Gene-targeted Markers Research Articles

Exploring the genetic diversity and population structure of Bacopa monnieri (L.) using random amplified (RAPD and ISSR) and gene-targeted (SCoT and CBDP) markers

Exploring the genetic diversity and population structure of Bacopa monnieri (L.) using random amplified (RAPD and ISSR) and gene-targeted (SCoT and CBDP) markers

PlantPathMarks (PPMdb): an interactive hub for pathways-based markers in plant genomes

AbstractOver the past decade, the problem of finding an efficient gene-targeting marker set or signature for plant trait characterization has remained challenging. Many databases focusing on pathway mining have been released with one major deficiency, as they lack to develop marker sets that target only genes controlling a specific pathway or certain biological process. Herein, we present the PlantPathMarks database (PPMdb) as a comprehensive, web-based, user-friendly, and interactive hub for pathway-based markers in plant genomes. Based on our newly developed pathway gene set mining approach, two novel pathway-based marker systems called pathway gene-targeted markers (PGTMs) and pathway microsatellite-targeted markers (PMTMs) were developed as a novel class of annotation-based markers. In the PPMdb database, 2,690,742 pathway-based markers reflecting 9,894 marker panels were developed across 82 plant genomes. The markers include 691,555 PGTMs and 1,999,187 PMTMs. Across these genomes, 165,378 enzyme-coding genes were mapped against 126 KEGG reference pathway maps. PPMdb is furnished with three interactive visualization tools (Map Browse, JBrowse and Species Comparison) to visualize, map, and compare the developed markers over their KEGG reference pathway maps. All the stored marker panels can be freely downloaded. PPMdb promises to create a radical shift in the paradigm of the area of molecular marker research. The use of PPMdb as a mega-tool represents an impediment for non-bioinformatician plant scientists and breeders. PPMdb is freely available at http://ppmdb.easyomics.org.

Multiple DNA marker-assisted diversity analysis of Indian mango (Mangifera indica L.) populations

Arbitrary (65 RAPD, 25 ISSR, 23 DAMD), gene-targeted (22 SCoT, 33 CBDP) and co-dominant sequence specific (40 SSR) markers were used individually, or in combinations, to examine the genetic variability within and among 70 selected Indian mango genotypes based on geographic origin (East India, West India, North India, South India) and fruit status (Selection, Hybrid, Landrace). The highest genetic variability was demonstrated by the East Indian populations, followed by those from South India, West India, and North India, when measured in terms of Na, Ne, H, I, PB%, Ht and Hs. Interestingly, the local genotypes of Odisha, which forms a part of East Indian populations, showed the highest diversity compared to hybrid or selection groups, suggesting that the indigenous genotypes hold a greater potential for exploiting the unique and favourable alleles. The maximum genetic variability was detected in geographical/fruit status populations with SSRs (Na—1.76/1.88, Ne—1.48/1.51, H—0.28/0.30, I—0.41/0.45, PB%—76.1/86.9, Ht—0.31/0.32 and Hs—0.28/0.30), followed by CBDPs and SCoTs, reflecting their preeminence for examining the level of genetic polymorphism and diversity. Outcome of AMOVA based analyses as well as low-to-moderate coefficient of genetic differentiation (Gst) and high gene flow (Nm) indicated a greater amount of intra-population genetic variation compared to heterogeneity at inter-population level. Information generated through this investigation could facilitate conservation and further exploitation of mango germplasm including genetic improvement through breeding.

Evaluation of genetic variability and relatedness among eight Centaurea species through CAAT-box derived polymorphism (CBDP) and start codon targeted polymorphism (SCoT) markers

Centaurea is a value-ultimate genus of medicinal plants showing high diversification levels, especially within the Mediterranean basin, and is still traditionally recognized as a complicated taxon. So far, few studies utilizing molecular markers have been done on Centaurea spp. towards a better dissection of its phylogeny and accurate assessment of genetic diversity. Here, two functional marker systems, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP), were implemented to assess the genetic diversity between eight wild Centaurea species in Egypt. Seventeen SCoT and 19 CBDP primers generated 197 and 179 bands, respectively. These primers generated 158 (80.2%) and 131 (73.18%) polymorphic amplicons with an average of 9.29 and 6.89 amplicons per primer, respectively. SCoT primers exhibited higher levels in % of polymorphism, % of heterozygosity, effective multiplex ratio, discriminating power, resolving power parameters compared to the CBDP. In contrast, no significant differences were observed between SCoT and CBDP in polymorphism information content, marker index and mean heterozygosity parameters. The UPGMA (unweighted pair group method with arithmetic mean) dendrogram of the combined data classified the Centaurea species under two major clades; the first comprised two species, whereas the second grouped five species. C. pallescens was kept separated as the most diverged among the eight Centaurea species. Principal component analysis (PCA) topology revealed highly similar results to those obtained from the cluster analysis. Ultimately, our results represent the first report utilising two gene-targeting marker systems as powerful techniques for assessing the genetic variability and relatedness among these eight valuable Centaurea species.

