Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants

Species-specific LTR retrotransposons were first cloned in five rare relic species of drug plants located in the Perm’ region. Sequences of LTR retrotransposons were used for PCR analysis based on amplification of repeated sequences from LTR or other sites of retrotransposons (IRAP). Genetic diversity was studied in six populations of rare relic species of plants Adonis vernalis L. by means of the IRAP method; 125 polymorphic IRAP-markers were analyzed. Parameters for DNA polymorphism and genetic diversity of A. vernalis populations were determined.     

A high-density cytogenetic map of the Aegilops tauschii genome incorporating retrotransposons and defense-related genes: insights into cereal chromosome structure and function

Aegilops tauschii (Coss.) Schmal. (2n=2x=14, DD) (syn. A. squarrosa L.; Triticum tauschii) is well known as the D-genome donor of bread wheat (T. aestivum, 2n=6x=42, AABBDD). Because of conserved synteny, a high-density map of the A. tauschii genome will be useful for breeding and genetics within the tribe Triticeae which besides bread wheat also includes barley and rye. We have placed 249 new loci onto a high-density integrated cytological and genetic map of A. tauschii for a total of 732 loci making it one of the most extensive maps produced to date for the Triticeae species. Of the mapped loci, 160 are defense-related genes. The retrotransposon marker system recently developed for cultivated barley (Hordeum vulgare L.) was successfully applied to A. tauschii with the placement of 80 retrotransposon loci onto the map. A total of 50 microsatellite and ISSR loci were also added. Most of the retrotransposon loci, resistance (R), and defense-response (DR) genes are organized into clusters: retrotransposon clusters in the pericentromeric regions, R and DR gene clusters in distal/telomeric regions. Markers are non-randomly distributed with low density in the pericentromeric regions and marker clusters in the distal regions. A significant correlation between the physical density of markers (number of markers mapped to the chromosome segment/physical length of the same segment in m) and recombination rate (genetic length of a chromosome segment/physical length of the same segment in m) was demonstrated. Discrete regions of negative or positive interference (an excess or deficiency of crossovers in adjacent intervals relative to the expected rates on the assumption of no interference) was observed in most of the chromosomes. Surprisingly, pericentromeric regions showed negative interference. Islands with negative, positive and/or no interference were present in interstitial and distal regions. Most of the positive interference was restricted to the long arms. The model of chromosome structure and function in cereals with large genomes that emerges from these studies is discussed.     

A movable feast: diverse retrotransposons and their contribution to barley genome dynamics

Cellular genes comprise at most 5% of the barley genome; the rest is occupied primarily by retrotransposons. Retrotransposons move intracellularly by a replicative mechanism similar to that of retroviruses. We describe the major classes of retrotransposons in barley, including the two nonautonomous groups that were recently identified, and detail the evidence supporting our current understanding of their life cycle. Data from analyses of long contiguous segments of the barley genome, as well as surveys of the prevalence of full-length retrotransposons and their solo LTR derivatives in the genus Hordeum, indicate that integration and recombinational loss of retrotransposons are major factors shaping the genome. The sequence conservation and integrative capacity of barley retrotransposons have made them excellent sources for development of molecular marker systems.     

Application of BARE-1 retrotransposon markers to the mapping of a major resistance gene for net blotch in barley

Net blotch, which is caused by the fungus Pyrenophoral teres Drechs. f. teres Smedeg., presents a serious problem for barley production worldwide, and the identification and deployment of sources of resistance to it are key objectives for many breeders. Here, we report the identification of a major resistance gene, accounting for 65% of the response variation, in a cross between the resistant line C19819 and the susceptible cv. Rolfi. The resistance gene was mapped to chromosome 6H with the aid of two recently developed systems of retrotransposon-based molecular markers, REMAP and IRAP. A total of 239 BARE-1 and Sukkula retrotransposon markers were mapped in the cross, and the 30-cM segment containing the locus with significant resistance effect contained 26 of the markers. The type and local density of the markers should facilitate future map-based cloning of the resistance gene as well as manipulation of the resistance through backcross breeding.     

High-throughput retrotransposon-based genetic diversity of maize germplasm assessment and analysis

Maize is one of the world’s most important crops and a model for grass genome research. Long terminal repeat (LTR) retrotransposons comprise most of the maize genome; their ability to produce new copies makes them efficient high-throughput genetic markers. Inter-retrotransposon-amplified polymorphisms (IRAPs) were used to study the genetic diversity of maize germplasm. Five LTR retrotransposons (Huck, Tekay, Opie, Ji, and Grande) were chosen, based on their large number of copies in the maize genome, whereas polymerase chain reaction primers were designed based on consensus LTR sequences. The LTR primers showed high quality and reproducible DNA fingerprints, with a total of 677 bands including 392 polymorphic bands showing 58% polymorphism between maize hybrid lines. These markers were used to identify genetic similarities among all lines of maize. Analysis of genetic similarity was carried out based on polymorphic amplicon profiles and genetic similarity phylogeny analysis. This diversity was expected to display ecogeographical patterns of variation and local adaptation. The clustering method showed that the varieties were grouped into three clusters differing in ecogeographical origin. Each of these clusters comprised divergent hybrids with convergent characters. The clusters reflected the differences among maize hybrids and were in accordance with their pedigree. The IRAP technique is an efficient high-throughput genetic marker-generating method.

