Target Region Amplification Polymorphism Research Articles

Sequence-related Amplified Polymorphism and Target Region Amplification Polymorphism Markers-based Profiling of Sodium Azide and Ethyl Methanesulfonate-derived Black Rot-resistant Dendrobium sp. ‘Earsakul’ Mutants from In Vitro Mutagenesis and Selection

To reveal the applicability of sequence-related amplified polymorphism (SRAP) and target region amplification polymorphism (TRAP) markers to mutant genotyping and marker identification for resistance to black rot (Phytophthora palmivora) in orchids (Dendrobium sp. ‘Earsakul’), we fingerprinted four nonmutagenized controls and 12 black rot-resistant mutants obtained with in vitro sodium azide (NaN3) and ethyl methanesulfonate (EMS) mutagenesis and in vitro selection using culture filtrate of P. palmivora. Each of the 20 SRAP and TRAP primer combinations yielded 375 and 384 scorable DNA bands, respectively, of which 94 (24.42%) and 88 (22.91%) were polymorphic, respectively. Mantel’s test cophenetic correlation coefficients of SRAP, TRAP, and SRAP/TRAP were 0.750, 0.921, and 0.861, respectively, indicating the efficiency of these markers, especially TRAP and SRAP/TRAP, for Dendrobium sp. ‘Earsakul’ mutant characterization. Moreover, the correlations between the matrices of cophenetic correlation values for the dendrograms of SRAP with TRAP, SRAP with SRAP/TRAP, and TRAP with SRAP/TRAP were 0.399, 0.566, and 0.793, respectively, and the dendrograms based on SRAP vs. TRAP and SRAP vs. SRAP/TRAP, with lower correlations, had more variations, i.e., the number of clusters, the members of clusters, and the placement of the materials, than the ones based on TRAP vs. SRAP/TRAP. Among the three dendrograms, all nonmutagenized controls were clustered together, whereas all the highly resistant and the most resistant mutants were distributed separately as individuals. Interestingly, the four SRAP and TRAP markers were significantly associated with black rot resistance. Overall, our results will be useful for facilitating future Dendrobium sp. ‘Earsakul’ breeding programs.

Male heterogametic sex determination in Rana dybowskii based on sex-linked molecular markers.

Identifying the mechanism for sex determination in amphibians is challenging. Very little is known about sex determination mechanisms of Rana dybowskii, a species of importance to evolutionary and conservation biology. We screened for sex‐linked molecular markers in R. dybowskii in China using target region amplification polymorphism with 2 fixed primers against the sequences of Dmrt1. We found 2 male‐linked molecular markers in R. dybowskii, which were 222 bp and 261 bp long. The detection rates of 222 bp marker in males form Xinglong, Huadian, and Dandong were 93.79%, 69.64%, and 13.64%, respectively, while the rate in females from Huadian was 27.50%. Besides, the detection rates of 261 bp marker in the above 3 regions were only observed in males at the rate of 93.79%, 87.50%, and 32.73%, respectively. The inheritance patterns of sex‐linked molecular markers showed that the 2 sex‐linked molecular markers were heterozygous. Compared to the XY‐male parent, progeny from XX‐pseudo‐male parent possessed lower sex reversal ratio at the same rearing temperature, and the proportion of female froglets from an XX‐pseudo‐male parent was more than 95% at low rearing temperature (15°C). Our findings suggest that R. dybowskii displays male heterogamety, and the 2 sex‐linked molecular markers may have a guiding significance for the protection and utilization of R. dybowskii.

Detecting Genetic Mobility Using a Transposon-Based Marker System in Gamma-Ray Irradiated Soybean Mutants.

