Retrotransposon-microsatellite Amplified Polymorphism Markers Research Articles

High genetic diversity in Aegilops tauschii Coss. accessions from North Iran as revealed by IRAP and REMAP markers

BackgroundAegilops tauschii Coss. as a donor of wheat D genome has an important role in wheat breeding programs. Genetic and phylogeographic diversity of 79 Ae. tauschii accessions collected from north and northwest of Iran were analyzed based on retroelement insertional polymorphisms using inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers. ResultsIn total, 306 and 151 polymorphic bands were amplified in IRAP and REMAP analyses, respectively. As a result, a high level of polymorphism was observed among the studied accessions as revealed by an average of 25.5 bands per primer/primer combination and mean PIC value of 0.47 in IRAP and an average of 25.16 bands per primer combination and mean PIC value of 0.47 in REMAP. Genetic relationships of the accessions were analyzed using distance- and model-based cluster analyses. ConclusionThe result showed that genetic distance did not seem to be related to geographic distribution, and the accessions could be divided into three groups, which was further supported by principal coordinate analysis. These results on genetic diversity and population structure of Ae. tauschii in Iran should provide important knowledge on genetic resources and their applications in wheat breeding programs.

Biosystematics, fingerprinting and DNA barcoding study of the genus Lallemantia based on SCoT and REMAP markers

Lallemantia is a medicinally important plant in the world. Due to interspecific hybridization and horizontal gene transfer, spices relationship and delimitation on the genus Lallemantia is difficult based on different molecular markers. Therefore, selecting the appropriate marker can be important. Fingerprinting techniques continue to be used for genomic profiling for the characterization of germplasm and the establishment of the identity of varieties/hybrids/parental sources of aromatic and medicinal plants. For this, we need to produce detailed information on genetic diversity available in Lallemantia as well as investigate spices relationship and delimitation. Therefore, the present study was performed on Lallemantia species in Iran. We used the start codon targeted and retrotransposon-microsatellite amplified polymorphism molecular marker for our genetic investigation with the following aims: 1- To reveal the species delimitation and species relationship in Lallemantia, and 2- To investigate discriminating power of the start codon targeted and retrotransposon-microsatellite amplified polymorphism markers by Gst and NM analysis. The results obtained revealed that the start codon targeted marker is the best to show the relationships between species while the retrotransposon-microsatellite amplified polymorphism marker is the best for species delimitation. we found the loci with the high value of Gst (1.00) in start codon targeted and retrotransposon-microsatellite amplified polymorphism markers that can be used in barcoding and fingerprinting of L. royleana.

Development and use of retrotransposons-based markers (IRAP/REMAP) to assess genetic divergence among table grape cultivars

AbstractFor more than four decades after the introduction of cv. Italia (Vitis vinifera L.) in Brazil, several somatic mutations in the genome of cv. Italia and its somatic mutants gave rise to phenotypes which generated at least five new cultivars of fine table grapes. Since no molecular marker proved to be effective in discriminating cv. Italia (V. vinifera L.) and its coloured mutants (Rubi, Benitaka, Brasil, Black Star), primers for the long terminal repeat (LTR) sequences were developed to analyse Inter Retrotransposon Amplified Polymorphism (IRAP) and Retrotransposon-Microsatellite Amplified Polymorphism (REMAP), and investigate how the coloured cultivars derived from clonal propagations of somatic mutations are genetically structured. Primers for LTR sequences of IRAP and REMAP markers were edited from grape sequence databases available at a GenBank. Twenty-four primers, denominated DKS001–DKS024, were edited. Three hundred and forty-nine DNA segments were amplified by individual DKS primers and DKS/ISSR (Inter Simple Sequence Repeats) primer combinations, at an average of 13.96 amplicons per primer pair. High genetic divergence between the five cultivars was inferred from polymorphism in retrotransposons IRAP and REMAP. The analysis of polymorphism of IRAP and REMAP retrotransposons was crucial to show that clonal propagation of somatic mutations may lead towards the formation of genetically divergent cultivars by the formation of genetically structured vineyards and show the mixture of genomes within each cultivar.

IRAP and REMAP based genetic diversity among varieties of Lallemantia iberica.

