Restriction Fragment Length Polymorphism Markers Research Articles

Comparative Analysis of Molecular RFLP and SNP Markers in Assessing and Understanding the Genetic Diversity of Various Chicken Breeds.

Monitoring the genetic diversity of small populations is important with respect to conserving rare and valuable chicken breeds, as well as discovery and innovation in germplasm research and application. Restriction fragment length polymorphisms (RFLPs), the molecular markers that underlie multilocus DNA fingerprinting (MLDF), have historically been employed for this purpose, but over the past two decades, there has been an irreversible shift toward high-throughput single-nucleotide polymorphisms (SNPs). In this study, we conducted a comparative analysis of archived MLDF results and new data from whole-genome SNP genotyping (SNPg) among 18 divergently selected breeds representing a large sample of the world gene pool. As a result, we obtained data that fit the general concept of the phylogenetic distribution of the studied breeds and compared them with RFLP and SNP markers. RFLPs were found to be useful markers for retrospective assessment of changes in the genetic architecture and variability underlying the phenotypic variation in chicken populations, especially when samples from previous generations used for MLDF are unavailable for SNPg. These results can facilitate further research necessary to assess the possibility of extrapolating previous MLDF results to study the long-term dynamics of genetic diversity in various small chicken germplasm populations over time. In general, the whole-genome characterization of populations and breeds by multiple SNP loci will further form the basis for the development and implementation of genomic selection with the aim of effective use of the genetic potential of the domestic gene pool in the poultry industry.

Study of Plant Genetic Variation through Molecular Markers: An Overview

This article refers to viewing the role of molecular markers during analyzing the genome of plants and their importance in plant biotechnology. In recent years, we observed the role of molecular techniques in programs for improving plant breeding and preserving genetic resources has been observed, and molecular and biochemical indicators which represent basic material through determining the diversity between genotypes for indicators it is never affected by external surrounding conditions as always in the phenotype features. Molecular markers of DNA have been widely applied to answer a range of questions related to taxonomy, molecular evolution, population genetics, and genetic diversity, as well as monitoring trade in plants and food products , in addition to its having a role in studying gene expression , genetic mapping, and studies of species evolution providing fast and accurate results. In this work, the advantages and limitations of the molecular techniques applied in plant sciences such as: RAPD (Random Amplification Polymorphic DNA Marker); ISSR (Inter Simple Sequence Repeat Marker); SSR (Simple Sequence Repeat Marker); AFLP (Amplified Fragment Length Polymorphic Marker); RFLP (Restriction Fragment Length Polymorphism Marker); SNP (Single Nucleotide Polymorphism) and Real Time PCR.

Utilization of Molecular Markers for Rice Breeding

Rice is the staple food for more than half of the world's population. Rice production in 2050 must increase by at least 50% to keep up with the population growth. Efforts to increase rice production continue using various strategies. Breeders apply multiple approaches including application of molecular markers in developing varieties better than the previous ones. Since the discovery of the restriction fragment length polymorphism (RFLP) markers in 1980s and the development of polymerase chain reaction (PCR) method, many types of molecular markers have been developed and applied to various crops including rice. Various molecular approaches to map genetic loci associated with rice superior traits were conducted. The mapped loci are very useful for rice breeding purposes. This paper reports the results of mapping and breeding economically important traits in rice, mainly those related to abiotic stresses, agronomic traits, yield, and yield quality. These included characters of semidwarf stature, aromatic grain, high yield potential, eating quality, higher Zn and Fe grain, more tolerant to abiotic stresses, such as salinity, drought, phosphate deficiency, Al toxicity and Fe toxicity, submergence, as well as early maturity character. The mapped characters can be transferred using marker-assisted backcrossing (MABC) method into cultivated rice genotypes well-adopted by farmers. Several countries including Indonesia have benefited from this breeding method, and Indonesia have released several rice varieties developed through MABC. These include rice varieties such as Code, Angke, Inpari 30, Inpari Blas, Inpari HDB, Bio Patenggang Agritan, and Bioni 63 Ciherang Agritan

Genetic Identification of all Four Mangrove Mud Crab Species (genus Scylla) Using Multiple Molecular Markers.

Misleading identification and subsequent publications on biological, molecular, and aquaculture data of mangrove mud crab (genus Scylla de Hann 1833) is a major concern in many countries. In this study, multiple molecular markers were used for genetic identification of all four known mud crab species under genus Scylla collected from India, Philippines, Myanmar, Malaysia and Indonesia. Internal Transcribed Spacer (ITS-1), Polymerase chain reaction (PCR)-Restriction Fragment Length Polymorphism (PCR-RFLP) and PCR-based species-specific markers were used to resolve taxonomic ambiguity. PCR-RFLP techniques using NlaIV and BsaJI restriction endonucleases were efficient to differentiate four different mud crab species under genus Scylla with specific fragment profile. The results also justified the use of ITS-1 and PCR-based species-specific markers to identify mud crab species available in many countries quite rapidly and effectively. Several new molecular markers generated during the study are reported here to resolve the taxonomic ambiguity of Scylla species and the results reconfirmed that India is only having two commonly available mud crab species which was reported by the authors earlier.