Genetic Characterization, Agro-Morphological and Physiological Evaluation of Grafted Tomato under Salinity Stress Conditions

This study aims to determine grafting’s efficiency to improve tomato growth and production under salinity stress conditions. A commercial tomato hybrid (cv. Bark) and eight wild tomato accessions were evaluated at molecular, physiological and agronomic levels. At the molecular level, two robust gene-targeting marker systems (Conserved DNA-Derived Polymorphism; CDDP and Start Codon Targeted Polymorphism; SCoT) were employed. Bark cv. was grafted as a scion onto the four tomato genotypes’ roots as stocks. The rootstocks effect was evaluated by growing plants at 0, 100 and 200 mM NaCl. Our results showed that grafting enhanced plant shoots and roots growth (plant height, number of branches, plant fresh weight, root length, and root fresh and dry weight), fruit yield (total yield, number and weight of fruits) and fruit quality (Vitamin C, firmness and total soluble solids) in Bark on most tested rootstocks. A significant interaction between salinity levels and rootstocks for all measured hormones, antioxidants and proline was observed. In conclusion, our consistent results from the three approaches (molecular, physiological and agronomical) revealed that the four genotypes (LA1995, LA2711, LA2485 and LA3845) were found to be grouped and exhibit better performance under salinity stress conditions. Furthermore, grafting could be a low-cost alternative method to improve salt tolerance in sensitive tomato genotypes.

Gene-targeted molecular phylogeny, phytochemical analysis, antibacterial and antifungal activities of some medicinal plant species cultivated in Egypt.

Medicinal plants have been used in healthcare since time immemorial, as have their therapeutic activities and the production of plant-based medicines. This study aims to use gene-targeted molecular markers for genetic diversity analysis of 16 medicinal plants. Besides, phytochemical analysis antibacterial and antifungal activities of some medicinal plant extracts commonly used in Egypt are compared to major compounds. DNA-based classification of 16 medicinal species using Conserved DNA-Derived Polymorphism (CDDP) and Start Codon Targeted (SCoT) primers. Three species representing three orders (Pelargonium graveolens, Matricaria chamomilla, and Hyoscyamus muticus were analysed [high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS)] and evaluated for their antibacterial and antifungal activities against (Escherichia coli O157: H7 ATCC 93111, Salmonella typhimurium ATCC 14028, Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Bacillus ceruse ATCC 33018, and Sclerotinia sclerotiorum in comparison with some of their antimicrobial components. Our results revealed 309 and 349 polymorphic bands with 100% polymorphism. Among them, 51 and 57 were unique loci for CDDP and SCoT, respectively. The 16 species were categorised into three groups depending on the similarity matrix. The results of antibacterial and antifungal activities revealed that Pelargonium oil showed significant antifungal and antibacterial activities against the tested pathogens. Gallic acid severely reduced all tested bacteria's growth, but atropine severely reduced the growth of the B. ceruse only. Molecular modelling revealed their activity against sclerotium development. The gene-targeted marker techniques were highly useful tools for the classification of the 16 medicinal plant species, despite displaying high similarities at morphological and phytochemical analyses but, have antifungal and antibacterial activities.

Assessment of the Suitability of Molecular SCoT Markers for Genetic Analysis of Coffee Species

AbstractStart codon targeted polymorphism (SCoT), a novel and gene-targeted marker, has recently become the marker of choice in genetic diversity studies. In the present study, 31 SCoT primers were tested for their suitability in the genetic analysis of 21 coffee genotypes representing 18 species. A total of 647 distinct PCR amplified fragments were produced with a mean of 20.9 fragments per primer and 80.80% of which were polymorphic. The polymorphic information content of SCoT primers ranged from 0.16 to 0.86, with a mean value of 0.63. Resolving power ranged from 6.19 to 28.29, with a mean value of 20.2. Species-specific unique PCR amplified fragments were identified for 16 species, which could be used as genetic fingerprints. The genetic similarity among various coffee species calculated using the Dice similarity coefficient ranged between 0.60 and 0.89. The dendrogram constructed using the unweighted pair group of arithmetic means (UPGMA) clustered the 21 coffee genotypes into two major groups. The study revealed that Coffea jenkinsii, an indigenous species from India, showed the highest similarity with C. arabica, which is of Ethiopian origin. The results proved the suitability of SCoT markers in genetic analysis of coffee genotypes.