This study describes the genetic relationships among 34 varieties of Lallemantia iberica using inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP). Samples were collected from Agriculture Research Center of Urmia city (northwest Iran). Ten IRAP and REMAP primers generated 76 scorable electrophoretic bands with 78.94% polymorphism. The pair-wise Jacquard genetic similarity varied from 0.48 to 0.94 for IRAP and REMAP data combined. Average PIC values for IRAP and REMAP markers were 0.38. The retro-elements marker system produced 76 alleles in range of 100- 3000 bp. The cophenetic correlation coefficient between Jaccard's similarity matrix and the plotted dendrogram was 0.66. A dendrogram constructed based on COMPLETE LINKAGE. Cluster analysis of IRAP and REMAP data using the NTSYSpc 2.02 resulted in five clusters. The present study represents high genetic distance at genotype level suggesting that IRAP and REMAP markers are useful for Lallemantia iberica genetic diversity analysis.

Assessments of genetic diversity in Iranian flax populations using retrotransposon microsatellite amplification polymorphisms (REMAP) markers

Flax (Linum usitatissimum L., 2n = 30), is an annual self-pollinated crop. It is used in oil and fiber industries, and also valued for pharmaceutical and nutraceutical applications. Characterization of flax gene pool and evaluation of genetic diversity are crucial for germplasm conservation and breeding. In present study, eighteen retrotransposon microsatellite amplified polymorphism markers were used to evaluate the genetic diversity of cultivated flax within and among populations. Thirty samples of flax plants from three different climates and geographic regions in Iran were used for present investigation. Analysis of molecular variance test showed significant genetic difference (PhiPT = 0.595, P = 0.001) among the studied populations in L. usitatissimum. 41% of total genetic variability was due to within population diversity, whereas 59% was due to among population genetic differentiation. The PCoA plot and cluster analysis grouped plants of Saveh population in a cluster entirely separated from the other two populations and showed high genetic affinity between the two Shiraz and Orumieh populations. STRUCTURE plot identified two distinct gene pools for flax, so that the population of Saveh is different from other populations, but some degree of shared alleles occurred between them.

Patterns of Evolutionary Trajectories and Domestication History within the Genus Hordeum Assessed by REMAP Markers.

The patterns of genetic diversity related to the taxonomy and domestication history of 85 accessions representing the main four species of the genus Hordeum were examined by retrotransposon-microsatellite amplified polymorphism (REMAP) markers based on the retrotransposon BARE-1. A substantial level of genetic polymorphisms at among- and within-species level was observed showing that this retrotransposon family and its adjacent genomic regions has been a target for genome dynamics during the evolution and domestication of barley. The obtained data are consistent with the current taxonomic status within the genus Hordeum. Similar level of genetic diversity was observed between the wild and the domesticated barley accessions suggesting that transposable elements` activity and accumulation may counteract the decrease of genome-wide diversity following domestication. In addition, eco-geographical sub-genome pools of the cultivated barley were identified in support to the theory of multiple origins of domestication within the genus Hordeum. We also provide conclusions about the relationship between accessions of different species and the putative routes of barley domestication. In conclusion, the retrotransposon BARE-1 stands as a reliable and perspective DNA marker for the assessment of the phylogenetic and domestication history in the genus Hordeum and other crop species.

Retrotransposon-related genetic distance among inbred lines of sweet corn (Zea mays var. saccharata) and hybrid performance

AbstractHeterosis is a main force underlying the hybrid seed industry in maize. Our experimental approach consists of a correlation study between retrotransposon-related genetic distances between parental inbred lines and hybrid performance. The assumption is that, at least for certain traits, heterosis results from genome rearrangements, largely related to retrotransposon insertions and/or removals. Fifteen maize inbred lines and one F1 hybrid, representative of the genetic diversity among sweet corn and field corn lines were screened for polymorphism by retrotransposon microsatellite amplified polymorphism markers. DNA fingerprints served as row data for estimating genetic diversity of maize inbred lines and its correlation with the heterotic effect in their hybrids. A correlation between phenotypic and molecular distances was evident only at the level of individual inbred lines. Weak correlation between genetic distances and heterosis effect was observed for the average of all inbred lines. Phenotypic distances negatively correlated with heterosis for insertion height, diameter of the ear and number of kernel rows per ear. The relative contribution of each inbred line to heterosis in its derived hybrids was also estimated. Accordingly, we identified inbred lines with increased genetic distances that mostly add to the heterosis effect in their hybrids and that we recommend as prospective to be used in maize breeding programmes.

Use of IRAP and REMAP markers to interpret the population structure of Linum usitatissimum from Iran

AbstractFlax (

High genetic variability in endophytic fungi from the genus Diaporthe isolated from common bean (Phaseolus vulgaris L.) in Brazil.

