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Molecular Breeding

, Volume 31, Issue 3, pp 517–528 | Cite as

Functional molecular markers and high-resolution melting curve analysis of low phytic acid mutations for marker-assisted selection in rice

  • Yuan-Yuan Tan
  • Hao-Wei Fu
  • Hai-Jun Zhao
  • Sha Lu
  • Jun-Jie Fu
  • You-Fa Li
  • Hai-Rui Cui
  • Qing-Yao ShuEmail author
Article

Abstract

Phytic acid (PA, myo-inositol-1,2,3,4,5,6-hexakis-phosphate) and its salt form (phytate) are the principal storage forms of phosphorus in cereal grains. Since PA and phytates cannot be efficiently digested by monogastric animals, the abundance of PA in cereal and legume grains causes nutritional and environmental problems. The present study aimed at developing breeder-friendly functional molecular markers of five low phytic acid (LPA) mutant alleles of three rice (Oryza sativa L.) genes: viz., XQZ-lpa (a 1,475-bp deletion) and KBNT-lpa (a C→T single nucleotide polymorphism [SNP]) of LOC_Os02g57400, Z9B-lpa (a 6-bp deletion) and MH-lpa (a 1-bp deletion) of LOC_Os04g55800, and XS-lpa (a C→T SNP) of LOC_Os03g04920. First, markers for gel-based length polymorphism analysis were developed: viz., two insertion–deletion markers for XQZ-lpa and Z9B-lpa, two cleaved amplified polymorphic sequence (CAPS) markers for KBNT-lpa and XS-lpa, and one derived CAPS marker for MH-lpa. Second, the high-resolution melting (HRM) curve analysis method was explored for distinguishing plants with wild-type (WT) and LPA alleles (except XQZ-lpa). Plants of genotypes with homozygous mutant allele and WT, and with heterozygous alleles, could be directly differentiated by HRM for KBNT-lpa, XS-lpa and MH-lpa; only heterozygous individuals could be directly distinguished from homozygous WT and mutant plants for Z9B-lpa. However, by adding 15 % WT DNA templates to test samples before PCR, amplicons of three genotypes of the Z9B-lpa allele could also be differentiated by HRM analysis. Third, it was demonstrated that these markers could be effectively used for marker-assisted selection of LPA rice, and breeding lines with two non-allelic LPA mutations were developed with PA contents significantly lower than their respective parental LPA lines. Taken together, the present study developed functional molecular markers for efficient selection of LPA plants and demonstrated that double mutant LPA lines with significantly lower PA levels than primary LPA mutants (with single mutations) could be developed by pyramiding two non-allelic LPA mutations.

Keywords

Low phytic acid Functional marker High-resolution melting curve analysis Marker-assisted selection Oryza sativa L. 

Notes

Acknowledgments

This research was financially supported by the Sino-Swiss Joint Research Project (2009 DFA32040) and the Natural Science Foundation of China through research contracts No. 31071481 and No. 30900887, and in part supported by the Fundamental Research Funds for Central Universities, the Special Fund for Agro-scientific Research in the Public Interest (201103007). The technical assistance of Ms Liquan Mao for determination of phytic acid content is highly appreciated.

Supplementary material

11032_2012_9809_MOESM1_ESM.pdf (444 kb)
Supplementary material 1 (PDF 444 kb)

