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.     

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.     

Effectiveness of AFLPs and Retrotransposon-Based Markers for the Identification of Portuguese Grapevine Cultivars and Clones

Grapevine germplasm, including 38 of the main Portuguese cultivars and three foreign cultivars, Pinot Noir, Pinot Blanc and Chasselas, used as a reference, and 37 true-to-type clones from the Alvarinho, Arinto, Loureiro, Moscatel Galego Branco, Trajadura and Vinh?o cultivars were studied using AFLP and three retrotransposon-based molecular techniques, IRAP, REMAP and SSAP. To study the retrotransposon-based polymorphisms, 18 primers based on the LTR sequences of Tvv1, Gret1 and Vine-1 were used. In the analysis of 41 cultivars, 517 IRAP, REMAP, AFLP and SSAP fragments were obtained, 83% of which were polymorphic. For IRAP, only the Tvv1Fa primer amplified DNA fragments. In the REMAP analysis, the Tvv1Fa-Ms14 primer combination only produced polymorphic bands, and the Vine-1 primers produced mainly ISSR fragments. The highest number of polymorphic fragments was found for AFLP. Both AFLP and SSAP showed a greater capacity for identifying clones, resulting in 15 and 9 clones identified, respectively. Together, all of the techniques allowed for the identification of 54% of the studied clones, which is an important step in solving one of the challenges that viticulture currently faces.     

Molecular Diversity in some Ghanaian Cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> L. (Walp)] Accessions

Cowpea [Vigna unguiculata L. (Walp)] is grown mainly for its protein-rich grains and is consumed in various forms in sub-Saharan Africa. Average grain yield in farmers’ fields is generally low due to a number of biotic and abiotic stresses. One hundred and six cowpea accessions from Ghana, which had previously been evaluated for seedling drought tolerance, were used for this study. This paper attempts to use three multi-locus PCR-based molecular markers; simple sequence repeats (SSR), inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphisms (REMAP), to analyse genetic diversity in the cowpea accessions. Analysis of the polymorphic bands data indicated that 101 alleles were amplified among 121 cowpea genotypes (83.4%) from 16 SSR primer pairs out of a total of 30 SSR primer pairs. Likewisely, a total of 66 (54.5%) polymorphic bands were obtained from IRAP and a total of 114 (94.2%) highly polymorphic bands obtained from REMAP analysis. The outcome indicated the highly polymorphic nature of the DNA markers, as small groups of these molecular markers were found to be able to identify each of the accessions used. Microsatellite markers (SSRs) and retrotransposon-based markers, like IRAP and REMAP, were found to be highly polymorphic and informative, suggesting that genomic fingerprinting has a major role in characterizing populations.     

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 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.     

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.     

S-SAP分子标记开发及其在苹果芽变鉴别上的应用

S-SAP(特异序列扩增多态性,Ssequence specific amplification polymorphism)是一种基于反转录转座元件的广泛应用于生物遗传多样性研究的分子标记。本研究从34对引物中筛选出7对具有谱带清晰、多态性高的引物组合,成功地开发了苹果基因组的S-SAP分子标记。27个元帅系芽变品种中,共扩增出588条谱带,每对引物组合平均扩增出84条谱带,其中多态性谱带48条,多态性谱带占总扩增出谱带数的8.2%,遗传相似系数介于0.73～0.90之间。对15个苹果芽变品种进行S-SAP分析,相似系数在0.42～0.94之间,以相似系数0.80为阈值,可以区分各芽变品系。开发的S-SAP分子标记可以有效地将苹果芽变品种区分,并为苹果生物遗传多样性与系统进化、品种鉴定提供新方法。     

Genetic relationships among a collection of Musa germplasm by fluorescent-labeled SRAP