Transposable elements (TEs)—major components of eukaryotic genomes—have the ability to change location within a genome. Because of their mobility, TEs are important for genome diversification and evolution. Here, a simple rapid method, using the consensus terminal inverted repeat sequences of PONG, miniature inverted-repeat transposable element (MITE)-Tourist (M-t) and MITE-Stowaway (M-s) as target region amplification polymorphism (TE-TRAP) markers, was employed to investigate the mobility of TEs in a gamma-irradiated soybean mutant pool. Among the different TE-TRAP primer combinations, the average polymorphism level and polymorphism information content value were 57.98% and 0.14, respectively. Only the PONG sequence separated the mutant population into three major groups. The inter-mutant population variance, determined using the PONG marker (3.151 and 29%) was greater than that of the M-t (2.209 and 20%) and M-s (2.766 and 18%) markers, whereas the reverse was true for the intra-mutant population variations, with M-t and M-s values, being 15.151 (82%) and 8.895 (80%), respectively, compared with the PONG marker (7.646 and 71%). Thus, the MITE markers revealed more dynamic and active mobility levels than the PONG marker in gamma-ray irradiated soybean mutant lines. The TE-TRAP technique associated with sensitive MITEs is useful for investigating genetic diversity and TE mobilization, providing tools for mutant selection in soybean mutation breeding.

Assessment of Heat Tolerance in Some Wheat Species and Interspecific Hybrids

Twelve accessions including two diploid, four tetraploid and six hexaploid genotypes were used in this study to assess heat tolerance in wheat. All possible crosses were made and the resulted hybrids with their parents were subsequently evaluated for heat tolerance in two sowing dates. Evaluation was based on 1000 grain weight, grain yield per plant, cell membrane thermostability and tetrazolium chloride reduction. Results revealed significant variability among wheat genotypes and hybrids in all evaluated traits under favorable and heat stress conditions. However, tetraploid genotypes showed the highest performance under heat stress followed by hexaploid and diploid genotypes, respectively. According to results of heat tolerance index, one tolerant and one susceptible parent from each ploidy level along with 6 related interspecific hybrids were chosen to be involved in molecular analysis. Target region amplification polymorphism (TRAP) markers were used based on fixed primers of heat shock protein genes. TRAP clearly confirmed the morpho-physiological findings and generated a dendrogram which was quietly like that of morpho-physiological data. TRAP was able to generate one and five specific bands for diploid and hexaploid genotypes respectively, while no specific bands were generated for tetraploid. In addition, the different genomes showed some shared bands between each other revealing their relationship. Interestingly, two specific bands for tolerant genotypes of different ploidy level were generated which were absent in all susceptible genotypes. Findings herein are of high importance and could help in successive breeding programs for wheat improvement.

Suitability of target region amplified polymorphism (TRAP) markers to discern genetic variability in sweet sorghum

BackgroundSweet sorghum is an emerging biofuel candidate crop with multiple benefits as a source of biomass energy. Increase of biomass and sugar productivity and quality is a central goal in its improvement. Target region amplified polymorphism (TRAP) is a polymerase chain reaction (PCR) based functional marker system that can detect genetic diversity in the functional region of target genes. Thirty sweet sorghum genotypes were used to study the potential of 24 pairs of TRAP marker system in assessing genetic diversity with regard to three lignin and three sucrose biosynthesis genes. ResultsA total of 1638 bands were produced out of which 1161 (70.88%) were polymorphic at least at one locus. The average polymorphic information content (PIC), resolving power (RP), marker index (MI), Shannon’s diversity index (H), and gene diversity values were 0.32, 8.86, 1.74, 3.25, and 0.329, respectively. Analysis of molecular variance (AMOVA) revealed a highly significant genetic variation both within and among accessions studied (P = 0.01). However, the variation within the population was higher than among the populations (accessions). Bootstrap analysis showed that the number of loci amplified using this marker system is sufficient to estimate the available genetic diversity. The thirty genotypes were categorized into five clusters using a similarity matrix at 0.72 coefficient of similarity. The genotypes were also grouped mostly according to their geographic origin where the Ethiopian and Egyptian genotypes tend to fall in specific clusters. Moreover, the genotypes reflected the same pattern of distribution when ordinated using principal coordinate analysis. ConclusionsIn conclusion, TRAP marker can be used as a powerful tool to study genetic diversity in sweet sorghum.

