Target Region Amplification Polymorphism Primer 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.

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.

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.

Development of SCAR markers linked to sin-2, the stringless pod trait in pea (Pisum sativum L.)

With increasing consumer demand for vegetables, edible-podded peas have become more popular. Stringlessness is one of most important traits for snap peas. A single recessive gene, sin-2, controls this trait. Because pollen carrying the stringless gene is less competitive than pollen carrying the stringy gene, there are fewer than expected stringless plants recovered in segregating generations. Marker-assisted selection (MAS) is a valuable tool to identify plants with the traits of interest at an early stage in the breeding process. The objective of this study was to identify robust, user-friendly molecular markers tightly linked to sin-2. A total of 144 target region amplification polymorphism (TRAP) primer combinations were used to screen four DNA bulks, which were constructed from 32 pea breeding lines based on their phenotypes. Sixty polymorphic TRAP primer combinations were identified between bulks of stringless and stringy pods. Five primer combinations, F6_Trap03_168, F6_SA12_145, F10_ODD8_130, F11_GA5_850, and F12_SA12_190, showed more than 90 % association with the stringless phenotype in 32 pea breeding lines. Two of the TRAP markers, F10_ODD8_130 and F12_SA12_190, were cloned, sequenced, and successfully converted to sequence characterized amplified region (SCAR) markers. These two SCAR markers were validated using 20 F5 recombinant inbred lines derived from a cross between Bohatyr (a dry pea variety with strings) and S1188 (a stringless snap pea variety) and showed strong marker-trait association. The results will have direct application in MAS of stringless edible-podded peas.

New Sugarcane Microsatellites and Target Region Amplification Polymorphism Primers Designed from Candidate Genes Related to Disease Resistance

Many types of molecular marker systems have been developed to detect and map genomic regions related to disease resistance in many major agronomic crops. However, in sugarcane, molecular marker systems for genetic mapping of genomic regions associated to disease resistance is not yet established mainly due to its complex polyploid genome. In addition, there is a lack of information to develop molecular marker systems (e.g., TRAP and EST–SSR) targeting specific genic regions related to disease resistance. In the present work, a set of 10 EST–SSR primer pairs and 16 fixed TRAP primers designed from sugarcane-derived sequence related to disease resistance are reported. Most of the primers were amplifiable and polymorphic in the sugarcane genotypes tested. In addition, these set of primers were also tested in sorghum, Miscanthus and a cross between sugarcane commercial varieties showing good cross-transferability. This new set of primers, in particular the TRAP ones, will be useful in genetic mapping of genome regions related to disease resistance in sugarcane and related genera.

Start codon targeted (SCoT) and target region amplification polymorphism (TRAP) for evaluating the genetic relationship of Dendrobium species

Two molecular marker systems, start codon targeted (SCoT) and target region amplification polymorphism (TRAP), were used for genetic relationship analysis of 36 Dendrobium species collected from China. Twenty-two selected SCoT primers produced 337 loci, of which 324 (96%) were polymorphic, whereas 13 TRAP primer combinations produced a total of 510 loci, with 500 (97.8%) of them being polymorphic. An average polymorphism information content of 0.953 and 0.983 was detected using the SCoT and TRAP primers, respectively, showing that a high degree of genetic diversity exists among Chinese Dendrobium species. The partition of clusters in the unweighted pair group method with arithmetic mean dendrogram and principal coordinate analysis plot based on the SCoT and TRAP markers was similar and clustered the 36 Dendrobium species into four main groups. Our results will provide useful information for resource protection and will also be useful to improve the current Dendrobium breeding programs. Our results also demonstrate that SCoT and TRAP markers are informative and can be used to evaluate genetic relationships between Dendrobium species.

Genetic diversity analysis of Hypsizygus marmoreus with target region amplification polymorphism.

Hypsizygus marmoreus is an industrialized edible mushroom. In the present paper, the genetic diversity among 20 strains collected from different places of China was evaluated by target region amplification polymorphism (TRAP) analysis; the common fragment of TRAPs was sequenced and analyzed. Six fixed primers were designed based on the analysis of H. marmoreus sequences from GenBank database. The genomic DNA extracted from H. marmoreus was amplified with 28 TRAP primer combinations, which generated 287 bands. The average of amplified bands per primer was 10.27 (mean polymorphism is 69.73%). The polymorphism information content (PIC) value for TRAPs ranged from 0.32 to 0.50 (mean PIC value per TRAP primer combination is 0.48), which indicated a medium level of polymorphism among the strains. A total of 36 sequences were obtained from TRAP amplification. Half of these sequences could encode the known or unknown proteins. According to the phylogenetic analysis based on TRAP result, the 20 strains of H. marmoreus were classified into two main groups.

