Sequence Characterized Amplified Region Research Articles

Characterizing and marker-assisting a novel chili pepper (Capsicum annuum L.) yellow bud mutant with cytoplasmic male sterility.

Cytoplasmic male sterility (CMS) in pepper is a better way to produce hybrid seeds compared to manual production. We used the two sequence characterized amplified region (SCAR) markers (CRF-SCAR and CMS-SCAR130) in CMS pepper, to identify the genotype. We assembled two CMS yellow bud mutants (YBM; YBM12-A and YBM12-B). This mutation in leaf color is controlled by a single dominant nuclear gene. The aim was to create a new hybrid seed production method that reduces the costs and increases F1 hybrid seed purity. The results suggest that the CRF-SCAR and CMS-SCAR130 markers can be used together in multiple generations to screen for restorer or maintainer genes. We found the marker linked to the restorer gene (Rf) in the C-line and F1 hybrids, as well as partially in the F2 generation, whereas it was not found in the sterile YBM12-A or the maintainer line YBM12-B. In the F2 population, sterility and fertility segregated at a 3:1 ratio based on the CRF-SCAR marker. A 130 bp fragment was produced in the YBM12-A, F1, and F2 populations, suggesting that these lines contained sterile cytoplasm. A 140 bp fragment present in the YBM12-B and C-line indicated that these lines contained normal cytoplasm. In addition, we identified some morphological characters distinguishing sterile and fertile buds and flowers that may be linked to the sterility gene. If more restorer lines are identified, CMS expressing the YBM trait can be used in hybrid seed production.

Identification of a Causal Pathogen of Watermelon Powdery Mildew in Korea and Development of a Genetic Linkage Marker for Resistance in Watermelon (Citrullus lanatus)

Watermelon production is often limited by powdery mildew in areas with a large daily temperature range. Development of resistant watermelon cultivars can protect against powdery mildew; however, little is known about the characteristics of its causal agents. Here, we identified the genus and race of a causal pathogen of powdery mildew in Ansung province of South Korea, and developed molecular markers for the generation of resistant watermelon cultivars. The causal pathogen was determined to be Podosphaera xanthii based on multiple sequence alignments of internal transcribed spacers (ITS) of rDNA. The physiological race was identified as 1W, and the Ansung isolate was named P. xanthii 1W-AN. Following inoculation with the identified P. xanthii 1W-AN, we found inheritance of the resistant gene fitting a single dominant Mendelian model in a segregated population (‘SBA’ × PI 254744). To develop molecular markers linked to fungus-resistant loci, random amplified polymorphic DNA (RAPD) was accomplished between DNA pooled from eight near-isogenic lines (NILs; BC4F6), originated from PI 254744 and susceptible ‘SBB’ watermelon. After sequencing bands from RAPD were identified in all eight NILs and PI254744, 42 sequence-characterized amplifiedregion (SCAR) markers were developed. Overall, 107 F2 plants derived from BC4F6 NIL- 1 × ‘SBB’ were tested, and one SCAR marker was selected. Sequence comparison between the SCAR marker and the reference watermelon genome identified three Nco I restriction enzyme sites harboring a single nucleotide polymorphism, and codominant cleavage-amplified polymorphic site markers were subsequently developed. A CAPS marker was converted to a high-resolution melt (HRM) marker, which can discriminate C/T SNP (254PMR-HRM3). The 254PMR-HRM3 marker was evaluated in 138 F2:3 plants of a segregating population (‘SBA’ × PI254744) and was presumed to be 4.3 cM from the resistance locus. These results could ensure P. xanthii 1W-AN resistance in watermelon germplasm and aid watermelon cultivar development in marker-assist breeding programs.

A report on extensive lateral genetic reciprocation between arsenic resistant Bacillus subtilis and Bacillus pumilus strains analyzed using RAPD-PCR

The study involves isolation of arsenic resistant bacteria from soil samples. The characterization of bacteria isolates was based on 16S rRNA gene sequences. The phylogenetic consanguinity among isolates was studied employing rpoB and gltX gene sequence. RAPD-PCR technique was used to analyze genetic similarity between arsenic resistant isolates. In accordance with the results Bacillus subtilis and Bacillus pumilus strains may exhibit extensive horizontal gene transfer. Arsenic resistant potency in Bacillus sonorensis and high arsenite tolerance in Bacillus pumilus strains was identified. The RAPD-PCR primer OPO-02 amplified a 0.5kb DNA band specific to B. pumilus 3ZZZ strain and 0.75kb DNA band specific to B. subtilis 3PP. These unique DNA bands may have potential use as SCAR (Sequenced Characterized Amplified Region) molecular markers for identification of arsenic resistant B. pumilus and B. subtilis strains.