Target Region Amplification Polymorphism (TRAP) for Assessing Genetic Diversity and Relationships Among Cultivated Populations of Dendrobium officinale

Dendrobium officinale (Orchidacesae) is one of the rare and endangered species of herbs in China. Therefore, it will be beneficial to investigate the genetic diversity and relationships of cultivated populations of D. officinale for quality improvement. In this study, eight target region amplification polymorphism (TRAP) primer combinations were selected from fifty-four combinations, which were designed based on the related genes of the polysaccharides and alkaloids. A total of 148 fragments were scored in nine cultivated populations of D. officinale, including 130 (87.84%) polymorphic fragments. The analysis of genetic diversity revealed high level of genetic diversity in cultivated populations of D. officinale (H = 0.4125, I = 0.5985). Based on analysis of genetic structure, there was a moderate variation (Gst = 0.4706) and lower gene flow (Nm = 0.5625) among the cultivated populations due to some isolated measures, and domestication of excellent cultivars. Moreover, UPGMA dendrogram and Principal Coordinate Analysis (PCoA) indicated that nine cultivated populations were divided into four major groups. The results suggested that genetic relationships were associated with geographical germplasm sources instead of cultivation locations. Therefore, TRAP markers can be effectively employed to analyze genetic diversity and relationships among cultivated populations of D. officinale.

Construction of Soybean Mutant Diversity Pool (MDP) Lines and an Analysis of Their Genetic Relationships and Associations Using TRAP Markers

Mutation breeding is useful for improving agronomic characteristics of various crops. In this study, we conducted a genetic diversity and association analysis of soybean mutants to assess elite mutant lines. On the basis of phenotypic traits, we chose 208 soybean mutants as a mutant diversity pool (MDP). We then investigated the genetic diversity and inter-relationships of these MDP lines using target region amplification polymorphism (TRAP) markers. Among the different TRAP primer combinations, polymorphism levels and polymorphism information content (PIC) values averaged 59.71% and 0.15, respectively. Dendrogram and population structure analyses divided the MDP lines into four major groups. According to an analysis of molecular variance (AMOVA), the percentage of inter-population variation among mutants was 11.320 (20.6%), whereas mutant intra-population variation ranged from 0.231 (0.4%) to 14.324 (26.1%). Overall, intra-population genetic similarity was higher than that of inter-populations. In an analysis of the association between TRAP markers and agronomic traits using three different statistical approaches based on the single factor analysis (SFA), the Q general linear model (GLM), and the mixed linear model (Q+K MLM), we detected six significant marker–trait associations involving five phenotypic traits. Our results suggest that the MDP has great potential for soybean genetic resources and that TRAP markers are useful for the selection of soybean mutants for soybean mutation breeding.

Genetic Structure Analysis in Sugarcane (Saccharum spp.) Using Target Region Amplification Polymorphism (TRAP) Markers Based on Sugar- and Lignin-Related Genes and Potential Application in Core Collection Development

Sugarcane is a main bioenergy crop and is highlighted worldwide in sugar, ethanol, and biomass production. Sugar and lignin contents are important quality traits for traditional and energy cane cultivar development, respectively. In the present study, genetic variability of a broad group of sugarcane basic germplasm accessions encompassing wild relatives and traditional and modern cultivars was assessed using target region amplification polymorphism (TRAP) markers derived from candidate genes involved in sugar and lignin metabolism. In total, 823 polymorphic markers (483 for sugar and 340 for lignin metabolism genes) were amplified; the average polymorphism information content values were highest for sugarcane wild relatives followed by traditional cultivars and modern cultivars. Genetic variability of the 96 genotypes captured by TRAP candidate genes for sugar and lignin metabolism was structured into two and three subpopulations, respectively. Based on the membership proportion (Q), modern cultivars inherited variability for genes involved in sugar metabolism from both S. officinarum and S. spontaneum. The genetic differentiation index based on sugar and lignin metabolism genes suggests moderate genetic differentiation among wild relatives, traditional cultivars, and modern cultivars. A core collection was established for sugar and lignin TRAP markers. Values for average genetic distance for the core collection based on sugar (0.761) and lignin (0.804) TRAP-derived markers were higher than those observed for all accessions, indicating that the core collections retained the most divergent accessions.

