Article | 04. 2016 Vol. 34, Issue. 2
Development of molecular markers for varietal identification of Brassica juncea on the basis of the polymorphic sequence of ITS regions and MITE families



Department of Horticulture, Sunchon National University1
Agricultural Technology Center of Yeosu City2
Department of Agricultural Education, Sunchon National Universtiy3




2016.04. 305:313


PDF XML




Brassica juncea (2n = 4x = 36, AABB genome, 1,068 Mb) is a U’s triangle species and an amphidiploid derivative of B. rapa and B. nigra. Fifteen varieties were used to study the ITS (internal transcribed spacer) regions of ribosomal DNA and MITEs (miniature inverted-repeat transposable elements) with a view of developing specific molecular markers. ITSs and MITEs are an excellent resource for developing DNA markers for genomics and evolutionary studies because most of them are stably inherited and present in high copy numbers. The ITS (ITS1 and ITS2) sequence was compared with the consensus sequence of B. rapa and B. nigra. Variation in ITS1 created two separate groups among 15 varieties, with 10 varieties in one group and 5 in the other. Phylogenetic analysis revealed two major clusters for those 10 and 5 varieties. Among the 160 different MITE primers used to evaluate the selected 15 varieties of B. juncea, 70 were related to the Stowaway, 79 to the Tourist, 6 to the hAT, and 5 to the Mutator super-families of MITEs. Of 160 markers examined, 32 were found to be polymorphic when fifteen different varieties of B. juncea were evaluated. The variety ‘Blackgat’ was different from the other mustard varieties with respect to both phenotype and genotype. The diversity of 47 additional accessions could be verified using eight selected molecular markers derived from MITE family sequences. The polymorphic markers identified in this study can be used for varietal classification, variety protection, and other breeding purposes.



1. Al-Shehbaz I, Beilstein M, Kellogg E (2006) Systematics and phylogeny of the Brassicaceae (Cruciferae): an overview. Plant Syst Evol 259:89-120. doi:10.1007/s00606-006-0415-z  

2. Amundsen K, Rotter D, Li HM, Messing J, Jung G, Belanger F, Warnke S (2011) Miniature inverted-repeat transposable element identification and genetic marker development in. Crop Science 51:854-861. doi:10.2135/cropsci2010.04.0215  

3. Casa AM, Nagel A, Wessler SR (2004) MITE display. Methods Mol Biol 260:175-188. doi:10.1385/1-59259-755-6:175  

4. Casacuberta JM, Santiago N (2003) Plant LTR-retrotransposons and MITEs: control of transposition and impact on the evolution of plant genes and genomes. Gene 311:1-11. doi:10.1016/S0378-1119(03)00557-2   

5. Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, et al (2010) Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PloS One 5:e8613. doi:10.1371/journal.pone.0008613   

6. Chen J, Hu Q, Zhang Y, Lu C, Kuang H (2014) P-MITE: a database for plant miniature inverted-repeat transposable elements. Nucleic Acids Res 42: D1176-D1181. doi:10.1093/nar/gkt1000  

7. Downie SR, Katz-Downie DS (1996) A molecular phylogeny of Apiaceae subfamily Apiaceae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences. Am J Bot 83:234-251. doi:10.2307/2445943  

8. Fu J, Zhang MF, Qi XH (2006) Genetic diversity of traditional chinese mustard crops as revealed by phenotypic differences and RAPD markers. Genet Resour Crop Evol 53:1513-1519. doi:10.1007/s10722-005-7763-3  

9. Han Y, Wessler SR (2010) MITE-Hunter: a program for discovering miniature inverted-repeat transposable elements from genomic sequences. Nucleic Acids Res 38:e199. doi:10.1093/nar/gkq862   

10. Johnston JS, Pepper AE, Hall AE, Chen ZJ, Hodnett G, Drabek J, Lopez R, Price HJ (2005) Evolution of genome size in Brassicaceae.Ann Bot 95:229-235. doi.org/10.1093/aob/mci016  

11. Kim H, Yeo SS, Han DY, Park YH (2015) Interspecific transferability of watermelon EST-SSRs assessed by genetic relationship analysis of cucurbitaceous crops. Korean J Hortic Sci Technol 33:93-105. doi:10.7235/hort.2015.14120   

