Variations in BARE-1 insertion patterns in barley callus cultures

The stability of aging barley calli was investigated with the barley retroelement 1 (BARE-1) retrotransposon specific inter-retrotransposon amplified polymorphism (IRAP) technique. Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were cultured on callus induction MS medium supplemented with 3 mg/L 2,4-D and maintained on the same medium for 60 days. Ten IRAP primers were used in 25 different combinations. The similarity index between 30-day-old and 45-day-old calli was 84%; however, the similarity index between mature embryos and 45-day-old calli was 75%. These culture conditions caused BARE-1 retrotransposon alterations to appear as different band profiles. This is the first report of the use of the IRAP technique in barley in an investigation of callus development.     

Development of retrotransposon primers and their utilization for germplasm identification in Diospyros spp. (Ebenaceae)

Retrotransposons play an important role in plant genetic instability and genome evolution. Retrotransposon-based molecular markers are valuable tools to reveal the behavior of retrotransposons in their host genome. In this study, suppression polymerase chain reaction was used, for the first time, to develop retrotransposon long terminal repeat (LTR) and polypurine tract (PPT) primers in Japanese persimmon (Diospyros kaki Thunb.), which were then employed for germplasm identification by means of interretrotransposon-amplified polymorphism (IRAP), sequence-specific amplified polymorphism (SSAP) and retrotransposon-microsatellite-amplified polymorphism (REMAP) molecular markers. The results showed that 16 out of 26 primers produced expected amplifications and abundant polymorphisms by IRAP in 28 genotypes of Diospyros. Moreover, some of these primers were further successfully used in REMAP and SSAP analysis. Each type of molecular markers produced unique fingerprint in 28 genotypes analyzed. Among the primers/primer combinations, two IRAP primers and four SSAP primer combinations could discriminate all of the germplasm solely. Further comparative analysis indicated that IRAP was the most sensitive marker system for detecting variability. High level of retrotransposon insertion polymorphisms between bud sports were detected by IRAP and SSAP, and the primers/primer combinations with powerful discrimination capacity for two pairs of bud sports lines were further obtained. Additionally, possible genetic relationships between several Japanese persimmon were discussed. To our knowledge, this is the first report on the development of retrotransposon LTR and PPT primers in Diospyros, and the retrotransposon primers developed herein might open new avenue for research in the future.     

IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.     

Retrotransposon insertional polymorphism in sunflower (Helianthus annuus L.) lines revealed by IRAP and REMAP markers

Retrotransposons are ubiquitous components of plants genomes, making them useful molecular markers for genetic diversity studies. We used inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers to assess genetic diversity and survey activity of LTR retrotransposon elements in 106 sunflower (Helianthus annuus L.) genotypes from different research centers. We found 118 (out of 128) and 113 (out of 120) polymorphic loci using 14 IRAP and 14 REMAP primers, respectively. The Mantel test between IRAP and REMAP cophenetic matrices revealed low correlation (r = 0.55) between them. Dice similarities based on combined (IRAP + REMAP) data ranged from 0.34 to 0.93 among (“11 × 12” and “F1250/03”) and (“HA335B” and “TMB51”) genotypes, respectively. Classification of genotypes using the Dice similarity matrix derived from IRAP+REMAP data based on the un-weighted pair-group method using the arithmetic average algorithm resulted in nine distinct groups. The studied genotypes were divided into seven groups considering their origins (research centers). Classification of genotypes can be useful to assess the genetic variation and gene flow between and within research centers. Analysis of molecular variance based on IRAP+REMAP data revealed a higher level of genetic variation within (94%) than between (6%) research centers. A high amount of gene flow was detected among USDA, ASGROW, and ENSAT groups. Because environmental factors have no influence on molecular markers, the construction of heterotic groups based on retrotransposon markers will be useful for the selecting of parents with a high probability of producing superior hybrids.     