References

  1. Bregitzer PP, Raboy V, Obert DE, Windes J, Whitmore J (2008) Registration of ‘Clearwater’ low-phytate hulless spring barley. J Plant Regist 2:1–4CrossRefGoogle Scholar
  2. Chen PS, Toribara TY, Warner H (1956) Microdetermination of phosphorus. Anal Chem 28:1756–1758CrossRefGoogle Scholar
  3. Collard BCY, Mackill D (2008) Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Philos Trans R Soc B 363:557–572CrossRefGoogle Scholar
  4. Fischer AJ, Cheetham DP, Laca EA et al (2004) Outcrossing study between transgenic herbicide-resistant rice and non-transgenic rice in California. In: Ferrero A, Vidotto F (eds) Proceedings of the conference on challenges and opportunities for sustainable rice-based production systems. International rice conference, September 13–15, 2004, Turin, Italy, pp 407–408Google Scholar
  5. Fu HW, Li YF, Shu QY (2008) A revisit of mutation induction by gamma rays in rice (Oryza sativa L.): implications of microsatellite markers for quality control. Mol Breed 22:281–288CrossRefGoogle Scholar
  6. Hofinger BJ, Jing HC, Kosack KEH, Kanyuka K (2009) High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley. Theor Appl Genet 119:851–865PubMedCrossRefGoogle Scholar
  7. Josefsen L, Bohn L, Sorensen MB, Rasmussen SK (2007) Characterization of a multifunctional inositol phosphate kinase from rice and barley belonging to the ATP-grasp superfamily. Gene 397:114–125PubMedCrossRefGoogle Scholar
  8. Kim SI, Tai TH (2010) Genetic analysis of two OsLpa1-like genes in Arabidopsis reveals that only one is required for wild-type seed phytic acid levels. Planta 232:1241–1250PubMedCrossRefGoogle Scholar
  9. Kim SI, Andaya CB, Goyal SS, Tai TH (2008a) The rice OsLpa1 gene encodes a novel protein involved in phytic acid metabolism. Theor Appl Genet 117:769–779PubMedCrossRefGoogle Scholar
  10. Kim SI, Andaya CB, Newman JW, Goyal SS, Tai TH (2008b) Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK. Theor Appl Genet 117:1291–1301PubMedCrossRefGoogle Scholar
  11. Koeyer DD, Douglass K, Murphy A, Whitney S, Nolan L, Song Y, Jong WD (2010) Application of high-resolution DNA melting for genotyping and variant scanning of diploid and autotetraploid potato. Mol Breed 25:67–90CrossRefGoogle Scholar
  12. Larson SR, Rutger JN, Young KA, Raboy V (2000) Isolation and genetic mapping of a non-lethal rice (Oryza sativa L.) low phytic acid 1 mutation. Crop Sci 40:1397–1405CrossRefGoogle Scholar
  13. Li YD, Chu ZZ, Liu XG, Hao DY (2010) A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants. J Integr Plant Biol 52(12):373–384CrossRefGoogle Scholar
  14. Li JS, Wang XM, Dong RX, Yang Y, Zhou J, Yu C et al (2011) Evaluation of high-resolution melting for gene mapping in rice. Plant Mol Biol Rep 29:979–985CrossRefGoogle Scholar
  15. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C (2004) Genotyping of single-nucleotide polymorphisms by high resolution melting of small amplicons. Clin Chem 50(7):1156–1164PubMedCrossRefGoogle Scholar
  16. Liu QL, Xu XH, Ren XL, Fu HW, Wu DX, Shu QY (2007) Generation and characterization of low phytic acid germplasm in rice (Oryza sativa L.). Theor Appl Genet 114:803–814PubMedCrossRefGoogle Scholar
  17. Lott JNA, Ockenden I, Raboy V, Batten GD (2000) Phytic acid and phosphorus in crop seeds and fruits: a global estimate. Seed Sci Res 10:11–33Google Scholar
  18. Maupin LM, Rosso ML, Rainey KM (2011) Environmental effects on soybean with modified phosphorus and sugar composition. Crop Sci 51(2):642–650CrossRefGoogle Scholar
  19. O’Dell BL, de Boland AR, Koirtyohann SR (1972) Distribution of phytate and nutritionally important elements among the morphological components of cereal grains. J Agric Food Chem 20:718–721CrossRefGoogle Scholar
  20. Panzeri D, Cassani E, Doria E, Tagliabue G, Forti L, Campion B, Bollini R, Brearley CA, Pilu R, Nielsen E, Sparvoli F (2011) A defective ABC transporter of the MRP family, responsible for the bean lpa1 mutation, affects the regulation of the phytic acid pathway, reduces seed myo-inositol and alters ABA sensitivity. New Phytol 191:70–83PubMedCrossRefGoogle Scholar
  21. Raboy V (1997) Accumulation and storage of phosphate and minerals. In: Larkin BA, Vasil IK (eds) Cellular and molecular biology of plant seed development. Kluwer Publishers, Netherlands, pp 441–477CrossRefGoogle Scholar
  22. Raboy V (2001) Seeds for a better future: ‘low phytate’ grains help to overcome malnutrition and reduce pollution. Trends Plant Sci 6:458–462PubMedCrossRefGoogle Scholar
  23. Raboy V (2009) Approaches and challenges to engineering seed phytate and total phosphorus. Plant Sci 177:281–296CrossRefGoogle Scholar
  24. Reed GH, Kent JO, Wittwer CT (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics J 8:597–608CrossRefGoogle Scholar