Identification of new TRAP markers linked to chlorophyll content, leaf senescence, and cell membrane stability in water-stressed wheat

In order to identify target region amplification polymorphism (TRAP) markers linked to three physiological traits in wheat (Triticum aestivum L.), the segregating F4 population from the cross between drought-sensitive (Yecora Rojo) and drought-tolerant (Pavon 76) genotypes was made. The parents and 150 F4 families were evaluated phenotypically for drought tolerance using two irrigation treatments [2.5 and 7.5 m3(H2O) m-2(soil)]. Using 40 different TRAP primer combinations tested for polymorphism in parental and F4 family genotypes, the results revealed that quantitative trait locus (QTL) for chlorophyll content was associated with TRAP 5, TRAP 14, and TRAP 20 and explained 18, 16, and 23 % phenotypic variation, respectively. The genetic distance between chlorophyll content QTL and TRAP 5, TRAP 14, and TRAP 20 were 12.3, 19.8, and 13.6 cM, respectively. QTL for flag leaf senescence was associated with TRAP 2, TRAP 3, TRAP 15, and TRAP 16 and explained 33, 27, 28, and 23 % phenotypic variations, respectively. The genetic distance between flag leaf senescence QTL and TRAP 2, TRAP 3, TRAP 15, and TRAP 16 were 9.4, 14.7, 18.1, and 17.3 cM, respectively. QTL for cell membrane stability was associated with TRAP 8, TRAP 9, and TRAP 37 and explained 27, 30, and 24 % phenotypic variation, respectively. The markers TRAP 8, TRAP 9, and TRAP 37 had genetic distances of 17.0, 10.0, and 9.0 cM, respectively. Therefore, these TRAP markers can be used in breeding for drought tolerance in wheat.

Genetic diversity of Liriope Muscari by TRAP analysis

Target region amplification polymorphism (TRAP) marker was occupied to study on the genetic diversity of fifty Liriope Muscari clones. Total eight genes, one lectin gene and seven related to fructose, photosynthesis and steroid saponin metabolism, were selected as target genes and used to design thirteen the anchored primers for pairing with nineteen arbitrary primers. And eleven combinations of primers were screened to be able to produce clear banding patterns and polymorphisms. The results showed that 335 bands were amplified totally by 11 pair TRAP primers, of which 323 bands (96.41%) were polymorphic in the species level The average of polymorphism information content (PIC) was 0.930. gene differentiation index (Gst) was 0.610. The results of cluster analysis based on UPGMA revealed genetic coefficient ranged from 0.52 to 0.98. A relatively high genetic diversity existed in L. muscari, a certain level of genetic differentiation among populations.

Target Region Amplification Polymorphism (TRAP) for Assessing Genetic Diversity in Sugarcane Germplasm Collections

Target region amplification polymorphism (TRAP) is a fairly new PCR‐based molecular marker technique which uses gene‐based information for primer design. The objective of this study was to evaluate the utility of TRAP markers for assessing genetic diversity and interrelationships in sugarcane germplasm collections. Thirty genotypes from the genera Saccharum, Miscanthus, and Erianthus were used in the study. Among the genus Saccharum were the species, S. officinarum L., S. barberi Jesw., S. sinense Roxb, S. spontaneum L, S. robustum Brandes and Jeswiet ex Grassl, cultivars, cultivar‐derived mutants and interspecific hybrids between S. officinarum and S. spontaneum Six fixed primers, designed from sucrose‐ and cold tolerance‐related EST (expressed sequence tags) sequences, paired with three arbitrary primers, were used to characterize the germplasm. Both the cluster and principal coordinate (PCoA) analyses placed the Erianthus spp. and Miscanthus spp. genotypes distinctly from each other and from the Saccharum species, thus, supporting their taxonomic classification as separate genera. Genotypes of the low sucrose and cold tolerant species, S. spontaneum, formed one distinct group, while the rest of the Saccharum species formed one interrelated cluster with no distinct subgroups. Sequence analysis of TRAP bands derived from a S. spontaneum genotype revealed homology with known gene sequences from other grass species including Sorghum A BLASTn search using the homologous sequences from Sorghum matched with the S. officinarum GenBank accession from which the fixed TRAP primer was designed. These results ratify TRAP as a potentially useful marker technique for genetic diversity studies in sugarcane.