A TaqMan real-time PCR assay for Rhizoctonia cerealis and its use in wheat and soil

Rhizoctonia cerealis causes sharp eyespot in cereals and the pathogen survives as mycelia or sclerotia in soil. Real-time Polymerase Chain Reaction (qPCR) assays based on TaqMan chemistry are highly suitable for use on DNA extracted from soil. We report here the first qPCR assay for R. cerealis using TaqMan primers and a probe based on a unique Sequence Characterised Amplified Region (SCAR). The assay is highly specific and did not amplify DNA from a range of other binucleate Rhizoctonia species or isolates of anastomosis groups of Rhizoctonia solani. The high sensitivity of the assay was demonstrated in soils using a bulk DNA extraction method where 200 μg sclerotia in 50 g of soil were detected. DNA of the pathogen could also be amplified from asymptomatic wheat plants. Using the assay on soil samples from fields under different crop rotations, R. cerealis was most frequently detected in soils where wheat was grown or soil under pasture. It was detected least frequently in fields where potatoes were grown. This study demonstrates that assays derived from SCAR sequences can produce specific and sensitive qPCR assays.

Detection of Powdery Mildew (Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff) Resistance Gene in Melon (Cucumis melo L.) Cultivar Tacapa Green Black

Melon (Cucumis melo L.) is one of excellent horticultural commodities in Indonesia. The seed's demand rises in every year due to the increase of people's avocation. However, in the production, melon faces many problems. One of them is Powdery Mildew attack. Consequently, it is needed to create a new cultivar that has gene against the attack. Tacapa Green Black (GB) was one of cultivars developed by Laboratory of Genetics and Breeding, Faculty of Biology, Universitas Gadjah Mada. Tacapa GB was derived between PI 371795 and Act3 434 with a test cross Act3 434. The goal of this research was detection of a powdery mildew resistance gene. It was conducted using Sequence Characterized Amplified Regions (SCARs) analysis. The comparator cultivars used in this research were Tacapa Silver, Action 434, and Aramis. The result showed that Tacapa GB had a resistance gene of powdery mildew. It was proven with DNA band at 1058 base pair (bp). Based on this result, it could be concluded that Tacapa GB was excellent melon because of an ability in facing Powdery Mildew problems naturally. Furthermore, because of the presence of that gene, it could reduce the using of pesticide which was commonly used by Farmers to handle fungi attack.

Molecular screening of tomato (Solanum lycopersicum L.) genotypes for resistance alleles against important biotic stresses

Molecular markers are vastly used as tool for screening of disease resistant/tolerant plant genotypes in early stage of growth in an environment-independent manner. In tomato (Solanum lycopersicum L.), the major biotic stresses like tomato yellow leaf curl virus, Fusarium wilt, tomato spotted wilt virus and root knot nematode cause severe crop loss. Recently, DNA based molecular markers for the screening of resistance alleles for the abovementioned diseases have been reported in tomato. In the present study, a total of eighteen tomato genotypes have been screened for the presence of possible resistance alleles, using sequence characterized amplified region(SCAR) molecular markers. Resistance allele-specific bands for Fusarium wilt disease, tomato spotted wilt disease and partial resistance allele-specifc band for root knot disease have been identified in some of the genotypes used in the present study. However, none of the genotypes was found to contain Ty3 resistance allele-specific band for resistance to tomato yellow leaf curl disease. Thus, possible resistance sources have been identified for three out of the four biotic stresses, mentioned earlier. Thus, the present study has screened the 18 tomato genotypes at molecular level for presence of resistance alleles for biotic stress, which might be further evaluated and explored in future tomato breeding programmes, targeting biotic stress resistance in tomato. At the same time, the study documents the applicability of molecular markers for rapid disease screening in tomato in an environment independent manner.

Efficiency of Sequence Characterized Amplified Region Markers for Selecting Non-Astringent Persimmon (Diospyros kaki Thunb.)