Evaluating the genetic relationship of Lilium species/cultivars based on target region amplification polymorphism (TRAP)

Most of the plant species from the genus Lilium L. have great ornamental and edible value. In this study, target region amplification polymorphism (TRAP) polymorphic marker technique was applied to assess the genetic relationships among 13 Lilium species/cultivars. The technique was also used to evaluate genetic diversity among two population of L. regale E. H. Wilson, cultivar of ‘Regale’ and ‘Regale Album’, and genetic diversity among two population of L. henryi Baker and cultivar of ‘Henryi’. In total, 221 fragments were amplified by 14 pairs of primers for 13 Lilium species/cultivars, of which 59 (26.70%) showed polymorphism. 1160 fragments were amplified for two population of L. regale, cultivar of ‘Regale’ and ‘Regale Album’, of which 943 (81.25%) were polymorphic. 798 fragments were amplified for two population of L. henryi and ‘Henryi’, of which 574 (71.93%) were polymorphic. A cluster analysis was performed based on UPGMA and PCoA by relying on the Nei’s genetic distance. Our results indicated that TRAP marker technique is informative and could be used to evaluate the genetic relationships among Lilium species/cultivars and genetic diversity among populations. The results will also provide more valuable information for Lilium resource protection and current plant breeding programs.

Utility of TRAP markers to determine indel mutation frequencies induced by gamma-ray irradiation of faba bean (Vicia faba L.) seeds

Purpose: The aims were to determine the optimal gamma ray dose for faba bean (Vicia faba) and to identify genetic variation and indel mutation frequency among mutants using target region amplification polymorphism (TRAP) markers.Materials and methods: Seeds from 10 elite lines were irradiated with gamma rays (50–700 Gy), and germination, survival rate, and representative morphological traits were measured. The extent of DNA damage was investigated using comet assays, and TRAP markers were used to evaluate genetic variation, genetic diversity, and mutation frequencies.Results: Germination percentages significantly decreased at doses >100 Gy. Survival percentages and morphological traits decreased with elevation in dose. The comet assays revealed that high irradiation doses decreased head DNA levels. Phylogenetic and principal component analysis of 555 individual faba bean plants resolved eight major groups. Genetic variation between controls and mutants was limited to within groups. Mutation frequencies were associated with gamma dose in each mutant line.Conclusions: The optimal gamma dose was 100–150 Gy on the basis of survival percentage and morphological response analysis. The TRAP markers distinguished mutant lines and showed association between mutation frequency and gamma doses. This study will be useful for faba bean mutation breeding and may be applicable to other crops.

Construction of a genetic linkage map of Lentinula edodes based on SSR, SRAP and TRAP markers.

Genetic mapping is a basic tool for eukaryotic genomic research. It allows the localization of genes or quantitative trait loci (QTLs) and map-based cloning. In this study, we constructed a linkage map based on DNA samples from a commercial strain L808, including two parental monokaryons and 93 single spore isolates considered with segregating to 1:1:1:1 at four mating types (A1B1, A1B2, A2B1 and A2B2). Using Simple Sequence Repeats (SSR), Sequence Related Amplified Polymorphism (SRAP), Target Region Amplified Polymorphism (TRAP) molecular markers, 182 molecular markers and two mating factors were located on 11 linkage groups (LGs). The total length of the map was 948.083 centimorgan (cM), with an average marker interval distance of 4.817 cM. Only two gaps spanning more than 20 cM was observed. The probability of 20 cM, 10 cM, 5 cM genetic distance cover one marker was 99.68%, 94.36%, 76.43% in our genetic linkage map, respectively. This is the first linkage map of Lentinula edodes using SSR markers, which provides essential information for quantitative trait analyses and improvement of genome assembly.