The goals of the present study were to identify, to analyse the phylogenetic relations and to evaluate the genetic variability in Diaporthe endophytic isolates from common bean. Diaporthe sp., D. infecunda and D. phaseolorum strains were identified using multilocus phylogeny (rDNA ITS region; EF1-α, β-tubulin, and calmodulin genes). IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) molecular markers reveal the existence of high genetic variability, especially among D. infecunda isolates. It was concluded that the multilocus phylogenetic approach was more effective than individual analysis of ITS sequences, in identifying the isolates to species level, and that IRAP and REMAP markers can be used for studying the genetic variability in the genus Diaporthe particularly at the intraspecific level. The combined use of molecular tools such as multilocus phylogenetic approach and molecular markers, as performed in this study, is the best way to distinguish endophytic strains of Diaporthe isolated from common bean (Phaseolus vulgaris L.).

Development of retrotransposon-based markers IRAP and REMAP for cassava (Manihot esculenta).

Retrotransposons are abundant in the genomes of plants. In the present study, inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers were developed for the cassava genome (Manihot esculenta Crantz). Four cassava cultivars (Fécula Branca, IPR-União, Olho Junto, and Tamboara, two samples per cultivar) were used to obtain IRAP and REMAP fingerprints. Twelve designed primers were amplified alone and in combinations. The 42 IRAP/REMAP primer combinations amplified 431 DNA segments (bands; markers) of which 36 (8.36%) were polymorphic. The largest number of informative markers (16) was detected using the primers AYF2 and AYF2xAYF4. The number of bands for each primer varied from 3 to 16, with an average of 10.26 amplified segments per primer. The size of the amplified products ranged between 100 and 7000 bp. The AYF2 primer generated the highest number of amplified segments and showed the highest number of polymorphic bands (68.75%). Two samples of each cassava cultivar were used to illustrate the usefulness and the polymorphism of IRAP/REMAP markers. IRAP and REMAP markers produced a high number of reproducible bands, and might be informative and reliable for investigation of genetic diversity and relationships among cassava cultivars.

Endophytic fungi from the genus Colletotrichum are abundant in the Phaseolus vulgaris and have high genetic diversity.

To evaluate the diversity of endophytic fungi from the leaves of the common bean and the genetic diversity of endophytic fungi from the genus Colletotrichum using IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism) analyses. The fungi were isolated by tissue fragmentation and identified by analysing the morphological features and sequencing the internal transcribed spacer (ITS) regions and the rDNA large subunit (LSU). Twenty-seven different taxa were identified. Colletotrichum was the most commonly isolated genera from the common bean (32.69% and 24.29% of the total isolates from the Ouro Negro and Talismã varieties, respectively). The IRAP and REMAP analyses revealed a high genetic diversity in the Colletotrichum endophytic isolates and were able to discriminate these isolates from the phytopathogen Colletotrichum lindemuthianum. Fungi from the genus Colletotrichum are abundant in the Phaseolus vulgaris endophytic community, and the IRAP and REMAP markers can be used to rapidly distinguish between C. lindemuthianum and other Colletotrichum members that are frequently found as endophytes. This is the first report of the diversity of endophytic fungi present in the common bean and the use of IRAP and REMAP markers to assess the genetic diversity of endophytic fungi from the genus Colletotrichum.

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.

Assessment of genetic diversity among Indian potato (Solanum tuberosum L.) collection using microsatellite and retrotransposon based marker systems

