R-ISSR-for fingerprinting, mapping and identification of new genomic loci in rye (Secale cereale L.)

R-ISSR-for fingerprinting, mapping and identification of new genomic loci in rye (Secale cereale L.) The results of the research confirming the possibility of applying various combinations of RAPD and ISSR primers in one multiplex PCR and the generation of a new type of R-ISSR products for the rye genome were presented in this work. The following was applied in the research: five rye genotypes including two inbred lines (153/79-1 and Ot1-3), hybrid F1 and two bulks (tolerant and susceptible) formed from recombinant inbred lines—RILs (F9) varying in the response to abiotic stress caused by nutrient deficiencies at the seedling stage. While evaluating the possibility of applying R-ISSR to the assessment of the rye variability, five of its genotypes were amplified separately with the RAPD and ISSR primers in each PCR reaction. These primers were combined in R-ISSR amplifications. The products of RAPD, ISSR and R-ISSR amplification were separated in 1.5% agarose gel. 32 R-ISSR combinations were examined, combining 20 and 8 selected RAPD and ISSR primers, respectively. 658 loci were amplified, including 230 RAPD, 180 ISSR and 271 R-ISSR, including 157 new loci. Over 91 loci were found, with an identical electrophoretic mobility for three methods. It was shown that R-ISSR products with electrophoretic mobility on agarose gels, identical to the co-migrating RAPD or ISSR, are not products of RAPD or ISSR, but they possess sequences of heteroamplicons—R-ISSR. The occurrence of sequences of primers used to R-ISSR was demonstrated while sequencing seven selected products of the above type. The ISSR primers with a low T m were proven to generate repeatable fingerprints in the thermal profile of the reaction specific for RAPD and combined with the RAPD primer—repeatable R-ISSR profiles. A similar range of variability as described in RAPD or ISSR was observed in the R-ISSR profiles. The correlation coefficient between genetic similarity matrices for five rye genotypes, calculated with the Mantel test, amounted to r AB.C = 0.870. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Russian Journal of Genetics Springer Journals

R-ISSR-for fingerprinting, mapping and identification of new genomic loci in rye (Secale cereale L.)

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Publisher
Springer Journals
Copyright
Copyright © 2013 by Pleiades Publishing, Ltd.
Subject
Biomedicine; Human Genetics; Animal Genetics and Genomics; Microbial Genetics and Genomics
ISSN
1022-7954
eISSN
1608-3369
D.O.I.
10.1134/S1022795413020117
Publisher site
See Article on Publisher Site

Abstract

The results of the research confirming the possibility of applying various combinations of RAPD and ISSR primers in one multiplex PCR and the generation of a new type of R-ISSR products for the rye genome were presented in this work. The following was applied in the research: five rye genotypes including two inbred lines (153/79-1 and Ot1-3), hybrid F1 and two bulks (tolerant and susceptible) formed from recombinant inbred lines—RILs (F9) varying in the response to abiotic stress caused by nutrient deficiencies at the seedling stage. While evaluating the possibility of applying R-ISSR to the assessment of the rye variability, five of its genotypes were amplified separately with the RAPD and ISSR primers in each PCR reaction. These primers were combined in R-ISSR amplifications. The products of RAPD, ISSR and R-ISSR amplification were separated in 1.5% agarose gel. 32 R-ISSR combinations were examined, combining 20 and 8 selected RAPD and ISSR primers, respectively. 658 loci were amplified, including 230 RAPD, 180 ISSR and 271 R-ISSR, including 157 new loci. Over 91 loci were found, with an identical electrophoretic mobility for three methods. It was shown that R-ISSR products with electrophoretic mobility on agarose gels, identical to the co-migrating RAPD or ISSR, are not products of RAPD or ISSR, but they possess sequences of heteroamplicons—R-ISSR. The occurrence of sequences of primers used to R-ISSR was demonstrated while sequencing seven selected products of the above type. The ISSR primers with a low T m were proven to generate repeatable fingerprints in the thermal profile of the reaction specific for RAPD and combined with the RAPD primer—repeatable R-ISSR profiles. A similar range of variability as described in RAPD or ISSR was observed in the R-ISSR profiles. The correlation coefficient between genetic similarity matrices for five rye genotypes, calculated with the Mantel test, amounted to r AB.C = 0.870.

Journal

Russian Journal of GeneticsSpringer Journals

Published: Feb 16, 2013

References

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