Retrotransposon-microsatellite Amplified Polymorphism Research Articles

Epigenetic changes detected in micropropagated hop plants

Micropropagation is a widely used technique in hops (Humulus lupulus L.). However, to the best of our knowledge, the genetic and epigenetic stability of the microplants has never been tested before. In the present study, two hop accessions were established in vitro and micropropagated for 2 years. The genetic and epigenetic stability of the in vitro plants was analyzed with several molecular techniques: random amplified DNA polymorphism (RAPD), retrotransposon microsatellite amplified polymorphism (REMAP), and methylation-sensitive amplification polymorphism (MSAP). No genetic variation among control and treated plants was found, even after 12 cycles of micropropagation. Epigenetic variation was detected, first, when field and in vitro samples were compared. Nearly a 30% of the detected fragments presented the same pattern of alterations in all the vitroplants. Second, lower levels of epigenetic variation were detected among plants from the different subcultures. Part of this detected variation seemed to be accumulated along the 12 sequential subcultures tested.

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.

Retrotransposon Microsatellite Amplified Polymorphism Strain Fingerprinting Markers Applicable to Various Mushroom Species

The retrotransposon marY1 is a gypsy family retroelement, which is detected ubiquitously within the fungal taxonomic groups in which mushrooms are included. To utilize marY1 as a molecular marker for the DNA fingerprinting of mushrooms, oligonucleotides marY1-LTR-L and marY1-LTR-R were designed on the basis of highly conserved regions from the multiple sequence alignment of 30 marY1 sequences retrieved from a nucleotide sequence database. In accordance with Retrotransposon Microsatellite Amplified Polymorphism (REMAP) fingerprinting methodology, the two oligonucleotides were utilized together with the short sequence repeat primers UBC807 and UBC818 for polymerase chain reaction using templates from different mushroom genomic DNAs. Among the tested oligonucleotides, the marY1-LTR-L and UBC807 primer set yielded the greatest amount of abundance and variation in terms of DNA band numbers and patterns. This method was successfully applied to 10 mushroom species, and the primer set successfully discriminated between different commercial mushroom cultivars of the same strains of 14 Pleurotus ostreatus and 16 P. eryngii. REMAP reproducibility was superior to other popular DNA fingerprinting methodologies including the random amplified polymorphic DNA method.

The first doubled haploid linkage map for cultivated oat

To date, all linkage maps of hexaploid oat (Avena sativa L.) have been constructed using recombinant inbred lines (RILs). Doubled haploids (DHs), however, have the advantage over RILs of their comprehensive homozygosity. DHs have been used for mapping in several cereal species, but in oats the production of large DH populations has only recently become an option. A linkage map of hexaploid oat was constructed using an anther culture-derived DH population (137 individuals) from the F1 individuals of a cross between the Finnish cultivar 'Aslak' and the Swedish cultivar 'Matilda'. The map is composed of 28 linkage groups containing 625 DNA markers: 375 AFLPs (amplified fragment length polymorphisms), 3 IRAPs (inter-retrotransposon amplified polymorphisms), 12 ISSRs (inter simple sequence repeats), 12 microsatellites, 57 RAPDs (random amplified polymorphic DNAs), 59 REMAPs (retrotransposon-microsatellite amplified polymorphisms), 105 SRAPs (sequence-related amplified polymorphisms), and 2 SNPs (single-nucleotide polymorphisms). The total map size is 1526 cM. Over half of the markers in the map showed distorted segregation, with alleles from 'Aslak' usually prevailing. This is explained by the better performance of 'Aslak' in anther culture. Quantitative trait loci affecting some important quality and agronomic traits are being localized on the map.

Genetic variation in natural populations of Hordeum brevisubulatum native to the Songnen Prairie in northeastern China: Comparison of four nuclear DNA markers

Hordeum brevisubulatum (Trin) Link, a perennial wild grass, shows high tolerance to salinity- and/or alkaline-eroded soil, and is an important forage grass. Nonetheless, the genetic variation within and among natural populations of this species has not been investigated. In this study, four PCR-based dominant nuclear DNA markers, i.e., amplified fragment length polymorphism (AFLP), inter-simple sequence repeat (ISSR), retrotransposon-microsatellite-amplifiedpolymorphism (REMAP), and random amplified polymor phic DNA (RAPD) were used to investigate the genetic variation of a total of 80 plants with an average of 20 individual plants randomly collected from each of the four natural populations of H. brevisubulatum distributed in the Songnen Prairie in northeastern China. Results indicated that all four markers detected a surprisingly high level of genetic variation among the individual genotypes both within and among populations. Specifically, (i) of the 708 bands produced by 10 AFLP primer pairs, 223 (32%) were polymorphic, corresponding to a genetic similarity coefficient (Dice) range of 0.703 to 0.895 with a mean of 0.793; (ii) of the 174 bands produced by 12 ISSR primers, 149 (85%) were polymorphic, corresponding to a genetic similarity coefficient range of 0.524 to 0.826 with a mean of 0.676; (iii) of the 104 bands produced by REMAP (one retrotransopon-specific primer in combination with each of 8 ISSR primers), 89 (85%) were polymorphic, leading to an average similarity coefficient range of 0.494–0.840 with a mean of 0.688, and; (iv) of the 150 bands produced by 17 RAPD primers, 117 (77%) were polymorphic, giving a similarity coefficient range of 0.526–0.909 with a mean of 0.713. Thus, all four markers are highly informative in detecting genetic variations among the individual genotypes in natural populations of H. brevisubulatum. Nevertheless, no correlation was detected among the markers, suggesting that they likely targeted to different genomic regions of H. brevisubulatum. Whereas ISSR, REMAP and RAPD showed significantly higher levels of polymorphism than AFLP, only the latter was capable of grouping some of the plants largely in accordance with their geographic localities, suggesting population differentiation probably occurred preferentially at genomic regions targeted to by AFLP primers. Key words: Genetic diversity, Hordeum brevisubulatum, Molecular markers, Population differentiation