Potato (Solanum tuberosum) is an important non-cereal crop throughout the world and is highly recommended for ensuring global food security. Owing to the complexities in genetics and inheritance pattern of potato, the conventional method of cross breeding for developing improved varieties has been difficult. Identification and tagging of desirable traits with informative molecular markers would aid in the development of improved varieties. Insertional polymorphism of copia-like and gypsy-like long terminal repeat retrotransposons (RTN) were investigated among 47 potato varieties from India using Inter-Retrotransposon Amplified Polymorphism (IRAP) and Retrotransposon Microsatellite Amplified Polymorphism (REMAP) marker techniques and were compared with the DNA profiles obtained with simple sequence repeats (SSRs). The genetic polymorphism, efficiency of polymorphism and effectiveness of marker systems were evaluated to assess the extent of genetic diversity among Indian potato varieties. A total of 139 polymorphic SSR alleles, 270 IRAP and 98 REMAP polymorphic bands, showing polymorphism of 100%, 87.9% and 68.5%, respectively, were used for detailed characterization of the genetic relationships among potato varieties by using cluster analysis and principal coordinate analysis (PCoA). IRAP analysis resulted in the highest number of polymorphic bands with an average of 15 polymorphic bands per assay unit when compared to the other two marker systems. Based on pair-wise comparison, the genetic similarity was calculated using Dice similarity coefficient. The SSRs showed a wide range in genetic similarity values (0.485–0.971) as compared to IRAP (0.69–0.911) and REMAP (0.713–0.947). A Mantel’s matrix correspondence test showed a high positive correlation (r=0.6) between IRAP and REMAP, an intermediate value (r=0.58) for IRAP and SSR and the lowest value (r=0.17) for SSR and REMAP. Statistically significant cophenetic correlation coefficient values, of 0.961, 0.941 and 0.905 were observed for REMAP, IRAP and SSR, respectively. The widespread presence and distinct DNA profiles for copia-like and gypsy-like RTNs in the examined genotypes indicate that these elements are active in the genome and may have even contributed to the potato genome organization. Although the three marker systems were capable of distinguishing all the 47 varieties; high reproducibility, low cost and ease of DNA profiling data collection make IRAP and REMAP markers highly efficient whole-genome scanning molecular probes for population genetic studies. Information obtained from the present study regarding the genetic association and distinctiveness provides an useful guide for selection of germplasm for plant breeding and conservation efforts.

English

Potato dextrose and malt extract agar were found to be suitable for culturing different isolates of Pyricularia oryzae. Colonies of P. oryzae appeared as white on oat meal, rice polish and malt extract agar, grey on potato dextrose agar and whitish grey on rice agar. Spore induction was hastened on maize stem pieces than on rice and Panicum repens. Spore density on maize was 3420/ml of water as against 500/ml on P. repens. When spores of 11 isolates of P. oryzae were compared, conidia of the isolate from Pennisetum purpureum were significantly bigger than the other isolates. The spores of rice isolates from Erode and Gopichettipalayam were significantly smaller in length and width.Fingerprinting of 11 isolates of P. oryzae by retrotransposon - microsatellite amplified polymorphism (REMAP) showed a high level of variability and polymorphism among them. Phylogenetic analysis using REMAP markers grouped out one rice isolate from others. Two isolates of Tirunelveli region were grouped together like Erode and Gopichettipalayam isolates. Isolates causing leaf and neck blast showed 52.9% similarity. Isolates from Coimbatore and Gudalore were 32.7% similar; likewise two non-rice host isolates were grouped together with 27.6% similarity.   Key words: Variability, culture media, rice blast, Pyricularia oryzae, retrotransposon, microsatellite.

Construction of the first genetic linkage map of Japanese gentian (Gentianaceae)

BackgroundJapanese gentians (Gentiana triflora and Gentiana scabra) are amongst the most popular floricultural plants in Japan. However, genomic resources for Japanese gentians have not yet been developed, mainly because of the heterozygous genome structure conserved by outcrossing, the long juvenile period, and limited knowledge about the inheritance of important traits. In this study, we developed a genetic linkage map to improve breeding programs of Japanese gentians.ResultsEnriched simple sequence repeat (SSR) libraries from a G. triflora double haploid line yielded almost 20,000 clones using 454 pyrosequencing technology, 6.7% of which could be used to design SSR markers. To increase the number of molecular markers, we identified three putative long terminal repeat (LTR) sequences using the recently developed inter-primer binding site (iPBS) method. We also developed retrotransposon microsatellite amplified polymorphism (REMAP) markers combining retrotransposon and inter-simple sequence repeat (ISSR) markers. In addition to SSR and REMAP markers, modified amplified fragment length polymorphism (AFLP) and random amplification polymorphic DNA (RAPD) markers were developed. Using 93 BC1 progeny from G. scabra backcrossed with a G. triflora double haploid line, 19 linkage groups were constructed with a total of 263 markers (97 SSR, 97 AFLP, 39 RAPD, and 30 REMAP markers). One phenotypic trait (stem color) and 10 functional markers related to genes controlling flower color, flowering time and cold tolerance were assigned to the linkage map, confirming its utility.ConclusionsThis is the first reported genetic linkage map for Japanese gentians and for any species belonging to the family Gentianaceae. As demonstrated by mapping of functional markers and the stem color trait, our results will help to explain the genetic basis of agronomic important traits, and will be useful for marker-assisted selection in gentian breeding programs. Our map will also be an important resource for further genetic analyses such as mapping of quantitative trait loci and map-based cloning of genes in this species.