Androgenic Diploids of Erianthus arundinaceus with Paternal Chloroplast and Mitochondrial Genomes in Backcross Progeny of Saccharum × Erianthus Intergeneric Hybrids

Erianthus arundinaceus (Retz.) Jesweit is a tropical wild grass used in sugarcane (Saccharum L.) breeding for introgression of disease resistance and high cane yield traits. In the backcross (BC2) hybrids of Saccharum officinarum×E. arundinaceus and S. spontaneum×E. arundinaceus, with E. arundinaceus as pollen parent, the progeny had plants morphologically resembling E. arundinaceus. These plants were with chromosome number 2n = 60, same as that of E. arundinaceus. Through genomic in situ hybridization it was found that all the chromosomes were of E. arundinaceus. PCR-RFLP studies of the chloroplast and mitochondrial DNA polymorphism revealed that these plants had Erianthus type cytoplasmic genomes rather than the expected maternal Saccharum type. Target region amplification polymorphism of molecular markers, comparison of morphological characters and pollen size deviation studies confirmed the possible origin of E. arundinaceus type progeny. The diploid pollen from E. arundinaceus plants is considered to be resulted in the diploid androgenesis or paternal apomixis. The diploid androgenesis with paternal transmission of chloroplast and mitochondrial genomes along with the nuclear genome of E. arundinaceus in backcross progeny of Saccharum×Erianthus intergeneric hybrids is the first report in angiosperms.

Genetic variability and relationships among Salvia ecotypes/species revealed by TRAP-CoRAP markers

In this study, target region amplification polymorphism (TRAP) and conserved region amplification polymorphism (CoRAP) markers were used for genetic diversity and relationship analysis of 25 Salvia ecotypes/species. Twelve TRAP and CoRAP primer combinations (four arbitrary primers and three fixed primers from Salvia miltiorrhiza expressed sequence tag sequences) amplified 180 loci, of which all were polymorphic. The obtained high average PIC and MI values revealed high capacity of TRAP and CoRAP primers to detect polymorphic loci among Salvia species. TRAPs and CoRAPs had similar efficiency in detecting polymorphisms with excellent ability to differentiate the closely related Salvia ecotypes. Based on both TRAP and CoRAP data, tetraploid Salvia virgata (2n = 4x = 32) showed the highest values for Na (1.17, 1.29), Ne (1.29, 1.35), Shannon index (0.26, 0.32) and heterozygosity (0.172, 0.212) in all 11 studied species. The Nei’s genetic distances with TRAPs and CoRAPs ranged from 0.23 to 0.69 and 0.19 to 0.64, respectively. The tree pattern based on combined CoRAP + TRAP data with the neighbour joining method clustered 25 Salvia species/ecotypes into five major groups that showed good alignment with the population structure analysis results. In both TRAPs and CoRAPs data analysis of molecular variance (AMOVA), the estimated variance within Salvia species was greater than that among Salvia species.