Persimmon (Diospyros kaki Thunb.) is classified into four types based on the fruit traits, astringency and flesh color. Of the four types, the pollination-constant non-astringent (PCNA) fruit is typically most desirable for consumption. In the present study, we used five sequence characterized amplified region (SCAR) markers associated with astringency in persimmon fruit, namely E4/E9r, E4/A2r, 7H9F/AST-R, AST-F/AST-R, and AST-F/PCNA-F/5R3R, to improve the efficiency of PCNA-type persimmon breeding via marker-assisted selection (MAS). A total of 84 cultivars of the four types and their segregating F1 progeny were used to evaluate the association of SCAR markers with the fruit astringency phenotype. Polymerase chain reaction evaluation of each SCAR marker showed that E4/E9r combined with AST-F/PCNA-F/5R3R was appropriate for selecting the ast allele responsible for PCNA-type fruit, as the phenotype-genotype match percentages of these two markers were 94% and 99%, respectively. This MAS was verified by the successful use of AST-F/PCNA-F/5R3R to select 107 PCNA-type individuals from 609 F1 hybrid progeny derived from various crosses.

Development of a diagnostic DNA marker for the geographic origin of Shorea leprosula

Abstract The development of sequence characterized amplified region (SCAR) markers derived from amplified fragment length polymorphisms (AFLPs) is described for Shorea leprosula. An AFLP fragment that showed nearly complete differentiation between Borneo and Sumatra was gel-extracted, sequenced, and converted into a SCAR marker using the inverse polymerase chain reaction (PCR) technique. The single nucleotide polymorphism (SNP) that originally caused the AFLP was found in the MseI restriction site. Differentiation between islands was detected either as size variation of the codominant SCAR marker or after digestion of the PCR products with the restriction enzyme MseI (PCR-RFLP). Size variation was due to insertions/deletions found within the sequenced region that flanked the original AFLP fragment. After genotyping 151 samples of S. leprosula from 14 populations in Sumatra and Borneo, all but one sample from Sumatra were homozygous for one size variant (427 bp), while S. leprosula populations from Borneo showed different genotypes than Sumatra populations and variation not only among populations but also within populations. Complete differentiation and fixation on alternative variants was found for the geographic regions of Sumatra and Borneo by the PCR-RFLP method. The SCAR marker did not amplify in Shorea parvifolia and thus can also be used to distinguish between S. leprosula and S. parvifolia. The marker was successfully amplified from wood DNA extracts suggesting its applicability to track the geographic origin of timber.

English

Cassava is an economically important crop in sub-Saharan Africa; however, its yield potential is constrained by cassava mosaic disease (CMD) infection. Classical genetics and biotechnology are being harnessed to overcome the disease and secure yields for farmers. The CMD2 resistance locus flanked by three simple sequence repeats (SSR) markers and one sequence characterized amplified region (SCAR) marker were mapped in West African genotypes and shown to impart qualitative resistant to all species of CMGs. However, gene(s) associated with the CMD2 locus and their mode of actions remains unknown. In an effort to discover gene(s) located in CMD2 locus region, TME3 BAC collections were screened for the presence of CMD2 flanking markers. CMD susceptible and resistant cassava genotypes were found to contain 100% of the markers flanking CMD2 locus. SNPs and nucleotide deletions were identified within the marker sequences but there was no evidence of trait and marker association. All the SSR markers flanking CMD2, and the more recently characterized CMD3 loci were to be located on chromosome 12. Through BAC pools library hybridization with marker probes, 130 BACs were identified, but only 23 BACs contained at least CMD2 specific two markers. Whole BAC sequencing identified five clones that mapped to the marker regions. BAC29 assembled into a 100 kb contig and encoded tandem repeats of three full length R genes (3.5 kb) and two partial repeats. These R genes were conserved and highly expressed in CMD susceptible and CMD resistant cassava genotypes. Promoter sequences derived from R genes showed similar transient expression of GUS as 35S promoter. On cassava genome V6.1 BAC29 sequences were mapped to chromosome 16, eliminating their potential role in CMD resistant. Key words: Bacteria artificial chromosome, CMD2, cassava, cassava mosaic disease.

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.

Development of SCAR Markers and an SYBR Green Assay to Detect Puccinia striiformis f. sp. tritici in Infected Wheat Leaves.