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     

Retrotransposon Insertional Polymorphism in Iranian Bread Wheat Cultivars and Breeding Lines Revealed by IRAP and REMAP Markers

Inter-retrotransposon amplified polymorphisms (IRAPs) and retrotransposon-microsatellite amplified polymorphisms (REMAPs) were used to detect retrotransposon integration events and genetic diversity in 101 Iranian bread wheat (Triticum aestivum L.) cultivars and breeding lines. The 9 IRAP primers amplified 128 loci, and 20 REMAP primers amplified 263 loci. Percentage of polymorphic loci, average expected heterozygosity, number of effective alleles, and Shannon’s information index for the REMAP markers were slightly higher than those for the IRAP markers. The same estimated parameters calculated for native and nonnative retrotransposons were not considerably different. A Mantel test between IRAP and REMAP cophenetic matrices evidenced no significant correlation. Cluster analysis based on the Dice similarity coefficient and complete linkage algorithm using IRAP+REMAP loci identified five groups among the genotypes studied that could be applied as crossing parents in T. aestivum breeding programs.     

Retrotransposon based genetic status of North-West Himalayan Zingiber officinale revealed high heterogeneity

Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were successfully applied, for the first time, to analyze genetic diversity among 92 ginger landraces collected from north-western Himalayan region of India. Six IRAP primer/combinations generated 75 loci with an average of 12 loci/primer displaying an overall polymorphism of 95.95 %. On the other hand, twenty five REMAP primer combinations produced 414 loci with 96.5 % polymorphism. IRAP showed maximum Rp (5.39) and PIC (0.28) values, while the same in REMAP was observed to be 10.92 and 0.34. Cluster analysis using Jaccard’s similarity coefficient for IRAP and REMAP data ranged between 0.21 to 1.0 and 0.21 to 0.85, respectively distinguishing all the genotypes with diverse genetic makup. The results also confirmed the presence of sukkula retrotransposon (RT6) in the ginger genome which effectively acted as genetic marker revealing high regional genetic diversity in the ginger gene pool. The study will help in giving insight to the genetic constitution of vegetatively grown ginger crop and for its further utilization in improvement, conservation and management programmes.     

IRAP,a retrotransposon-based marker system for the detection of somaclonal variation in barley

The retrotransposon-based marker system, inter-retrotransposon amplified polymorphism (IRAP), and inter-simple sequence repeats (ISSRs) were used to detect somaclonal variation induced by tissue culture. IRAPs use a single primer designed to amplify out from the 5′ LTR sequence of the BARE-1 retrotransposon combined with a degenerate 3′ anchor, similar to that of ISSR primers. We analysed DNA polymorphisms in 147 primary regenerants and parental controls from three cultivars of barley (Hordeum vulgare). The ISSR marker system generated an average of 218 bands per primer, with 29 polymorphisms of which 12 were novel non-parental bands. In comparison, the IRAP system generated an average of 121 bands per primer, with 15 polymorphisms of which nine were novel non-parental bands. Polymorphism detected for IRAP and ISSR markers was more than twofold higher in Golden Promise than Mackay and Tallon cultivars. However, there was no significant difference in the frequency of novel non-parental bands. Cluster analysis revealed that the level of polymorphism and genetic variability detected was comparable between IRAP and ISSR markers. This suggests that retrotransposon-based marker systems, such as IRAP, based on retrotransposons such as BARE-1, are valuable tools for the detailed characterisation of mutation profiles that arise during tissue culture. Their use should improve our understanding of processes influencing mutation and somaclonal variation and allow for the design of methods that yield fewer genome changes in applications where maintaining clonal integrity is important.     

Retrotransposon-based markers from potato monoploids used in somatic hybridization.

In an attempt to remove lethal and deleterious genes and enhance the heterozygosity of the potato genome, we developed several diverse somatic hybrids through the electrofusion of selected monoploids. Somatic hybrids and somaclones resulting from fused and unfused protoplasts, respectively, were verified with microsatellites. Molecular markers anchored in the Tst1 retrotransposon were used to examine polymorphisms in the regenerated plants and to reveal any somaclonal variation. Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon display (sequence-specific amplified polymorphism (S-SAP), anchored in a retransposon) were examined on an ALFexpress DNA sequencer. Because of inconsistencies in the number and quality of bands revealed by the combination of either class of marker in combination with the ALFexpress, we cloned and sequenced 11 S-SAP bands to use as restriction fragment length polymorphism (RFLP) probes in Southern blot analyses of genetic relationships in our potato populations and among related Solanaceae. Readily scorable bands (n = 27) that separated somatic hybrids from parental monoploids and somaclones and grouped monoploids according to known genetic relationships were produced. Some of the probes could be used to differentiate tomato and Datura from potato. Sequence analysis of 5 cloned IRAP and 11 cloned S-SAP markers confirmed that they were anchored in the Tst1 retrotransposon. BLAST searches within GenBank produced 10 highly significant hits (5 nucleotide, 4 expressed sequence tag (EST), and 1 protein) within closely related Solanaceae, suggesting that Tst1 represents an old retroelement that was inserted before the diversion of genera within Solanaceae; however, most sequences were undescribed.     