Somatic embryogenesis of a seedless sweet orange (Citrus sinensis (L.) Osbeck)

Somatic embryogenesis is an important in vitro technique used to obtain Citrus sinensis (L.) Osbeck (sweet orange) plantlets for conservation, sanitation, propagation, and breeding. The induction of somatic embryogenesis from adult tissues of sweet orange could be an alternative to in vitro organogenesis from epicotyl segments, especially in seedless cultivars, where the latter is not feasible. The aim of this study was to obtain plantlets from ovary-derived somatic embryos of sweet orange cv. ‘Washington Navel’, an important seedless cultivar for citrus fresh fruit production. The explants used were pistils from flower buds, pre-anthesis, from 20-y-old plants cultivated in the field. Forty plantlets from 47 somatic embryos were obtained, in vitro-grafted, and acclimatized in greenhouse conditions. Ploidy evaluation through flow cytometric analysis, as well as the results of target region amplification polymorphism (TRAP) molecular markers confirmed the somatic origin of embryos as genetically similar to donor plants. This technique could be used for obtaining embryogenic cell suspension cultures or regenerated plants from mature tissues other than seed-derived tissues, especially for seedless genotypes.

Breeding of the new Saccharina variety “Sanhai” with high-yield

Backcrossing, self-crossing and target region amplified polymorphism (TRAP) marker assisted selection were utilized to breed a new Saccharina variety in this work. After two continuous cycles of backcrossing using “Fujian” as the recurrent parent and three cycles of self-crossing, the new high-yield variety “Sanhai” with stable excellent traits was obtained. During the whole Saccharina growth period (from January to April), the blade length and width of “Sanhai” were always significantly larger than “Fujian” as the control at the level of 0.05, displaying the obvious growth advantage of the new variety. In addition, the blade width and fresh weigh of “Sanhai” have reached 32.69±3.36cm, 686.82±80.45g respectively which both superior to its two parents (“Fujian” 25.22±4.34cm, 508.51±96.27g and “Rongfu” 24.59±3.58cm, 521.23±68.13g). For this breeding process, TRAP combined with phenotypic traits analysis was used to select the second generation of backcrossing (BC2F1) for subsequent self-crossing. The gene diversities of “Sanhai” obtained in this work (H=0.1572) and “Fujian” (H=0.1687) were lower than that of “Rongfu” (H=0.2303) on the basis of TRAP markers, which showed the outstanding genetic stability of the new variety. The results of Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) cluster indicated variety “Sanhai” could be distinguished from “Fujian” and “Rongfu” and the genetic structure of new variety was stable.

Remarks on genetic diversity and relationship of Punica protopunica and P. granatum assessed by molecular analyses

Here, two Punica species, viz., P. protopunica Balf. fil., reported as native to Socotra, and P. granatum L., were compared for the first time. Analysis of one P. protopunica and eleven P. granatum accessions was performed using three molecular markers, i.e., sequence related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), and intron targeted amplified polymorphism (ITAP), along with analysis of pgWD40 sequences, a gene involved in anthocyanin biosynthesis. All markers revealed the relationship between the two species and placed them at 33% similarity. SRAP, TRAP, and ITAP generated a total of 299, 260, and 160 bands, respectively. Of these, 78, 74, and 41 bands were specific for P. protopunica, and 92, 85, and 57 bands, respectively, were shared between both species. Sequence analysis of pgWD40~870 bp amplicons exhibited 100% identity among P. granatum accessions and 98% identity to that of P. protopunica. Phylogenetic analysis of WD40 sequences from monocot and dicot species, including both Punica species confirmed the relation between P. protopunica and P. granatum, supporting earlier reports that P. protopunica could be an ancestral species of P. granatum. Furthermore, the genetic diversity among and within P. granatum accessions from Egypt (3), Mexico (5), and Yemen (3) was assessed. Molecular marker-based relationships among region-bulked accessions was approximately the same (~90% similarity), whereas the degree of genetic variation was altered within each region. Specific bands (alleles) for accessions of each region along with those shared among them were identified. Thus, these bands could be used for pomegranate genotyping and breeding programs.