Edible banana and plantains of the Musa genus are important staple food crops cultivated in humid tropical and subtropical climatic zones. These crops are important for subsistence farming in rural communities and also to generate significant employment and income. In an effort to increase the genetic variability of available cultivars, indexed accessions have been introduced into a regional collection in southeastern Mexico, through the Banana Bioversity International Program. The aim of this study was to use the fluorescently labeled sequence-related amplified polymorphism (SRAP) molecular marker system to characterize the genetic variability within 71 accessions of the existing collection and resolved uncertainties for the better management of the collection, as a preliminary step to establishing a breeding program. These accessions, which included wild species and cultivars of different subgroups, were consistently identified and separated by SRAP markers. A total of 330 polymorphic bands were detected using 12 primer combinations. The average number of polymorphic bands per primer pair was 27.5. The genetic similarity between accessions ranged between 0.44 and 0.97, as estimated using Jaccard's coefficient. Moreover, SRAP marker system probed to be useful to identify closely related accessions in the genus Musa and facilitated the recognition of duplicates to be eliminated and clarified uncertainties or mislabeled banana accessions introduced to the collection.     

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 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.     

Use of SSR and Retrotransposon-Based Markers to Interpret the Population Structure of Native Grapevines from Southern Italy

Native grapevines are the quintessential elements of Southern Italy winemaking, and genomic characterization plays a role of primary importance for preservation and sustainable use of these unexploited genetic resources. Among the various molecular techniques available, SSR and retrotransposons-based markers result to be the most valuable for cultivars and biotypes distinctiveness. A total of 62 accessions including 38 local grape cultivars were analyzed with 30 SSR, four REMAP and one IRAP markers to assess their genetic diversity and obtain a complete genomic profiling. The use of VrZAG79, VrZAG112, VVS2, VVMD25 and VVMD5 combined with retrotransposon-based markers proved to be the most discriminating and polymorphic markers for the rapid and unambiguous identification of minority grapevines from Campania region, which is considered one of the most appreciated Italian districts for wine production. Results revealed 58 SSR marker-specific alleles, 22 genotype-specific SSR alleles, and four REMAP and IRAP private bands. Cases of synonymy and homonymy were discovered. In conclusion, we provided evidences that the integrating SSR and retrotransposon-based markers is an effective strategy to assess the genetic diversity of autochthonous grapes, allowing their easy identification.     

Sequence related amplified polymorphism (SRAP) analysis for genetic diversity and micronutrient content among gene pools in mungbean [Vigna radiata (L.) Wilczek]

Vigna radiata (L.) Wilczek, commonly called mungbean is an important pulse crop. Commercial cultivars contain low levels of iron and zinc and it is important to assess genetic variability in the available germplasm for improving micronutrient content in commercial cultivars. The present study was undertaken to study molecular diversity using Sequence-related amplified polymorphism (SRAP) among 21 Vigna radiata genotypes. Twenty nine SRAP primer combinations produced a total of 121 amplified bands which were polymorphic with an average of 4.65 bands per primer. The size of amplified bands ranged from 70 bp to 3,000 bp and 6 out of 29 SRAP primers were most useful in fingerprinting Vigna radiata genotypes under study. The similarity coefficients between different genotypes ranged from 0.45 to 0.96 with an average similarity value of 0.71. At an arbitrary cut-off at 60 % similarity level on a dendrogram, the Vigna radiata accessions were categorized into two major clusters. ML1108 and 2KM115 were found to be genetically similar. SMH99-1A and ML776 showed high iron and zinc content while Satya was poor in iron as well as zinc content. Mapping population involving ML776 and Satya could be used for tagging gene(s) for micronutrient content. The results indicated that SRAP markers were efficient for identification of Vigna radiata genotypes and assessment of the genetic relationships among them.     