Stripe rust, caused by the pathogenic fungus Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. A rapid and reliable detection of the pathogen in latent infected wheat leaves is useful for accurate and early forecast of outbreaks and timely application of fungicides for managing the disease. Using the previously reported primer pair Bt2a/Bt2b, a 362-bp amplicon was obtained from P. striiformis f. sp. tritici and a 486-bp amplicon was obtained from both P. triticina (the leaf rust pathogen) and P. graminis f. sp. tritici (the stem rust pathogen). Based on the sequence of the 362-bp fragment, two pairs of sequence characterized amplified region (SCAR) primers were designed. PSTF117/PSTR363 produced a 274-bp amplicon and TF114/TR323 produced a 180-bp amplicon from P. striiformis f. sp. tritici, whereas they did not produce any amplicon from P. triticina, P. graminis f. sp. tritici, or any other wheat-infecting fungi. The detection limit of PSTF117/PSTR363 was 1 pg/µl and TF114/TR323 was 100 fg/µl. Both SCAR markers could be detected in wheat leaves 9 h post inoculation. An SYBR Green RT-PCR method was also developed to detect P. striiformis f. sp. tritici in infected leaves with the detection limit of 1.0 fg DNA from asymptomatic leaf samples of 6 h after inoculation. These methods should be useful for rapid diagnosis and accurate detection of P. striiformis f. sp. tritici in infected wheat leaves for timely control of the disease.

Development of a SCAR marker associated with salt tolerance in durum wheat (Triticum turgidum ssp. durum) from a semi-arid region

Durum wheat (Triticum turgidum ssp. durum) is one of the main species of cultivated wheat. In arid and semi-arid areas, salinity stress reduces durum wheat productivity. This study used 26 durum wheat accessions from semi-arid regions in Tunisia to analyze plant tolerance to salt stress. Salt stress was experimentally applied by regularly submerging pots in NaCl solution. The salt tolerance trait index (STTI) and salt tolerance index (STI) of various growth parameters were used as criteria to select for salt tolerance. Analysis of genetic relationships was carried out to determine the genetic distance between durum wheat accessions. Based on simple sequence repeats analysis, a molecular marker for salt stress resistance in durum wheat was developed. Salt-treated plants had reduced morphological traits compared to control plants. Most STTIs in all genotypes were below 100 %. Based on STI, 8 accessions were found to be salt-resistant, 16 were salt-moderate, two were salt-susceptible. Analysis of the genetic relationships among 28 Tunisian durum wheat accessions revealed that landraces of the same nominal type are closely related. Of the 94 SSR primers investigated, three were selected and used to design sequence characterized amplified region (SCAR) primers. One SCAR primer pair, KUCMB_Xgwm403_2, produced a 207 bp band that was present in salt-resistant durum wheat lines but absent in salt-susceptible lines. The results suggest that KUCMB_Xgwm403_2 could be a potential genetic tag for salt-tolerant durum wheats.

RAPD and SCAR markers as potential tools for detection of milk origin in dairy products: Adulterant sheep breeds in Serra da Estrela cheese production

Serra da Estrela Protected Designation of Origin (PDO) cheese is the most famous Portuguese cheese and has a high commercial value. However, the adulteration of production with cheaper/lower-quality milks from non-autochthones ovine breeds compromises the quality of the final product and undervalues the original PDO cheese. A Randomly Amplified Polymorphic DNA (RAPD) method was developed for efficient detection of adulterant breeds in milk mixtures used for fraudulent production of this cheese. Furthermore, Sequence Characterized Amplified Region (SCAR) markers were designed envisioning the detection of milk adulteration in processed dairy foods. The RAPD-SCAR technique is here described, for the first time, to be potentially useful for detection of milk origin in dairy products. In this sense, our findings will play an important role on the valorization of Serra da Estrela cheese, as well as on other high-quality dairy products prone to adulteration, contributing to the further development of the dairy industry.

A saturated SNP linkage map for the orange wheat blossom midge resistance gene Sm1.