Gene-targeted molecular phylogeny, phytochemical profiling, and antioxidant activity of nine species belonging to family Cactaceae

Cactaceae plant family comprises over 130 genera and 2000 species of succulent flowering plants. The genera Mammillaria and Notocactus (Parodia), which have medicinal and nutritional applications as well as aesthetic appeal, are considered to be among the major genera of the family. Several species of both genera show morphological and chemical similarities and diversities according to environmental conditions and genotypes. Here, we assessed the genetic relationships of nine species belonging to two major genera Mammillaria and Notocactus under the family Cactaceae, using two modern gene-targeting marker techniques, the Start Codon Targeted (SCoT) Polymorphism and the Conserved DNA-Derived Polymorphism (CDDP). Besides, we screened the various phytochemicals and evaluated the antioxidant activities of the nine species of cacti. Five out of the 10 SCoT and eight CDDP primers used to screen genetic variations within the nine species yielded species-specific reproducible bands. The entire 156 loci were detected, of which 107 were polymorphic, 26 were monomorphic, and 23 were unique loci. The nine species were categorized into two groups based on the dendrogram and similarity matrix. Phytochemical profiling revealed that sterols, triterpenes, flavonoids, and tannins were found in all the tested species. Additionally, two Notocactus species (N. shlosserii and N. roseoluteus) and one Mammillaria species (M. spinosissima) revealed a considerable antioxidant activity. Our results demonstrated that gene-targeting marker techniques were highly powerful tools for the classification and characterization of the nine investigated species, despite displaying high similarities at both morphological and phytochemical levels.

Gene-Targeted markers to assess genetic diversity and population structure within Tunisian Phoenix dactylifera L. cultivars

Abstract Genetic variability in date palm genotypes collected from different regions of southern Tunisia was analyzed using a Start Codon Targeted (SCoT) marker system. Thirty-one accessions collected from three locations were investigated. One hundred and nine amplicons were produced among which 84 % were polymorphic. The PIC value and the Rp values testified of the efficiency of used primers. The percentage of polymorphic loci (Pp) varied from 44.57 to 83.70 %, Nei‘s gene diversity (H) from 0.175 to 0.273 with a mean of 0.228, and Shannon‘s information index (I) values ranging from 0.257 to 0.409 with an average value of 0.338 were illustrated. The Tunisian date palm populations exhibited high genetic differentiation (GST = 0.319) and gene flow (Nm = 1.063). The AMOVA analysis presented 70 % of the variation within the population and 30 % of the variation between them. Phylogenetic analysis and the Bayesian clustering approach also revealed high genetic variation among genetic variants with a net divergence of the wild insular population of Kerkennah from other cultivars. The present investigation suggests the effectiveness of the SCoT marker system to estimate the genetic diversity of Phoenix dactylifera genotypes.

Genetic diversity and population structure analysis of landrace and improved safflower (Cartamus tinctorious L.) germplasm using arbitrary functional gene-based molecular markers

The genetic diversity and relationships among 48 landrace and improved safflower (Carthamus tinctorius L.) genotypes were analyzed using three gene-targeted markers, start codon-targeted (SCoT) polymorphism, conserved DNA-derived polymorphism (CDDP) and CAAT box-derived polymorphism (CBDP). A total of 30 primers (10 primers from each marker) detected genetic polymorphism among the safflower genotypes. Three different marker types showed high level of polymorphism and the CDDP markers produced a higher number of polymorphic bands (74) in comparisons with SCoT and CBDP. The average PIC values for SCoT, CDDP, and CBDP were 0.39, 0.43, and 0.41, respectively. The marker index (MI) in CDDP markers was higher than that in SCoT and CBDP markers. Cluster analysis using SCoT and CDDP markers grouped the safflower genotypes into three distinct groups, whereas the CBDP markers divided the genotypes into two clusters. There were positive correlations between the similarity matrices obtained by each marker type. The results from the cluster analysis and STRUCTURE analysis using combined data grouped the genotypes into two clusters, generally in agreement with their origins. To our knowledge, this is the first detailed report of using gene-targeted molecular markers for genetic diversity analysis in safflower. Our results showed the efficiency of these markers for genetic diversity analysis in safflower and their potential for genome diversity and germplasm conservation.