Genetic molecular analysis of Coffea arabica (Rubiaceae) hybrids using SRAP markers

In Coffea arabica (arabica coffee), the phenotypic as well as genetic variability has been found low because of the narrow genetic basis and self fertile nature of the species. Because of high similarity in phenotypic appearance among the majority of arabica collections, selection of parental lines for inter-varietals hybridization and identification of resultant hybrids at an early stage of plant growth is difficult. DNA markers are known to be reliable in identifying closely related cultivars and hybrids. Sequence Related Amplified Polymorphism (SRAP) is a new molecular marker technology developed based on PCR. In this paper, sixty arabica-hybrid progenies belonging to six crosses were analyzed using 31 highly polymorphic SRAP markers. The analysis revealed seven types of SRAP marker profiles which are useful in discriminating the parents and hybrids. The number of bands amplified per primer pair ranges from 6.13 to 8.58 with average number of seven bands. Among six hybrid combinations, percentage of bands shared between hybrids and their parents ranged from 66.29% to 85.71% with polymorphic bands varied from 27.64% to 60.0%. Percentage of hybrid specific fragments obtained in various hybrid combinations ranged from 0.71% to 10.86% and ascribed to the consequence of meiotic recombination. Based on the similarity index calculation, it was observed that F1 hybrids share maximum number of bands with the female parent compared to male parent. The results obtained in the present study revealed the effectiveness of SRAP technique in cultivar identification and hybrid analysis in this coffee species.     

Genetic variability and structure of Quercus brantii assessed by ISSR,IRAP and SCoT markers

Persian oak (Quercus brantii Lindl.) is one of the most important woody species of the Zagros forests in Iran. Three molecular marker techniques: start codon targeted (SCoT), inter-simple sequence repeat (ISSR) and inter-retrotransposon amplified polymorphism (IRAP) markers were compared for fingerprinting of 125 individuals of this species collected from different geographical locations of north-west of Iran. A total of 233 bands were amplified by 18 ISSR primers, of which 224 (96.10%) were polymorphic, and 126 polymorphic bands (97.65%) were observed in 129 bands amplified by 10 IRAP primers. Besides, 118 bands were observed for all 10 SCoT primers, of which 113 were polymorphic (95.71%). Average polymorphism information content (PIC) for ISSR, IRAP and SCoT markers was 0.30, 0.32 and 0.38, respectively, and this revealed that SCoT markers were more informative than IRAP and ISSR for the assessment of diversity among individuals. Based on the three different molecular types, cluster analysis revealed that 125 individuals taken for the analysis can be divided into three distinct clusters. The Jaccard's genetic similarity based on the combined data ranged from 0.23 to 0.76. These results suggest that efficiency of SCoT, IRAP and ISSR markers was relatively the same in fingerprinting of individuals. All molecular marker types revealed a low genetic differentiation among populations, indicating the possibility of gene flow between the studied populations. These results have an important implication for Persian oak (Q. brantii) germplasm characterization, improvement, and conservation.     

利用SRAP标记分析河南小麦栽培品种的遗传多样性

利用小麦SRAP标记对22个河南省小麦品种进行了遗传多样性分析,10对引物组合扩增获得169个条带,其中70个条带具有多态性,多态条带百分率为41．42％,每对引物平均产生7个多态性条带。22个供试材料的带型按照条带的有、无分别记录为1、0后,采用Nei72方法计算不同品种的遗传距离,利用NTSYS软件进行非加权组法（UPGMA）聚类分析。结果表明SRAP标记技术能较真实地反映小麦品种间的亲缘关系,可以用于小麦品种遗传多样性研究。     

利用SRAP标记分析河南小麦栽培品种的遗传多样性

利用小麦SRAP标记对22个河南省小麦品种进行了遗传多样性分析,10对引物组合扩增获得169个条带,其中70个条带具有多态性,多态条带百分率为41.42％,每对引物平均产生7个多态性条带。22个供试材料的带型按照条带的有,无分别记录为1,0后,采用Nei 72方法计算不同品种的遗传距离,利用NTSYS软件进行非加权成组法（UPGMA）进行了聚类分析。结果表明SRAP标记技术能较真实地反映小麦品种间的亲缘关系,可以用于小麦品种遗传多样性的研究。