Retrotransposon-based molecular markers for grapevine species and cultivars identification

Insertional polymorphisms of two copia-like (Vine-1, Tvv1) and one gypsy-like (Gret1) retrotransposon found in the grapevine genome were studied in 29 Vitis genotypes (Vitis arizonica, Vitis cinerea, Vitis labrusca, Vitis rupestis, Vitis rotundifolia, Vitis vinifera subsp. sylvestris and 23 V. vinifera subsp. sativa) using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and sequence-specific amplified polymorphism (SSAP) techniques. IRAP, REMAP and SSAP polymorphisms were compared with amplified fragment length polymorphism (AFLP), Inter-single sequence repeats (ISSR) and SSR polymorphisms by evaluating the information content, the number of loci simultaneously analysed per experiment, the effectiveness of the analyses in assessing the relationship between accessions and the number of loci needed to obtain a coefficient of variation of 10%. The UPGMA dendrograms of each molecular marker system were compared and the Mantel matrix correspondence test was applied. Furthermore, the corresponding insertion ages of the transposable elements were estimated for each retrotransposon subfamily analysed. The presence of Gret1, Tvv1 and Vine-1 retrotransposons in all analysed genotypes suggests that copia-like and gypsy-like retrotransposons are widespread in Vitis genus. The results indicate that these retrotransposons were active before Vitis speciation and contributed to Vitis genus evolution. IRAP, REMAP and SSAP markers allow the discrimination of Vitis species and V. vinifera subsp. sativa cultivars with certainty as has been shown with AFLP, ISSR and SSR analyses, but phylogenetic trees obtained by retrotransposon-based molecular markers polymorphisms show some significant differences in the allocation of the analysed accessions compare to those obtained by ISSR, AFLP and SSR molecular markers. The phylogenetic tree resulting from REMAP polymorphism appeared the most representative of the effective relationship between all analysed accessions.     

Characterization of fusarium wilt-resistant and fusarium wilt-susceptible somaclones of banana cultivar rastali (Musa AAB) by random amplified polymorphic DNA and retrotransposon markers

Two DNA fingerprinting techniques, random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP), were used to characterize somaclonal variants of banana. IRAP primers were designed on the basis of repetitive and genome-wide dispersed long terminal repeat (LTR) retrotransposon families for assessing the somaclonal variation in 2Musa clones resistant and susceptible toFusarium oxysporum f. sp.cubense race 4. RAPD markers successfully detected genetic variation within and between individuals of the clones. IRAP makers amplified either by a single primer or a combination of primers based on LTR orientation successfully amplified different retrotransposons dispersed in theMusa genome and detected new events of insertions. RAPD markers proved more polymorphic than IRAP markers. Somaclonal variation seems to be the result of numerous indels occurring genome-wide accompanied by the activation of retroelements, as a result of stress caused by micropropagation. It is concluded that characterization of the somaclonal variants requires more than one DNA marker system to detect variation in diverse components of the genome.     

Genetic variability of Old Portuguese bread wheat cultivars assayed by IRAP and REMAP markers

Retrotransposons (RTNs) constitute informative molecular markers for plant species as a result of their ability of integrating into a multitude of loci throughout the genome and thereby generating insertional polymorphisms between individuals. Inter-retrotransposon amplified polymorphisms (IRAPs) and the retrotransposon-microsatellite amplified polymorphisms (REMAPs) are marker systems based on long terminal repeats (LTRs) RTNs, developed for plants, that have been widely used for evolution, genetic diversity, DNA fingerprinting of cultivars and varieties, genetic mapping linkage and for detection of genetic rearrangements induced by polyploidisation. In the present study, we aimed to analyse the genetic variability among 48 Old Portuguese bread wheat cultivars using both IRAP and REMAP markers. Five IRAP and six REMAP primer combinations were used. IRAP produced 103 polymorphic fragments in a total of 113 bands. On average, 22.6 bands were amplified per IRAP primer combination. The bands ranged in size from 250 to 5000 bp. The REMAP primer combinations allowed the amplification of 53 bands, 51 of them polymorphic. An average of 8.8 REMAP bands was scored per primer combination. The REMAP bands ranged from 250 to 3000 bp. Both marker systems presented high percentages of polymorphism. However, IRAP markers were suitable for detecting genetic variability at the individual level and did not differentiate higher taxa. The REMAP maker system allowed the clustering by botanical variety and identified most of the homonym bread wheat cultivars.     