Development of a New Molecular Marker for the Resistance to Tomato Yellow Leaf Curl Virus.

Tomato yellow leaf curl virus (TYLCV) responsible for tomato yellow leaf curl disease (TYLCD) causes a substantial decrease in tomato (Solanum lycopersicum L.) yield worldwide. The use of resistant variety as a sustainable management strategy has been advocated. Tremendous progress has been made in genetically characterizing the resistance genes (R gene) in tomato. Breeding tomato for TYLCV resistance has been based mostly on Ty-3 as a race-specific resistance gene by introgression originating from wild tomato species relatives. Improvement or development of a cultivar is achievable through the use of marker-assisted selection (MAS). Therefore, precise and easy use of gene-targeted markers would be of significant importance for selection in breeding programs. The present study was undertaken to develop a new marker based on Ty-3 gene sequence that can be used for MAS in TYLCV resistant tomato breeding program. The new developed marker was named ACY. The reliability and accuracy of ACY were evaluated against those of Ty-3 linked marker P6-25 through screening of commercial resistant and susceptible tomato hybrids, and genetic segregation using F2 population derived from a commercial resistant hybrid AG208. With the use of bioinformatics and DNA sequencing analysis tools, deletion of 10 nucleotides was observed in Ty-3 gene sequence for susceptible tomato variety. ACY is a co-dominant indel-based marker that produced clear and strong polymorphic band patterns for resistant plant distinguishing it from its susceptible counterpart. The obtained result correlates with 3:1 segregation ratio of single resistant dominant gene inheritance, which depicted ACY as gene-tag functional marker. This marker is currently in use for screening 968 hybrids varieties and one thousand breeding lines of tomato varieties stocked in Jiangsu Green Port Modern Agriculture Development Company (Green Port). So far, ACY has been used to identify 56 hybrids and 51 breeding lines. These newly detected breeding lines were regarded as potential source of resistance for tomato breeding. This work exploited the sequence of Ty-3 and subsequently contributed to the development of molecular marker ACY to aid phenotypic selection. We thus recommend this marker to breeders, which is suitable for marker-assisted selection in tomato.

Assessment of genetic stability amongst micropropagated Ansellia africana, a vulnerable medicinal orchid species of Africa using SCoT markers

Micropropagation is an important tool for the conservation of threatened and commercially important plant species of which orchids deserve special attention. Ansellia africana is one such medicinally important orchid species having much commercial significance. However, for large-scale micropropagation to become not only successful but also acceptable by end-users, somaclonal variations occurring in the plantlets need to be eliminated. In the present study, the clonal integrity of micropropagated A. africana plants derived from two different pathways was assessed using a gene-targeted marker system, i.e. Start Codon Targeted polymorphism (SCoT). Our studies recorded a significantly higher gene flow value (Nm=1.596) amongst the generations with an increment in clonal variability. The developed protocol helps to understand the main start point of clonal variability in a model tissue culture system and the role played by the various plant growth regulators (PGRs). The protocol also documents a fast and cost-effective regeneration pathway for commercially important medicinal orchids with reproducible molecular detection system to monitor and detect clonal variability for obtaining clonally stable true-to-type plantlets for sustainable commercial use.

Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination.

Positive-negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive-negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice.

Molecular Characterisation of Fennel (Foeniculum vulgareMill.) Accessions using Start Codon-Targeted (SCoT) Markers

Fourteen accessions were collected from different locations in northern India. Seeds of defined varieties grown in Rajasthan were procured from the Department of Plant Breeding and Genetics, S.K.N. College of Agriculture, Jobner, Jaipur (Rajasthan), India and were studied using start codon-targeted (SCoT) markers. A total of 36 SCoT primers were screened to study the diversity, out of which, 35 primers could amplify reproducible bands. A part of that SCoT 20 primers could not amplify reproducible bands. These primers were selected for molecular characterisation of 14 Foeniculum vulgare accessions, which produced a total of 256 bands (7.0 bands per primer), of which, 240 were polymorphic. The analysis revealed 85.69% polymorphism for SCoT, respectively. Similarity coefficient was used to detect the phylogenetic relationship; subsequently, dendogram was constructed for SCoT data. The unweighted pair group method with arithmatic average cluster analysis revealed two distinct sub-clusters in the dendogram. In cluster 1, 13 accessions were grouped together, whereas variety 6 was present in cluster 2. Accession number 6 was different from all 13. The genetic diversity explained by gene-targeted markers shows that SCoT marker can be very effective for characterisation of genetic diversity in medicinal plant like F. vulgare.