SNP markers were developed for the OWBM resistance gene Sm1 that will be useful for MAS. The wheat Sm1 region is collinear with an inverted syntenic interval in B. distachyon. Orange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin) is an important insect pest of wheat (Triticum aestivum) in many growing regions. Sm1 is the only described OWBM resistance gene and is the foundation of managing OWBM through host genetics. Sm1 was previously mapped to wheat chromosome arm 2BS relative to simple sequence repeat (SSR) markers and the dominant, sequence characterized amplified region (SCAR) marker WM1. The objectives of this research were to saturate the Sm1 region with markers, develop improved markers for marker-assisted selection (MAS), and examine the synteny between wheat, Brachypodium distachyon, and rice (Oryza sativa) in the Sm1 region. The present study mapped Sm1 in four populations relative to single nucleotide polymorphisms (SNPs), SSRs, Diversity Array Technology (DArT) markers, single strand conformation polymorphisms (SSCPs), and the SCAR WM1. Numerous high quality SNP assays were designed that mapped near Sm1. BLAST delineated the syntenic intervals in B. distachyon and rice using gene-based SNPs as query sequences. The Sm1 region in wheat was inverted relative to B. distachyon and rice, which suggests a chromosomal rearrangement within the Triticeae lineage. Seven SNPs were tested on a collection of wheat lines known to carry Sm1 and not to carry Sm1. Sm1-flanking SNPs were identified that were useful for predicting the presence or absence of Sm1 based upon haplotype. These SNPs will be a major improvement for MAS of Sm1 in wheat breeding programs.

Targeted sequencing of a complex locus within a polyploid genome using reduced representation libraries

Apospory is a form of gametophytic apomixis in which embryos develop from unreduced embryo sacs derived from aposporous initials formed from nucellar cells of ovules to produce offspring genetically identical to the female plant. Apospory in Pennisetum squamulatum (8X) and Cenchrus ciliaris (4X) is a dominant trait controlled by a physically large, hemizygous, heterochromatic chromosomal block called the apospory-specific genomic region (ASGR). Both apomictic species are polyploid, with genome sizes estimated at 2600 to 3000 Mbp for C. ciliaris and 9400 to 10,300 Mbp for P. squamulatum. A study was conducted to determine whether duplex-specific nuclease (DSN) normalization of DNA from apomictic and sexual genotypes would reduce repetitive sequences and allow bioinformatic analysis to predict sequence contigs derived from the ASGR. DSN libraries from four genotypes were sequenced using Illumina® HiSeq 2000 technology. 39 out of 44 tested sequence characterized amplified region (SCAR) markers from in silico predicted ASGR-specific contigs were mapped to the ASGR in a Pennisetum F1 mapping population. Eighteen SCARs showed apomict-specific amplification in C. ciliaris. The successful mapping of ~90 % of the SCAR markers to the ASGR in the Pennisetum F1 mapping population shows that DSN normalization and Illumina sequencing can be used as an effective strategy for targeted mapping of a physically large locus rich in repetitive sequences, like that of the ASGR.

Development of a SCAR marker and a strain-specific genomic marker for the detection of the biocontrol agent strain CPA-8Bacillus amyloliquefaciens(formerlyB. subtilis)

In this work, reliable tools were developed to detect and identify the biocontrol strain CPA-8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA-8 that was sequenced and used to design SCAR (sequence-characterised amplified regions) primer pairs. The SCAR-4 marker amplified a semi-specific fragment of 665 bp not only for the strain CPA-8 but also for other 12 strains whose morphology was completely different from CPA-8. Another approach was developed to obtain a strain-specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA-8. Our results revealed that these two molecular markers, SCAR-4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA-8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA-8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.

Development of molecular markers for authentication of the medicinal plant species Patrinia by random amplified polymorphic DNA (RAPD) analysis and multiplex-PCR

The authentication of components from plant species is vital for the quality control and standardization of herbal medicines. Due to morphological similarities when dried, it is very difficult to distinguish authentic medicinal plants Patrinia villosa and P. scabiosifolia from adulterants of P. rupestris and P. saniculifolia in the herbal medicine Patriniae Radix. To establish a reliable authentication tool distinguishing authentic Patriniae Radix species from adulterants, we used random amplified polymorphic DNA (RAPD) genomic profiling and obtained several species-specific DNA fragments for each of the four Patrinia species. Based on the sequences of these amplicons, we developed sequence characterized amplified region (SCAR) markers as stable molecular authentication tools. SCAR markers to identify the authentic Patriniae Radix species, P. villosa and P. scabiosifolia, were developed. To accurately identify adulterants, we also developed SCAR markers able to identify the inauthentic P. rupestris and P. saniculifolia species. Furthermore, we established multiplex-PCR SCAR assays to simultaneously distinguish all four species using combinations of species-specific SCAR marker primer sets. These efforts resulted in reliable DNA-based molecular markers to identify authentic Patriniae Radix species from their adulterants and a rapid authentication assay for the efficient identification of Patrinia species. These markers will facilitate the standardization of Patriniae Radix preparations and therefore prevent the distribution of adulterants.