English

  ‘Wuzishatangju’(Citrus reticulata Blanco) is an excellent cultivar derived from a bud sport of a seedy ‘Shatangju’ cultivar found in Guangdong Province in the 1980s. In this study, six molecular markers including random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), simple sequence repeat (SSR), sequence-related amplified polymorphism (SRAP), inter-retrotransposn amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) were used to study the genetic variations between ‘Shatangju’ and ‘Wuzishatangju’. 1196 RAPD, seven SSR, 28 IRAP and 56 REMAP primers were used to detect the genetic variations between ‘Shatangju’ and ‘Wuzishatangju’. However, no difference was observed between the two cultivars. These results indicate that there was a very close genetic relationship between ‘Shatangju’ and ‘Wuzishatangju’ and RAPD, SSR, IRAP and REMAP markers could not distinguish them. Two and 21 specific bands were obtained using 100 ISSR and 153 SRAP primers, respectively. The present research could be a valuable tool for identification of Citrus bud sport clones, which laid the foundations for the further study of the mechanisms of Citrus bud sports.   Key words: Citrus reticulata Blanco, random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), simple sequence repeat (SSR), sequence-related amplified polymorphism (SRAP), inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), identification.

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.

Development of new molecular markers for the Colletotrichum genus using RetroCl1 sequences

A nonautonomous element of 624 bp, called RetroCl1 (Retroelement Colletotrichum lindemuthianum 1), was identified in the plant pathogenic fungus Colletotrichum lindemuthianum. RetroCl1 contains terminal direct repeats (223 bp) that are surrounded by CTAGT sequences. It has a short internal domain of 178 bp and shows characteristics of terminal-repeat retrotransposon in miniature (TRIM) family. We used RetroCl1 sequence to develop molecular markers for the Colletotrichum genus. IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) markers were used to analyze the genetic diversity of C. lindemuthianum. Fifty-four isolates belonging to different races were used. A total of 45 loci were amplified. The Nei index showed significant differences among the populations divided according to race, indicating that they are structured according to pathotype. No clear correlation between IRAP and REMAP markers with pathogenic characterization was found. C. lindemuthianum has high genetic diversity, and the analysis of molecular variance showed that 51% of variability is found among the populations of different races. The markers were also tested in different Colletotrichum species. In every case, multiple bands were amplified, indicating that these markers can be successfully used in different species belonging to the Colletotrichum genus.

Retroelement Insertional Polymorphism and Genetic Diversity in Medicago sativa Populations Revealed by IRAP and REMAP Markers

Retrotransposons (RTNs) are common components of plant genomes, showing activity at transcription and integration levels. Their abundance, dispersion, ubiquity, and ability to transpose make them useful as molecular markers. In this study, we used multilocus PCR-based techniques, inter-retrotransposon amplified polymorphism (IRAP), and retrotransposon-microsatellite amplified polymorphism (REMAP) to study the integration events of native (Tms1Ret1) and non-native RTN (LORE1, LORE2, Tps12a, and Tps19) families in M. sativa genome. IRAP and REMAP markers derived from these RTNs were used to assess genetic diversity among and within 80 M. sativa genotypes belonging to eight populations. Results indicated the presence and transpositional activation of RTNs Tms1Ret1, LORE1, LORE2, and even Tps12a in M. sativa genome. REMAP analysis showed the insertion of studied RTN families near the microsatellites in M. sativa genome except for RTN Tps19. A total of 101 and 119 loci were amplified using 10 and 14 IRAP and REMAP primers, respectively. The number of polymorphic loci was 66 and 62 for IRAP and REMAP, respectively. Mantel test between IRAP and REMAP cophenetic matrices evidenced no significant correlation (r = 0.15). Although populations could be relatively differentiated based on IRAP + REMAP data, overall genetic differentiation among populations was low (PhiPT = 0.082, P = 0.0010). Analysis of molecular variance based on IRAP + REMAP analysis revealed the higher level of genetic variation within populations (92%) compared to among populations (8%). IRAP + REMAP-based cluster analysis of 80 M. sativa genotypes using complete linkage algorithm identified five heterotic groups that could be applied as crossing parents in M. sativa alfalfa breeding programs.

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.