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.)

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), F 1 and two bulks (tolerant and susceptible), selected from a population of RILs (F 9 ) 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 comigrating 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 r AB.C = 0.964.

Chromosomal localization of selected SCARs converted from new RAPD, ISSR and R-ISSR markers linked to rye (Secale cereale L.) tolerance to nutrient deprivation stress identified using bulk segregant analysis

“…Detailed results describing this stage of the research are presented in Table 1. Polymorphic products differentiating inbred lines were identified in RAPD, ISSR and R-ISSR profiles (Smolik, 2012;Smolik, 2013b). Their presence in an F1 hybrid profile, as well as in various configurations in the profiles of two bulked DNA samples (bulks), both tolerant and susceptible to nutrient stress, are presented in Figure 1a.…”

“…However, in the base TIGR/TOGA/DFCI for rye it was matched to EST aquaropin PIP1 (DQ175869.1 -Sc01_05f04_A), and in the previous study as SCARECROW-like GRAS-family TF for Leymus sp.) (Smolik, 2012). Both HKT1 transporters and aquaporins (PIP1) have a significant relationship with plant response to stress, including nutrition stress (Maurel et al 2008;Alemán et al 2011).…”

“…Amplification of RAPD and ISSR was conducted according to the protocols presented by Smolik (2012); Smolik (2013b) and Zietkiewicz et al (1994), respectively. The papers by Smolik (2012) and Smolik (2013b) present combinations of primers RAPD+ISSR (R-ISSR) useful for the amplification of R-ISSR loci, and they were used in this study in the search for R-ISSR markers.…”

“…The papers by Smolik (2012) and Smolik (2013b) present combinations of primers RAPD+ISSR (R-ISSR) useful for the amplification of R-ISSR loci, and they were used in this study in the search for R-ISSR markers.…”

“…Primers were selected in the first stage of the study, and RAPD as well as ISSR products were generated in reactions with them. For R-ISSR combinations, this stage was presented in two papers (Smolik, 2012;Smolik, 2013b). The research was conducted on pooled DNA samples (bulks), searching for products distinguishing 'tolerant' bulks from 'susceptible' ones in both the examined RILs populations (Michelmore et al 1991, Masojć et al 2009).…”