Integration of Retrotransposons-Based Markers in a Linkage Map of Barley

A deeper understanding of random markers is important if they are to be employed for a range of objectives. The sequence specific amplified polymorphism (S-SAP) technique is a powerful genetic analysis tool which exploits the high copy number of retrotransposon long terminal repeats (LTRs) in the plant genome. The distribution and inheritance of S-SAP bands in the barley genome was studied using the Steptoe × Morex (S × M) double haploid (DH) population. Six S-SAP primer combinations generated 98 polymorphic bands, and map positions were assigned to all but one band. Eight putative co-dominant loci were detected, representing 16 of the mapped markers. Thus at least 81 of the mapped S-SAP loci were dominant. The markers were distributed along all of the seven chromosomes and a tendency to cluster was observed. The distribution of S-SAP markers over the barley genome concurred with the knowledge of the high copy number of retrotransposons in plants. This experiment has demonstrated the potential for the S-SAP technique to be applied in a range of analyses such as genetic fingerprinting, marker assisted breeding, biodiversity assessment and phylogenetic analyses.     

Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica C.E. Hubbard (Poaceae)

Spartina x townsendii arose during the end of the 19th century in England by hybridization between the indigenous Spartina maritima and the introduced Spartina alterniflora, native to the eastern seaboard of North America. Duplication of the hybrid genome gave rise to Spartina anglica, a vigorous allopolyploid involved in natural and artificial invasions on several continents. This system allows investigation of the early evolutionary changes that accompany stabilization of new allopolyploid species. Because allopolyploidy may be a genomic shock, eliciting retroelement insertional activity, we examined whether retrotransposons present in the parental species have been activated in the genome of S. anglica. For this purpose we used inter-retrotransposon amplified polymorphism (IRAP) and retrotransposons-microsatellite amplified polymorphism (REMAP) markers, which are multilocus PCR-based methods detecting retrotransposon integration events in the genome. IRAP and REMAP allowed the screening of insertional polymorphisms in populations of S. anglica. The populations are composed mainly of one major multilocus genotype, identical to the first-generation hybrid S. x townsendii. Few new integration sites were encountered in the young allopolyploid genome. We also found strict additivity of the parental subgenomes in the allopolyploid. Both these findings indicate that the genome of S. anglica has not undergone extensive changes since its formation. This contrasts with previous results from the literature, which report rapid structural changes in experimentally resynthesized allopolyploids.     

Genome merger: from sequence rearrangements in triticale to their elimination in wheat–rye addition lines

Genetic and epigenetic modifications resulting from different genomes adjusting to a common nuclear environment have been observed in polyploids. Sequence restructuring within genomes involving retrotransposon/microsatellite-rich regions has been reported in triticale. The present study uses inter-retrotransposon amplified polymorphisms (IRAP) and retrotransposon microsatellite amplified polymorphisms (REMAP) to assess genome rearrangements in wheat–rye addition lines obtained by the controlled backcrossing of octoploid triticale to hexaploid wheat followed by self-fertilization. The comparative analysis of IRAP and REMAP banding profiles, involving a complete set of wheat–rye addition lines, and their parental species revealed in those lines the presence of wheat-origin bands absent in triticale, and the absence of rye-origin and triticale-specific bands. The presence in triticale × wheat backcrosses (BC) of rye-origin bands that were absent in the addition lines demonstrated that genomic rearrangement events were not a direct consequence of backcrossing, but resulted from further genome structural rearrangements in the BC plant progeny. PCR experiments using primers designed from different rye-origin sequences showed that the absence of a rye-origin band in wheat–rye addition lines results from sequence elimination rather than restrict changes on primer annealing sites, as noted in triticale. The level of genome restructuring events evaluated in all seven wheat–rye addition lines, compared to triticale, indicated that the unbalanced genome merger situation observed in the addition lines induced a new round of genome rearrangement, suggesting that the lesser the amount of rye chromatin introgressed into wheat the larger the outcome of genome reshuffling.     

