R-ISSR - Tool for Generation of a New Type of Products, Applied for the Identification of Putative Molecular Marker Linked to QTL Determined Tolerance to Nutrient Deprivation Stress in Rye (Secale cereale L.)

Milosz Smolik

doi:10.15835/nbha4027535

Abstract

This study presents results confirming the possibility of the application of various combinations of RAPD and ISSR primers in one multiplex PCR for the rye genome and generating a new type of R-ISSR products. The plant material consisted of two inbred lines (Ot0-6 and Ot1-3), F1 and two bulks (tolerant and susceptible), selected from a population of RILs (F9) with different responses to stress caused by nutrient deprivation at the seedling stage. In one PCR reaction, the DNA of five genotypes was amplified separately, with RAPD, ISSR, and RAPD+ISSR primers. In total, 28 R-ISSR combinations were tested by combining 20 and 8 appropriately selected RAPD and ISSR primers. 567 loci were amplified, including 230 RAPD, 136 ISSR and 207 amplified R-ISSR loci, of which 114 were new. It was shown that only one ISSR product was identified amongst sequenced R-ISSR products with identical electrophoretic mobility as co-migrating RAPDs and ISSRs. The remaining ones had heteroamplicons (R-ISSR) sequences. A similar range of variability was observed both in R-ISSR profiles and in RAPD, as well as in ISSR. The correlation coefficient between the matrices of genetic similarity for five rye genotypes calculated by means of the Mantel test was highly significant rAB.C = 0.964.

Highlights

Identification of genes, factors and mechanisms regulating their expression enables their role in the functioning of organisms to be understood (Liu et al, 2008; Moller et al, 2011; Xu et al, 2011)
This study demonstrates the possibility of the application of the R-ISSR technique as a screening tool to describe the new range of variability in the rye genome
Twelve ISSR and thirty RAPD primers were used in multiplex PCR

Summary

Introduction

Identification of genes, factors and mechanisms regulating their expression enables their role in the functioning of organisms to be understood (Liu et al, 2008; Moller et al, 2011; Xu et al, 2011). In organic plant cultivation, including cultivation of plants with decreased nutrient requirements, such information may significantly facilitate selection of desired genotypes (Wolfe et al, 2008). Such research provides a wealth of biological information and it is becoming increasingly easier to gain access to it. It is increasingly easier to assign significance and sense to biological information obtained within experiments which are conducted based on information already collected using the synteny and collinearity of genomes (Devos and Gale, 2000). Rye is used as a source of resistance genes for many cereal diseases, winter hardiness, tolerance to sprouting, drought, and nutrient deficiencies that can be transferred to closely related cereal crops (Geiger and Miedaner, 2009; Hillel and Rosenzweig, 2005; Wolfe et al, 2008). Compared to wheat and barley, the data available on rye with various sequences collected at gene banks (primarily EST) remains modest (~10,000 sequences)

Methods

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Discussion

Conclusion