IRAP and REMAP assessments of genetic similarity in rice

Rice is a model genome for cereal research, providing important information about genome structure and evolution. Retrotransposons are common components of grass genomes, showing activity at transcription, translation and integration levels. Their abundance and ability to transpose make them good potential markers. In this study, we used 2 multilocus PCR-based techniques that detect retrotransposon integration events in the genome: IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism). Markers derived from Tos17, a copia-like endogenous retrotransposon of rice, were used to identify genetic similarity among 51 rice cultivars (Oryza sativa L.). Genetic similarity analysis was performed by means of the Dice coefficient, and dendrograms were developed by using the average linkage distance method. A cophenetic correlation coefficient was also calculated. The clustering techniques revealed a good adjustment between matrices, with correlation coefficients of 0.74 and 0.80, or lower (0.21) but still significant, between IRAP and REMAP-based techniques. Consistent clusters were found for Japanese genotypes, while a subgroup clustered the irrigated Brazilian genotypes.

A major gene for grain cadmium accumulation in oat (Avena sativa L.)

Cadmium (Cd) is a nonessential heavy metal that is highly toxic to living cells at very low concentrations. Most of the Cd in plants derives from soils. Owing to the large amounts consumed, cereals are the major source of dietary Cd, and Cd content in oat can exceed accepted limits. Plants have a set of mechanisms that control the uptake, accumulation, trafficking, and detoxification of Cd and other metals. Genetic factors affect the variation in Cd level between plant species and cultivars, and the development of cultivars that poorly accumulate Cd is a worthwhile goal. Because of the expense of Cd screening, the use of molecular markers linked to low Cd accumulation could be an alternative to phenotyping for selection. In this study, such markers were sought using bulked-segregant analysis in an F2 population from the cross between oat cultivars 'Aslak' and 'Salo', the second of which is known to be a high Cd accumulator. Four markers associated with grain Cd concentration were found: 2 RAPDs (random amplified polymorphic DNAs), 1 REMAP (retrotransposon-microsatellite amplified polymorphism), and 1 SRAP (sequence-related amplified polymorphism). The first 3 were converted into more reproducible SCAR (sequence-characterized amplified region) markers. The 4 markers were assigned to 1 linkage group that exhibited a QTL (quantitative trait locus) representing a major gene for grain Cd concentration.

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.

QTL mapping for economic traits based on a dense genetic map of cotton with PCR-based markers using the interspecific cross of Gossypium hirsutum × Gossypium barbadense

A high-density molecular marker linkage map of cotton based entirely on polymerase chain reaction-based markers is useful for a marker-assisted breeding program. Four kinds of markers—simple sequence repeats (SSRs), sequence-related amplified polymorphism (SRAP), random amplified polymorphic DNA (RAPD), and retrotransposon-microsatellite amplified polymorphism (REMAP)—were used to assay an F2 population from a cross between “Handan208” (Gossypium hirsutum) and “Pima90” (Gossypium barbadense). Sixty-nine F2 plants were used for map construction using 834 SSRs, 437 SRAPs, 107 RAPDs, and 16 REMAPs. Linkage analysis revealed that 1,029 loci could be mapped to 26 linkage groups that extended for 5,472.3 cM, with an average distance between 2 loci of 5.32 cM. The corresponding 69 F2:3 families were grown, arranged in two replicates, and scored for eight phenotypes. Quantitative trait loci (QTL) analysis was performed by means of composite interval mapping using WinQtlCart ver 2.0. A total of 52 distinct QTLs were detected: 4 QTLs for lint index, 8 for seed index, 11 for lint yield, 4 for seed cotton yield, 9 for number of seed per boll, 3 for fiber strength, 5 for fiber length, and 8 for micronaire value. The present map and QTL analysis may provide a useful tool for breeders to transfer desirable traits from G. barbadense to the mainly cultivated species, G. hirsutum.

Generation of SNP markers for short straw in oat (Avena sativa L.)