Molecular markers for tracking the origin and worldwide distribution of invasive strains of Puccinia striiformis.

Investigating the origin and dispersal pathways is instrumental to mitigate threats and economic and environmental consequences of invasive crop pathogens. In the case of Puccinia striiformis causing yellow rust on wheat, a number of economically important invasions have been reported, e.g., the spreading of two aggressive and high temperature adapted strains to three continents since 2000. The combination of sequence‐characterized amplified region (SCAR) markers, which were developed from two specific AFLP fragments, differentiated the two invasive strains, PstS1 and PstS2 from all other P. striiformis strains investigated at a worldwide level. The application of the SCAR markers on 566 isolates showed that PstS1 was present in East Africa in the early 1980s and then detected in the Americas in 2000 and in Australia in 2002. PstS2 which evolved from PstS1 became widespread in the Middle East and Central Asia. In 2000, PstS2 was detected in Europe, where it never became prevalent. Additional SSR genotyping and virulence phenotyping revealed 10 and six variants, respectively, within PstS1 and PstS2, demonstrating the evolutionary potential of the pathogen. Overall, the results suggested East Africa as the most plausible origin of the two invasive strains. The SCAR markers developed in the present study provide a rapid, inexpensive, and efficient tool to track the distribution of P. striiformis invasive strains, PstS1 and PstS2.

SCAR Marker for Identification and Discrimination of Commiphora wightii and C. myrrha.

Commercially important Commiphora species are drought-tolerant plants and they are leafless for most of the year. Therefore, it is necessary to develop some molecular marker for the identification. Intended for that, in the present study, species-specific, sequence-characterized amplified regions (SCAR) markers were developed for proficient and precise identification of closely related species Commiphora wightii and C. myrrha, which may ensure the quality, safety, and efficacy of medicines made from these plants through adulterous mixing of these plants. Two species-specific RAPD amplicons were selected, gel-purified, cloned, and sequenced after screening of 20 RAPD primers. The sequence of 979 and 590 nucleotides (Genebank accession numbers K90051 and K90052) was used for development of 4 SCAR markers, namely, Sc1P, Sc1Pm, Sc2P, and Sc2Pm. Out of them, the Sc1Pm was specific for C. wightii, while Sc2P discriminated both the Commiphora species. These markers are first reported and will be useful for rapid identification of closely related Commiphora wightii and C. myrrha species.

Gene-Based Markers for the Tomato Yellow Leaf Curl Virus Resistance Gene Ty-3

The viral disease induced by Tomato yellow leaf curl virus (TYLCV) reduces tomato (Solanum lycopersicum) yield significantly in tropical and subtropical regions. A number of loci, including Ty-1 to Ty-5, conferring resistance to TYLCV have been described and introgressed into modern tomato cultivars. The availability of molecular markers linked to these genes would expedite the introgression of TYLCV resistance into commercial cultivars. In the present study, we developed gene-based markers linked to the Ty-3 gene using a segregating population derived from a cross between the TYLCV-resistant line S. lycopersicum ‘A45’ and the susceptible line S. lycopersicum ‘A39’. Agrobacterium-mediated screening was used to test TYLCV resistance of plants in the segregating population, and the resistance was evaluated by a visual scoring method and polymerase chain reaction analysis. By comparing sequences of the Ty-3 genes of the resistant and susceptible lines, two high-resolution melting (HRM) markers (Ty3-HRM1 and Ty3-HRM2) and one sequence characterized amplified region (SCAR) marker (Ty3-SCAR1) were developed. The HRM markers were based on single nucleotide polymorphisms at the 13th exon and the 15th intron, whereas the SCAR marker was based on a 246-bp deletion in the 16th intron. These gene-based markers will be useful tools for marker-assisted selection in breeding programs to improve TYLCV resistance of tomato.