English

Screening of micropropagated banana (Musa spp.) cv. Grand Naine for somaclonal variants was carried out in the open field. The total detected variants were 26 somaclones (in addition to the normal plant) and were grouped into eight groups according to their morphological and yield characteristics. Sequence Related Amplified Polymorphism (SRAP) and Target Region Amplified Polymorphism (TRAP) techniques were used to study the differences among banana cv. Grand Naine and 26 somaclonal variants of the cultivar. SRAP markers amplified 1463 fragments while 841 fragments were resulted from TRAP markers. The somaclones “double bunching from peduncle”, “Giant plant” and “weak plant” somaclones were clustered with Grand Naine according to SRAP markers while “empty peduncle”, “horizontal bunch” and “angled bunch” somaclones clustered with Grand Naine using TRAP markers. According to principal coordinate analysis with SRAP markers, “pale green”, “black”, “wavy margins”, “double bunch from stem” and “vertical upward bunch” segregated from other variants; whereas, “pale green”, “black” and “vertical upward bunch” segregated from other variants using TRAP marker data. Although these markers were able to distinguish some of the somaclones derived from micropropagation of Grand Naine, additional markers would be needed to identify mutations generation during tissue culture propagation of banana.   Key words: Banana, sequence related amplified polymorphism, target region amplified polymorphism, cluster analysis, principal coordinate analysis, genetic diversity.

Study of Genetic Diversity using Morphological Traits and Target Region Amplified Polymorphism (TRAP) Marker in wheat

Study of Genetic Diversity using Morphological Traits and Target Region Amplified Polymorphism (TRAP) Marker in wheat

Genetic divergence in elite castor bean lineages based on TRAP markers.

Castor bean (Ricinus communis L.) is a tropical plant of great commercial interest and a potential source of biodiesel. The development of genetically improved cultivars with high amounts of oil in the seeds and low ricin toxicity is crucial to increase the productivity of this crop. The use of TRAP (target region amplification polymorphism) markers to develop elite lineages and study genetic divergence is fundamental to advance the genetic improvement of this species. The goal of this study was to evaluate the genetic divergence among 40 elite lineages of R. communis, which belong to the NBIO-UFRB Genetic Improvement Program, using TRAP markers involved in the biosynthesis of oil and ricin. Total DNA was extracted and quantified from the leaf tissue of the castor bean plants, and 70 TRAP combinations (fixed and arbitrary primers) were used to genotype the 40 lineages. Of the 580 fragments amplified, 335 were polymorphic (58%). The genetic dissimilarity among the lineages was calculated by the Jaccard dissimilarity index using the UPGMA grouping method. A dendrogram was generated, and four groups formed, showing divergence among the elite lineages that favors selection. The TRAP molecular markers were efficient at characterizing the genetic variability among the lineages and, because TRAP markers are functional markers involved in the biosynthesis of oil and ricin, they are important when studying the association between a marker and a candidate gene.

Development of TRAP primers for Ricinus communis L.

The objective of this article was to develop TRAP (target region amplification polymorphism) primers for castor bean, with the goal of making functional markers available for genetic studies about the species. To do this, oligonucleotides were designed based on ESTs, obtained from the NCBI (National Center for Biotechnology Information) databank, which code enzymes involved in metabolic routes of fatty acid synthesis, ricin synthesis, and resistance to castor bean pathogens. The forward primers were designed with the help of the Primer3 software and, for the reverse, six arbitrary primers were used. To standardize the amplification reactions, the following criteria were used to select the primers: sizes between 18 and 20 bp, guanine/cytosine (GC) in the range of 40 to 60%, and average annealing temperature between 55° and 62°C. The design quality of the primers was verified using the Net Primer application. Fifty-six primers were designed, which had an average GC percentage of 53.2%. A total of 336 combinations were obtained using the 56 fixed and 6 arbitrary primers. Based on polymerase chain reaction, 330 combinations (89%) presented good amplification patterns for the genomic DNA of castor bean. The size of the fragments amplified varied between 50 and 2072 bp. The TRAP primers designed and validated in this study are the first for castor bean and represent a significant increase in the molecular markers for this species.