     

Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers

Retrotransposon segments were characterized and inter-retrotransposon amplified polymorphism (IRAP) markers developed for cultivated flax (Linum usitatissimum L.) and the Linum genus. Over 75 distinct long terminal repeat retrotransposon segments were cloned, the first set for Linum, and specific primers designed for them. IRAP was then used to evaluate genetic diversity among 708 accessions of cultivated flax comprising 143 landraces, 387 varieties, and 178 breeding lines. These included both traditional and modern, oil (86), fiber (351), and combined-use (271) accessions, originating from 36 countries, and 10 wild Linum species. The set of 10 most polymorphic primers yielded 141 reproducible informative data points per accession, with 52% polymorphism and a 0.34 Shannon diversity index. The maximal genetic diversity was detected among wild Linum species (100% IRAP polymorphism and 0.57 Jaccard similarity), while diversity within cultivated germplasm decreased from landraces (58%, 0.63) to breeding lines (48%, 0.85) and cultivars (50%, 0.81). Application of Bayesian methods for clustering resulted in the robust identification of 20 clusters of accessions, which were unstratified according to origin or user type. This indicates an overlap in genetic diversity despite disruptive selection for fiber versus oil types. Nevertheless, eight clusters contained high proportions (70?C100%) of commercial cultivars, whereas two clusters were rich (60%) in landraces. These findings provide a basis for better flax germplasm management, core collection establishment, and exploration of diversity in breeding, as well as for exploration of the role of retrotransposons in flax genome dynamics.     

TRIM retrotransposons occur in apple and are polymorphic between varieties but not sports

Retrotransposon markers have been demonstrated to be powerful tools for investigating linkage, evolution and genetics diversity in plants. In the present study, we identified and cloned three full-size TRIM (terminal-repeat retrotransposon in miniature) group retrotransposon elements from apple (Malus domestica) cv. ‘Antonovka’, the first from the Rosaceae. To investigate their utility as markers, we designed primers to match the long terminal repeats (LTRs) of the apple TRIM sequences. We found that PCR reactions with even a single primer produced multiple bands, suggesting that the copy number of these TRIM elements is relatively high, and that they may be locally clustered or nested in the genome. Furthermore, the apple TRIM primers employed in IRAP (inter-retrotransposon amplified polymorphism) or REMAP (retrotransposon-microsatellite amplified polymorphism) analyses produced unique, reproducible profiles for 12 standard apple cultivars. On the other hand, all seven of the sport mutations in this study were identical to their mother cultivar. Genetic similarity values calculated from the IRAP/REMAP analyses or the STMS (sequence tagged microsatellite sites) analysis were generally comparable. PAUP cluster analysis based on IRAP and REMAP markers in apple and Japanese quince generated an NJ tree that is in good accordance with both a tree based on SMTS markers and the origin of the studied samples. Our results demonstrate that, although they do not encode the proteins necessary to carry out a life cycle and are thereby non-autonomous, TRIMs are at least as polymorphic in their insertion patterns as conventional complete retrotransposons. Kristiina Antonius-Klemola, Ruslan Kalendar are the first two authors contributed equally to this work     

Mapping of major spot-type and net-type net-blotch resistance genes in the Ethiopian barley line CI 9819.

Net blotch of barley (Hordeum vulgare L.), caused by the fungal phytopathogen Pyrenophora teres Drechs. f. teres Smedeg., constitutes one of the most serious constraints to barley production worldwide. Two forms of the disease, the net form, caused by P. teres f. teres, and the spot form, caused by P. teres f. maculata, are differentiated by the type of symptoms on leaves. Several barley lines with major gene resistance to net blotch have been identified. Earlier, one of these was mapped in the Rolfi x CI 9819 cross to barley chromosome 6H, using a mixture of 4 Finnish isolates of P. teres f. teres. In this study, we used the same barley progeny to map resistance to 4 spot-type isolates and 4 net-type isolates of P. teres. With all net-type isolates, a major resistance gene was located on chromosome 6H, in the same position as described previously, explaining up to 88% of the phenotypic variation in infection response in the progeny. We designate this gene Rpt5. Several minor resistance genes were located on chromosomes 1H, 2H, 3H, 5H, and 7H. These minor genes were not genuinely isolate-specific, but their effect varied among isolates and experiments. When the spot-type isolates were used for infection, a major isolate-specific resistance gene was located on chromosome 5H, close to microsatellite marker HVLEU, explaining up to 84% of the phenotypic variation in infection response in the progeny. We designate this gene Rpt6. No minor gene effects were detected in spot-type isolates. The Ethiopian 2-rowed barley line CI 9819 thus carries at least 2 independent major genes for net-blotch resistance: Rpt5, active against net-type isolates; and Rpt6, active against specific spot-type isolates.