Potential of start codon targeted (SCoT) markers for assessment of genetic diversity of arecanut (Areca catechu L.)

Gene-targeted markers constitute recent and novel marker systems, which are based on polymorphic sites existing within gene targeting regions. One among these marker systems is the Start Codon Targeted (SCoT), which is based on the short conserved region of plant genes, which neighbours the ATG start codon. SCoT markers, by virtue of both their higher length of the primers and annealing temperatures, are generally reproducible and have been reported to be highly polymorphic, compared to other dominant markers. In this study, analysis of genetic diversity among six arecanut accessions, viz., Mangala, Sumangala, Sreemangala, Mohitnagar, Swarnamangala and a natural dwarf mutant (Hirehalli Dwarf) was carried out using SCoT markers to evaluate the applicability of these markers in genetic diversity studies in arecanut. Using 10 SCoT primers, 82 band were produced among the accessions, of which 58 (70.73%) were found to be polymorphic. The highest genetic similarity value of 0.89 was found between the Swarnamangala and Mohitnagar and the lowest value of 0.63 was noticed between the Hirehalli Dwarf and Mohitnagar. The similarity coefficient values were then utilized to construct a dendrogram utilizing the unweighted pair group of arithmetic means (UPGMA). The cultivars were grouped depending on their geographical origins. The results obtained in this study indicate the suitability of SCoT marker system for genetic diversity analysis in arecanut.

Genetic relationship and diversity among coconut (Cocos nucifera L.) accessions revealed through SCoT analysis.

Coconut (Cocos nucifera L.) is one of the important palms grown both as a homestead and plantation crop in countries and most island territories of tropical regions. Different DNA-based marker systems have been utilized to assess the extent of genetic diversity in coconut. Advances in genomics research have resulted in the development of novel gene-targeted markers. In the present study, we have used a simple and novel marker system, start codon targeted polymorphism (SCoT), for its evaluation as a potential marker system in coconut. SCoT markers were utilized for assessment of genetic diversity in 23 coconut accessions (10 talls and 13 dwarfs), representing different geographical regions. Out of 25 SCoT primers screened, 15 primers were selected for this study based on their consistent amplification patterns. A total of 102 scorable bands were produced by the 15 primers, 88 % of which were polymorphic. The scored data were used to construct a similarity matrix. The similarity coefficient values ranged between 0.37 and 0.91. These coefficients were utilized to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA). The extent of genetic diversity observed based on SCoT analysis of coconut accessions was comparable to earlier findings using other marker systems. Tall and dwarf coconut accessions were clearly demarcated, and in general, coconut accessions from the same geographical region clustered together. The results indicate the potential of SCoT markers to be utilized as molecular markers to detect DNA polymorphism in coconut accessions.

Novel Set of Sex-Specific PCR-Based Markers Reveals New Hypothesis of Sex Differentiation in Date Palm

The date palm (Phoenix dactylifera L.), 2n=36, is a dioecious long-lived monocotyledonous plant, with separate male and female trees. The dioecy represents the major challenge in development of breeding programs as it is impossible to distinguish tree gander till they flower approximately five to eight years after planting. Although, during the past two decades numerous attempts to develop molecular markers can discriminate among male and female trees in date palm. But, to date, sex-differentiation mechanism is still uncertain and there is no reliable way to determine the sex of date palm plants before reproductive age across all cultivars. Here, we employed an effective three novel gene-targeting marker approaches (SCoT, CDDP and ITAP) in additions to AFLP, in an attempt to develop a novel set of reliable sex-specific PCR-based markers can helping in early gender determination in Egyptian date palm trees. A set of 26 SCoT, 21 CDDP, 18 ITAP and 14 AFLP primers/primer combinations (PCs) were applied against twelve date palm genotypes belonging to three superior Egyptian date palm cultivars to identify any sex-specific markers. Four SCoT (SCoT1, SCoT24, SCoT26 and SCoT35), two CDDP (CDDP4 and CDDP6), one ITAP (ITAP-8/1) and one AFLP (AFLP-4/1) primer/PC exhibited differential fragments/bands between males and females. These differential bands were gel extracted and cloned for subsequent sequencing analysis. Three of the sequenced bands found to be contain more than one sequence. BLAST analysis results indicated that the eleven sequences generated from different gene-targeting marker systems (SCoT, CDDP and ITAP) revealed main similarity with master transcription factors, transcriptional activators/repressors and regulatory proteins involved in plant hormone signal transduction pathways, plant development and biosynthesis of secondary metabolites, playing important role in different types of abiotic and biotic stresses in date palm or oil palm. We speculate that kind of similarity is not just a coincidence. Our results reveals hypothesis that sex differentiation is a complex but well-organized process that involves endogenous and exogenous factors regulate and control the changes in gene expression, physiology, metabolism and architecture of the plant. These results represent the first case-study focusing on the applications of CDDP, ITAP and SCoT techniques as a novel gene targeting markers in sex-determination in date palm. Moreover, indicate that sex-differentiation process have to be addressed at system biology level for deep and better understanding. This developed sex-specific markers expected to be helpful in distinguish the gander in date palm at earliest stages.