Genetic Diversity in Old Portuguese Durum Wheat Cultivars Assessed by Retrotransposon-Based Markers

Retrotransposon-based markers, such as the inter-retrotransposon-amplified polymorphism (IRAP) and the retrotransposon microsatellite-amplified polymorphism (REMAP) are highly informative, multilocus, and reveal insertional polymorphisms among plant individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, genetic mapping linkage, and for the detection of genetic rearrangements induced by polyploidization. In this study, we used IRAP and REMAP markers to assess the genetic diversity among 51 old Portuguese durum wheat cultivars belonging to 27 botanical varieties and to define their genetic relationships. Five IRAP and four REMAP primer combinations were used. IRAP markers revealed 66.3% of polymorphism and an average of 18.4 bands per primer combination which ranged in size from 450 to 3,100?bp. The REMAP technique allowed the detection of 86.36% of inter-cultivar polymorphism and an average of 11 bands per primer combination. The molecular weight of the REMAP bands ranged from 250 to 2,750?bp. The durum wheat cultivars analyzed here belong to 27 botanical varieties of the subspecies Triticum turgidum subsp. turgidum L. [syn. T. turgidum] and Triticum turgidum L. subsp. durum [syn. T. durum] (Desf.) Husn.. Our results showed that the genetic variability assessed by both the IRAP and REMAP markers did not allow the clustering of the durum wheat cultivars according to their taxonomical criteria (subspecies or botanical variety) or homonymy. Nonetheless, these markers were useful for the assessment of genetic diversity at the individual level, for the definition of genetic relationships among cultivars, and for estimation of the genetic structure of the Old collection under analysis. The achieved data could be valuable for future experiments of DNA fingerprinting, genetic improvement, and germplasm conservation in wheat.     

Comparison of ISSR,IRAP and REMAP markers for assessing genetic diversity in different species of <Emphasis Type="Italic">Brassica</Emphasis> sp.

Molecular markers provide facilities in order to study genetic diversity and relationship among genotypes. In this study, genetic diversity among 35 genotype of Brassica sp. (belonging B. napus, B. juncea, B. rapa, B. nigra) were determined using 13 ISSR, 3 IRAP markers and 18 REMAP (primer combinations of ISSR and retrotransposon primer). The percentage of polymorphism for ISSR, IRAP and REMAP was 96.38, 94 and 96%, respectively. By comparison between markers, ISSRs indicated the highest expected heterozygosity (He) and Shannon’s information index (I) with value of 0.34 and 0.51, respectively, while REMAP marker had by far the highest number of polymorphic bands (340) and marker index (7.1) among all fragments scored over all markers. In pattern of clustering based on Bayesian methods, K = 8 was resulted for combined data clustering that was more organized clustering for genotypes compared to others. This research suggests the combined data of ISSR, IRAP and REMAP markers are most reliable than each solely marker whilst have been clustered genotypes in their taxonomic classification of Brassica without any mixture. Principle coordinate analysis (PCoA) separated 35 genotypes in four groups which all of genotypes were clustered correctly based on their taxonomic classification. The findings of this study provide the valuable insight into the Brassica species relationships in terms of similarity among genotypes which can be helpful in breeding programs, and also demonstrate that retrotransposon markers are legible for genetic diversity and next genetic analysis in Brassica genus.     

Retrotransposon-based S-SAP as a platform for the analysis of genetic variation and linkage in globe artichoke.

A high copy number of retrotransposon sequences are present and widely dispersed in plant genomes. Their activity generates a considerable degree of sequence polymorphism. Here, we report the cloning of CYRE-5, a long-terminal repeat carrying retrotransposon-like sequence in Cynara cardunculus L., and its exploitation to develop a DNA fingerprinting assay across 22 accessions, including both cultivated (globe artichoke and cultivated cardoon) and wild (wild cardoon) types. The effectiveness of the sequence-specific amplified polymorphism (S-SAP) platform is compared with that of amplified fragment length polymorphism (AFLP). A genetic linkage analysis, based on a hybrid population between 2 globe artichoke varietal types, resulted in the inclusion of 29 S-SAP loci in the core genetic map, confirming their dispersed distribution across the globe artichoke genome.