Short straw is a desired trait in oat germplasm (Avena sativa L.). Marker-assisted selection, a key tool for achieving this objective, is limited by the presence and number of available markers. Here, we have attempted to develop markers sufficiently linked to a gene specifying short straw so that marker-assisted selection could be applied. Bulked-segregant analysis was used to identify anonymous PCR-based markers associated with the dwarfing gene Dw6 in an F2 population from the cross between A. sativa "Aslak" and A. sativa "Kontant". One random amplified polymorphic DNA (RAPD) and 1 retrotransposon-microsatellite amplified polymorphism (REMAP) marker were found to be associated with height. These were converted into codominant single-nucleotide polymorphism (SNP) markers. The SNP-REMAP and the SNP-RAPD markers were located 5.2 and 12.6 cM from Dw6, respectively. They can be used in future efforts both to enhance oat germplasm by application of molecular markers and to determine the nature of the gene through positional cloning.

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.

Genetic relationships of Diospyros kaki Thunb. and related species revealed by IRAP and REMAP analysis

A modified method of inter-retrotransposon amplified polymorphism (IRAP) with primers annealing to the conserved reverse transcriptase sites instead of the conserved long-terminal repeat (LTR) regions and retrotransposon–microsatellite amplified polymorphism (REMAP) markers were used to investigate genetic relationships of some Japanese persimmon ( Diospyros kaki Thunb.) genotypes from Japan and China and the related species. In total, 117 scorable amplification bands ranging from 250 to 2000 bp were generated by IRAP and REMAP analysis, among which 101 (86%) were polymorphic, with an average of six polymorphic bands per primer combination. The analysis of genetic relationships of 27 genotypes revealed that the Japanese, Chinese genotypes and related species were distinctly separated. The phylogenetic trees constructed by unweighted pair-group method with arithmetic averages (UPGMA) and Wagner parsimony cluster methods agreed well with the geographic origin information. The Japanese persimmon genotypes from Japan and China may have different genetic background, suggesting a long independent domestication, intercrossing and breeding history in both regions. ‘Baogaitianshi’ and ‘Xiaobaogaitianshi’ had a high similarity coefficient, implying that they possibly have the bud sport relationships. The related species were clearly clustered in a separated group, suggesting that they are very closely related.

Retrotransposon-microsatellite amplified polymorphism (REMAP) markers for genetic diversity assessment of the rice blast pathogen (Magnaporthe grisea)

This present study is the first report of the application of the retrotransposon-microsatellite amplified polymorphism (REMAP) technique in fungi. Genome fingerprinting has a major role in the characterization of population structure and in the analysis of the variability in fungi. Retrotransposon-microsatellite amplified polymorphism assay was used in virulent isolates of a rice blast pathogen (Magnaporthe grisea) as a new assay system for genetic variability studies that overcomes the limitations of previous techniques. The high polymorphism observed in REMAP could be due to past or recent actions of retrotransposon in M. grisea. Retrotransposon-microsatellite amplified polymorphism, with its superior marker utility, was concluded to be the marker of choice for characterizing M. grisea isolates.

Comparison of the utility of barley retrotransposon families for genetic analysis by molecular marker techniques.

The Sequence-Specific Amplification Polymorphism (S-SAP) method, and the related molecular marker techniques IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism), are based on retrotransposon activity, and are increasingly widely used. However, there have been no systematic analyses of the parameters of these methods or of the utility of different retrotransposon families in producing polymorphic, scorable fingerprints. We have generated S-SAP, IRAP, and REMAP data for three barley (Hordeum vulgare L.) varieties using primers based on sequences from six retrotransposon families (BARE-1, BAGY-1, BAGY-2, Sabrina, Nikita and Sukkula). The effect of the number of selective bases on the S-SAP profiles has been examined and the profiles obtained with eight MseI+3 selective primers compared for all the elements. Polymorphisms detected in the insertion pattern of all the families show that each can be used for S-SAP. The uniqueness of each transposition event and differences in the historic activity of each family suggest that the use of multiple retrotransposon families for genetic analysis will find applications in mapping, fingerprinting, and marker-assisted selection and evolutionary studies, not only in barley and other Hordeum species and related taxa, but also more generally.

IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques

The BARE-1 retrotransposon is an active, dispersed, and highly abundant component of the genome of barley (Hordeum vulgare) and other species in its genus. Like all retrotransposons of its kind, BARE-1 is bounded by long terminal repeats (LTRs). We have developed two amplification-based marker methods based on the position of given LTRs within the genome. The IRAP (Inter-Retrotransposon Amplified Polymorphism) markers are generated by the proximity of two LTRs using outward-facing primers annealing to LTR target sequences. In REMAP (REtrotransposon-Microsatellite Amplified Polymorphism), amplification between LTRs proximal to simple sequence repeats such as constitute microsatellites produces markers. The methods can distinguish between barley varieties and produce fingerprint patterns for species across the genus. The patterns indicate that although the BARE-1 family of retrotransposons is disperse, these elements are locally clustered or nested and often found near tandem arrays of a simple sequence repeat.