  25. Ren XL, Liu QL, Fu HW, Wu DX, Shu QY (2007) Density alteration of nutrient elements in rice grains of a low phytate mutant. Food Chem 102:1400–1406CrossRefGoogle Scholar
  26. Rossnagel BG, Zatorski T, Arganosa G, Beattie AD (2008) Registration of ‘CDC Lophy-1’ Barley. J Plant Regist 2(3):169–173CrossRefGoogle Scholar
  27. Rosso ML, Burleson SA, Maupin LM, Rainey KM (2011) Development of breeder-friendly markers for selection of MPIS1 mutations in soybean. Mol Breed 28:127–132CrossRefGoogle Scholar
  28. Rutger JN, Raboy V, Moldenhauer KAK, Bryant RJ, Lee FN, Gibbons JW (2004) Registration of KBNT lpa11 low phytic acid germplasm of rice. Crop Sci 44:363CrossRefGoogle Scholar
  29. Shi JR, Wang HY, Wu YS, Hazebroek J, Meeley RB, Ertl DS (2003) The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene. Plant Physiol 131:507–515PubMedCrossRefGoogle Scholar
  30. Shi JR, Wang HY, Hazebroek J, Ertl DS, Harp T (2005) The maize low-phytic acid 3 encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds. Plant J 42:708–719PubMedCrossRefGoogle Scholar
  31. Shi JR, Wang HY, Schelin K, Li BL, Faller M, Stoop JM, Meeley RB, Ertl DS, Ranch JP, Glassman K (2007) Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds. Nat Biotechnol 25:930–937PubMedCrossRefGoogle Scholar
  32. Spear JD, Fehr WR (2007) Genetic improvement of seedling emergence of soybean lines with low phytate. Crop Sci 47:1354–1360CrossRefGoogle Scholar
  33. Stevenson-Paulik J, Odom AR, York JD (2002) Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases. J Biol Chem 277:42711–42718PubMedCrossRefGoogle Scholar
  34. Sun YJ, Thompson M, Lin GF, Butler H, Gao ZF, Thornburgh S, Yau K, Smith DA, Shukla VK (2007) Inositol 1,3,4,5,6-pentakisphosphate 2-kinase from maize: molecular and biochemical characterization. Plant Physiol 144:1278–1291PubMedCrossRefGoogle Scholar
  35. Suzuki M, Tanaka K, Kuwano M, Yoshida KT (2007) Expression pattern of inositol phosphate-related enzymes in rice (Oryza sativa L.): implications for the phytic acid biosynthetic pathway. Gene 405:55–64PubMedCrossRefGoogle Scholar
  36. Thie T, Kota R, Grosse I, Stein N, Graner A (2004) SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development. Nucleic Acids Res 32(1):e5CrossRefGoogle Scholar
  37. Vossen RHAM, Aten E, Roos A, den Dunnen JT (2009) High-resolution melting analysis (HRMA)-more than just sequence variant screening. Hum Mutat 30:860–866PubMedCrossRefGoogle Scholar
  38. Wang YH, Ren L, Liu QL, Chen WY, Shen SQ, Wu DX, Shu QY (2005) Screening, selection and development of high inorganic phosphorus mutants in rice. Chin J Rice Sci 19:47–51Google Scholar
  39. Warkentin TD, Delgerjav O, Arganosa G, Rehman AU, Bett KE, Anbessa Y, Rossnagel B, Raboy V (2012) Development and characterization of low-phytate pea. Crop Sci 52:74–78CrossRefGoogle Scholar
  40. Wu SB, Wirthensohn M, Hunt P, Sedgley M, Gibson J (2008) High resolution melting analysis of almond SNPs derived from ESTs. Theor Appl Genet 118:1–14PubMedCrossRefGoogle Scholar
  41. Xu XH, Zhao HJ, Liu QL, Frank T, Engel KH, An G, Shu QY (2009) Mutations of the multi-drug resistance-associated protein ABC transporter gene 5 result in reduction of phytic acid in rice seeds. Theor Appl Genet 119:75–83PubMedCrossRefGoogle Scholar
  42. Yuan FJ, Zhu DH, Tan YY, Dong DK, Fu XJ, Zhu SN, Li BQ, Shu QY (2012) Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation. Theor Appl Genet 125:1413–1423. doi: 10.1007/s00122-012-1922-7 PubMedCrossRefGoogle Scholar
  43. Zhao HJ, Liu QL, Fu HL, Xu XH, Wu DX, Shu QY (2008a) Effect of non-lethal low phytic acid mutations on grain yield and seed viability in rice. Field Crops Res 108:206–211CrossRefGoogle Scholar
  44. Zhao HJ, Liu QL, Ren XL, Wu DX, Shu QY (2008b) Gene identification and allele-specific marker development for two low phytic acid mutations in rice. Mol Breed 22:603–612CrossRefGoogle Scholar
  45. Zhou XS, Shen SQ, Wu DX, Sun JW, Shu QY (2006) Introduction of a xantha mutation for testing and increasing varietal purity in hybrid rice. Field Crops Res 96:71–79CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Yuan-Yuan Tan
    • 1
    • 2
  • Hao-Wei Fu
    • 3
  • Hai-Jun Zhao
    • 1
    • 2
  • Sha Lu
    • 1
    • 2
  • Jun-Jie Fu
    • 2
  • You-Fa Li
    • 3
  • Hai-Rui Cui
    • 1
    • 2
  • Qing-Yao Shu
    • 1
    • 2
    Email author
  1. 1.National Key Laboratory of Rice BiologyZhejiang UniversityHangzhouChina
  2. 2.Key Laboratory of Nuclear-Agricultural Sciences of the Ministry of Agriculture and Zhejiang Province, Institute of Nuclear Agricultural SciencesZhejiang UniversityHangzhouChina
  3. 3.Jiaxing Academy of Agricultural SciencesJiaxingChina

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