Thomson MJ (2014) High-throughput SNP genotyping to accelerate crop improvement. Plant Breed Biotech 2:195–212CrossRefGoogle Scholar
  31. Thomson MJ, Septiningsih EM, Suwardjo F, Santoso TJ, Silitonga TS, McCouch SR (2007) Genetic diversity analysis of traditional and improved Indonesian rice (Oryza sativa L.) germplasm using microsatellite markers. Theor Appl Genet 114:559–568CrossRefPubMedGoogle Scholar
  32. Thomson MJ, Zhao K, Wright M, McNally KL, Rey J, Tung CW, Reynolds A, Scheffler B, Eizenga G, McClung A (2012) High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform. Mol Breeding 29:875–886CrossRefGoogle Scholar
  33. Upadhyay P, Neeraja CN, Kole C, Singh V (2012) Population structure and genetic diversity in popular rice varieties of India as evidenced from SSR analysis. Biochem Genet 50:770–783CrossRefPubMedGoogle Scholar
  34. Wright MH, Tung C-W, Zhao K, Reynolds A, McCouch SR, Bustamante CD (2010) ALCHEMY: a reliable method for automated SNP genotype calling for small batch sizes and highly homozygous populations. Bioinformatics 26:2952–2960CrossRefPubMedPubMedCentralGoogle Scholar
  35. Xie F, Guo L, Ren G, Hu P, Wang F, Xu J, Li X, Qiu F, dela Paz MA (2012) Genetic diversity and structure of indica rice varieties from two heterotic pools of southern China and IRRI. Plant Genet Resour 10:186–193CrossRefGoogle Scholar
  36. Yamamoto T, Nagasaki H, Yonemaru J, Ebana K, Nakajima M, Shibaya T, Yano M (2010) Fine definition of the pedigree haplotypes of closely related rice cultivars by means of genome-wide discovery of single-nucleotide polymorphisms. BMC Genom 11:267CrossRefGoogle Scholar
  37. Yu J, Hu S, Wang J, Wong GKS, Li S, Liu B, Deng Y, Dai L, Zhou Y, Zhang X (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296:79–92CrossRefPubMedGoogle Scholar
  38. Zhao K, Tung CW, Eizenga GC, Wright MH, Ali ML, Price AH, Norton GJ, Islam MR, Reynolds A, Mezey J, McClung AM, Bustamante CD, McCouch SR (2011) Genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa. Nat Commun 2:467CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Department of Environmental SciencesCOMSATS Institute of Information TechnologyAbbottabadPakistan
  2. 2.Institute of Molecular Biology and BiotechnologyBahauddin Zakariya UniversityMultanPakistan
  3. 3.National Institute for Biotechnology and Genetic Engineering (NIBGE)FaisalabadPakistan
  4. 4.International Rice Research Institute (IRRI)Los BañosPhilippines
  5. 5.Department of Soil and Crop SciencesTexas A&M UniversityCollege StationUSA

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