STUDIES ON THE GENETIC RELATIONSHIP OF CHINESE DIPLOID STRAWBERRY SPECIES BASED ON SCOT ANALYSIS

Start Codon Targeted Polymorphism (SCoT) is a novel gene-targeted marker with the advantages of both ISSR and RAPD. In this experiment, the SCoT analysis was conducted to identify genetic relationship between diploid strawberry species distributed in China. The results were analyzed by the genetic symmetric coefficients and UPGMA cluster analysis. It proved that F. nilgerrensis was the most ancient species of all diploid Fragaria species. Fragaria nubicola had relatively close relationship with F. vesca and F. mandschurica, but was, genetically, far from other diploid species. F. vesca perhaps was the origins of most diploid species in Fragaria except F. nilgerrensis. F. pentaphylla had plentiful diversities, and was in the middle way of the evolution from F. vesca or F. mandschurica to F. viridis.

Stacking of five favorable alleles for amylase content, fragrance and disease resistance into elite lines in rice (Oryza sativa) by using four HRM-based markers and a linked gel-based marker

Marker-assisted selection (MAS) for qualitative traits such as grain quality and resistance to certain diseases has proven to be highly effective. Multiple genes responsible for various quality components and disease resistances can be simultaneously stacked to boost the performance and to lengthen the commercial lifespan of high-yield varieties. Grain quality genes (fgr and Wx) and three disease resistance genes (Pita, Pik and Xa23) have been well characterized and used in MAS breeding. However, stacking all of them together into a single variety has not been reported. We reported here the stacking of the five genes into elite lines in rice. We achieved this through the development of functional markers from causal mutations at fgr, Wx and Pita, a gene-targeted marker at Pik and the use of a linked marker for Xa23. We employed and optimized the high-resolution melting (HRM) analysis method for use as the genotyping platform of fgr, Wx, Pik and Pita. By combining high-throughput DNA isolation, multiplex and nested-PCR methods, we showed that HRM could serve as cost-effective, highly automated, moderate-throughput and reliable non-gel genotyping platform for a small-scale MAS program.

Potential of Start Codon Targeted (SCoT) markers for DNA fingerprinting of newly synthesized tritordeums and their respective parents.

Hexaploid tritordeum (H(ch)H(ch)AABB; 2n = 42) results from the cross between Hordeum chilense (H(ch)H(ch); 2n = 14) and cultivated durum wheat (Triticum turgidum ssp. durum (AABB; 2n = 28). Morphologically, tritordeum resembles the wheat parent, showing promise for agriculture and wheat breeding. Start Codon Targeted (SCoT) polymorphism is a recently developed technique that generates gene-targeted markers. Thus, we considered it interesting to evaluate its potential for the DNA fingerprinting of newly synthesized hexaploid tritordeums and their respective parents. In this study, 60 SCoT primers were tested, and 18 and 19 of them revealed SCoT polymorphisms in the newly synthesized tritordeum lines HT27 and HT22, respectively, and their parents. An analysis of the presence/absence of bands among tritordeums and their parents revealed three types of polymorphic markers: (i) shared by tritordeums and one of their parents, (ii) exclusively amplified in tritordeums, and (iii) exclusively amplified in the parents. No polymorphism was detected among individuals of each parental species. Three SCoT markers were exclusively amplified in tritordeums of lines HT22 and HT27, being considered as polyploidization-induced rearrangements. About 70% of the SCoT markers of H. chilense origin were not transmitted to the allopolyploids of both lines, and most of the SCoTs scored in the newly synthesized allopolyploids originated from wheat, reinforcing the potential use of tritordeum as an alternative crop.