Identification of cultivars and accessions of Lolium, Festuca and Festulolium hybrids through the detection of simple sequence repeat polymorphism

Abstract

Molecular diversity and genetic affinity in the Lolium/Festuca grass complex have been assessed using simple sequence repeat (SSR) marker technology. The genotypic set was derived from three accessions of Lolium perenne, two cultivars of Lolium multiflorum, two cultivars of Festuca pratensis, two cultivars of Festuca arunidinacea and ten accessions from different intergeneric hybrid (Festulolium) combinations. The majority of the genomic DNA-derived SSR primer pairs from perennial ryegrass (LPSSR) and Italian ryegrass (LMSSR) produced clear, simple and distinctive amplification products from the majority of the genotypes.The efficiency of cross-specific amplification for LPSSR markers varied from 38% in F. pratensis to 93% in two cultivars of Festulolium and for LMSSR markers from 57% in F. pratensis to 87% in L. multiflorum. Of 40 amplified markers, fourteen (35%) produced species-difference alleles in the relation to cultivars used in the present study. Thirty-five LPSSR locus-derived alleles were found to be specific to Lolium species, 4 to F. pratensis and 6 to F. arundinacea. For LMSSR alleles, 8 were specific to Lolium species and 5 were only associated with L. multiflorum, and null alleles were detected for F. pratensis in all instances. These species-difference markers could clearly identify different accessions of In Festulolium hybrid breeding, it is desirable to be able to distinguish the DNA of the parental species, in order to monitor the extent of the introgressed 3 regions. Fluorescence in situ hybridisation (FISH) methodology using labelled genomic DNA (genomic in situ hybridisation: GISH) has been widely used to identify alien chromatin (Thomas et al. 1994, Humphreys et al. 1995, Humphreys and Pašakinskiené 1996. However, there are potential difficulties in the identification of small introgressed chromosome segments using this technique (Humphreys et al. 1998).The development of molecular genetic marker technologies provides alternative procedures for the assessment of genetic diversity, cultivar identification and the genomic composition of polyploid and hybrid taxa. Several Moreover, SSRs have been shown to provide a powerful means for discrimination between closely related genotypes in many plant species (Yang et al. 1994, Russell et al. 1997, Röder et al. 1998 The cycling regime for the PCR amplification of LMSSR markers consisted of an initial denaturation step of 10 min at 94°C, followed by denaturation for 1 min at 94°C followed by 30 s at 94°C, 30 s at 60°C, 2 min at 72°C; repeated for 35 cycles, and a final extension of 7 min at 72°C (Fujimori et al. in preparation). Detection of SSR polymorphisms:The samples were prepared for polyacrylamide gel electrophoresis (PAGE) by addition of 2 μl gel loading buffer to the reaction mixture. Ten µl of amplification products were loaded on 10% (w/v) non-denaturing polyacrylamide gels for electrophoresis in 1×TBE. Each gel was run for 2.5 h at constant voltage (250V). The banding patterns were visualised following staining with ethidium ...

Molecular Mapping and Breeding of Physiological Traits

“…Further, the markers could directly characterize the genome of organism. The microsatellites or simple sequence repeats (SSRs) are now favoured at length for diversity analysis due to their simplicity, repeatability, and high polymorphism levels [115][116][117][118][119][120] among the various PCR-based markers available for genetic characterization. Molecular markers detected the entire genome as a substitute of only the regions responsible for the manifestation of the traits of interest [121].…”

Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information

“…This therefore implies that many of the indel-type markers developed in this study contained non-SSR indels. Generally, SSR sites are highly polymorphic, even within a species, and are often used for genotyping at the individual level (Morgante and Olivieri 1993), although several SSR markers showing species specificity have been reported (Momotaz et al 2004). Festulolium cultivars are often bred as polyploids .…”

“…Several DNA marker systems that enable distinction between Lolium and Festuca genomes have been developed (Stammers et al 1995;Pašakinskiene et al 2000;Momotaz et al 2004). Simple sequence repeat (SSR) markers, in particular, are efficient tools for introgression mapping due to their abundance, ubiquitous distribution in plant genomes, ease of polymerase chain reaction (PCR)-based analysis and detection of co-dominant multi-allelic loci (Momotaz et al 2004;Lauvergeat et al 2005).…”

“…Simple sequence repeat (SSR) markers, in particular, are efficient tools for introgression mapping due to their abundance, ubiquitous distribution in plant genomes, ease of polymerase chain reaction (PCR)-based analysis and detection of co-dominant multi-allelic loci (Momotaz et al 2004;Lauvergeat et al 2005). Currently, however, only a limited number of SSR markers are publicly available for Lolium/Festuca, and the development of SSR makers is both costly and labor intensive.…”

Transformation of androgenic-derived Festulolium plants (Lolium perenne L. × Festuca pratensis Huds.) by Agrobacterium tumefaciens

“…Lolium and Festuca species hybridize naturally, and, as hybrids, regularly exchange genes with high frequency. Recently, in Europe and Japan, Festulolium plants ( Lolium-Festuca complex, intergeneric hybrids between L. multiflorum, L. perenne and F. pratensis, F. arundinacea) were developed as novel temperate forage grasses Momotaz et al 2004;Pašakinskienė et al 1997;Zwierzykowski et al 1999;Yamada et al 2005).…”