12. Kim HI, Hong CP, Im S, Choi SR, Lim YP (2014) Development of molecular markers and application for breeding in chinese cabbage.Korean J Hortic Sci Technol 32:745-752. doi:10.7235/hort.2014.12203  

13. Koch MA, Dobe C, Olds TM (2003) Multiple hybrid formation in natural populations: concerted evolution of the internal transcribed spacer of nuclear ribosomal DNA (ITS) in Northern American Arabis divaricarpa (Brassicaceae ). Mol Biol Evol 20:338-350. doi:10.1093/molbev/msg046  

14. Mo YJ, Kim KY, Shin WC, Lee GM, Ko JC, Nam JK, Kim BK, Ko JK, Yu Y, Yang TJ (2012) Characterization of imcrop, a mutator-like MITE family in the rice genome. Genes & Genomics 34:189-198. doi:10.1007/s13258-011-0193-z  

15. Qi XH, Zhang MF, Yang JH (2007) Molecular phylogeny of Chinese vegetable mustard (Brassica juncea ) based on the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Genetic Resources and Crop Evolution 54:709-1716. doi:10.1007/s10722-006-9179-0  

16. Sampath P, Murukarthick J, Izzah NK, Lee J, Choi HI, Shirasawa K, Choi BS, Liu S, Nou IS, Yang TJ (2014) Genome-wide comparative analysis of 20 miniature inverted-repeat transposable element families in Brassica rapa and B. oleracea . PloS One 94:e94499. doi:10.1371/journal.pone.0094499  

17. Smapath P, Lee SC, Lee J, Izzah NK, Choi BS, Jin M, Park BS, Yang TJ (2013) Characterization of a new high copy Stowaway family MITE, BRAMI-1 in Brassica genome. BMC plant biol 13:56. doi:10.1186/1471-2229-13-56  

18.   

19. Shirasawa K, Hirakawa H, Tabata S, Hassegawa M, Kiyoshima H, Suzuki S, Sasamoto S, Watanabe A, Fujishiro T, Isobe S (2012)Characterization of active miniature inverted-repeat transposable elements in the peanut genome. Theor Appl Genet 124: 1429- 1438. doi:10.1007/s00122-012-1798-6  

20. Song KM, Osborn TC, Williams PH (1988) Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs) 1.genome evolution of diploid and amphidiploid species. Theor Appl Genet 75:784-794. doi:10.1007/bf00265606   

21. Song KM, Osborn TC, Williams PH (1990) Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs) 3.genome relationships in Brassica and related genera and the origin of B. oleracea and B. rapa (syn. campestris). Theor Appl Genet 76:497-506. doi:10.1007/bf00226159  

22. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725-2729. doi:10.1093/molbev/mst197  

23. Turcotte K, Srinivasan S, Bureau T (2001) Survey of transposable elements from rice genomic sequences. Plant J 25:169-179.doi:10.1111/j.1365-313X.2001.00945.x  

24. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds, PCR protocols: a guide to methods and applications. Academic Press, San Diego, Calif,pp 315-322. doi:10.1016/b978-0-12-372180-8.50042-1  

25. Yang TJ, Kim JS, Kwon SJ, Lim KB, Choi BS, Kim JA, Jin M, Park JY, Lim MH, Kim HI, et al (2006) Sequence-level analysis of the diploidization process in the triplicated FLOWERING LOCUS C region of Brassica rapa . Plant Cell 18:1339-1347. doi:10.1105/tpc.105.040535   

26. Yang YW, Lai PY, Tai PY, Ma DP, Li WH (1999) Molecular phylogenetic studies of Brassica, Rorippa Arabidopsis , and allied genera based on the internal transcribed spacer region of 18S–25S rDNA. Mol Phylogenet Evol 13:455-462. doi:10.1006/ mpev.1999.0648  

27. Yuan YM, Kupfer P, Doyle JJ (1996) Infrageneric phylogeny of the genus gentiana inferred from nucleotide sequences of internal transcribed spacers of the nuclear ribosomal DNA. Am J Bot 83:641-652. doi:10.2307/2445924  

28. Zhang Q, Arbuckle J, Wessler SR (2000) Recent, extensive, and preferential insertion of members of the miniature inverted-repeat transposable element family heartbreaker into genic regions of maize. Proc Natl Acad Sci USA 97:1160-1165. doi:10.1073/