Molecular Species Identification of Six Forensically Important Iranian Flesh Flies (Diptera).

Background:Flesh flies (Diptera: Sarcophagidae) are considered as myiasis agents and important evidences in forensic investigations. However, their use has been restricted because, at all larval stages and female adults, morphological species identification is difficult or very challenging. This study investigated to test utility of mitochondrial cytochrome oxidase subunit I (mt-COI) sequences for differentiation of six forensically important Iranian flesh flies namely, Sarcophaga crassipalpis, S. flagellifera, S. hirtipes, S. aegyptica, S. africa and S. argyrostoma.Methods:Male specimens were morphologically identified to species level and then the genomic DNA of the flies were extracted and subjected to polymerase chain reaction (PCR) against mt-COI gene. The PCR products were sequenced and the obtained sequences were analyzed for the species specific restriction fragment length polymorphisms (RFLPs).Results:Rate of genetic variation between species was 6–10% which was enough to find restriction enzymes (RE) that were able to produce species-specific RFLP profiles. Combinations of three REs: BsrFI, RsaI and HinfI, provided diagnostic bands for identification of the six Sarcophaga species.Conclusion:The results of this study showed that molecular markers such as RFLPs enhancing the use of evidence from flesh flies in forensic investigation. However, lack proper restriction sites in the COI region inhibited introduction of a single restriction enzyme for easy species identification. It is recommended to apply larger part of DNA such as combination of COI and COII genes to provide better RFLP markers for species identification of flesh flies.

Marker-assisted selection: A smart biotechnological strategy for modern plant breeding

Plant breeders and geneticists use molecular marker-assisted selection also called as MAS as a useful approach for breeding of plant to make selection more efficient and speed up the breeding cycle. MAS can be more efficient, effective, and reliable than phenotypic selection. Molecular markers are useful to identify the economically important traits in the breeding population for further manipulation in a short time. Due to the applicability of markers at the seedling stage ensuring high precision at the reduced level of cost, marker-assisted selection offer the chances to improve responses from selection. The MAS using DNA level polymorphism accelerate the pace of selection. The main marker technologies applied are chiefly co-dominant markers i.e. microsatellite markers/SSR (Simple Sequence Repeats) marker, RFLP (Restriction Fragment Length Polymorphism) marker and SNPs (Single nucleotide polymorphisms). This review overviews the various MAS technologies and their applications in crop improvement programs.

Comparative Molecular Description of a Novel GST Gene in Culex pipiens (Diptera: Culicidae).

Repeated exposure to insecticides, particularly pyrethroids and organophosphates, has resulted in the development of insecticide resistance in the mosquito Culex pipiens, a primary disease vector. Glutathione S-transferase (GST) is involved in the phase II detoxification of numerous xenobiotics, including insecticides. In this study, a GST gene (CPIJ002678) was amplified, sequenced, and used in comprehensive molecular analyses ending up in development of a rapid assay to distinguish more tolerant individuals from susceptible Culex pipiens using the Restriction Fragment Length Polymorphism (RFLP) technique. Field collected Culex pipiens strains from untreated areas, organophosphates-treated areas and a lab strain reared for many generations, all were used in CDC bottle bioassays to evaluate the susceptibility status of the studied individuals to malathion insecticide. Interestingly, both field sites collected groups showed high levels of resistance at the malathion diagnostic time. Gene amplification, and bidirectional direct sequencing results were analyzed. Compared with the reference genome sequence, the pairwise alignment of the amplified sequences showed 96.6% similarity to the reference sequence in the GenBank database. The confirmed gene sequences were assembled and aligned using various bioinformatic softwares. The assembled contigs were used in NEBcutter V2.0 for constructing restriction maps and checked for the availability of differences (if present) between susceptible and more tolerant strains. Specific molecular RFLP markers were successfully recognized to differentiate the more tolerant from the susceptible Culex pipiens phenotypes.

Identification and Validation of Candidate Genes Conferring Resistance to Downy Mildew in Maize (Zea mays L.).

Downy mildew (DM) is a major disease of maize that causes significant yield loss in subtropical and tropical regions around the world. A variety of DM strains have been reported, and the resistance to them is polygenically controlled. In this study, we analyzed the quantitative trait loci (QTLs) involved in resistance to Peronosclerospora sorghi (sorghum DM), P. maydis (Java DM), and Sclerophthora macrospora (crazy top DM) using a recombinant inbred line (RIL) from a cross between B73 (susceptible) and Ki11 (resistant), and the candidate genes for P. sorghi, P. maydis, and S. macrospora resistance were discovered. The linkage map was constructed with 234 simple sequence repeat (SSR) and restriction fragment length polymorphism (RFLP) markers, which was identified seven QTLs (chromosomes 2, 3, 6, and 9) for three DM strains. The major QTL, located on chromosome 2, consists of 12.95% of phenotypic variation explained (PVE) and a logarithm of odds (LOD) score of 14.12. Sixty-two candidate genes for P. sorghi, P. maydis, and S. macrospora resistance were obtained between the flanked markers in the QTL regions. The relative expression level of candidate genes was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) using resistant (CML228, Ki3, and Ki11) and susceptible (B73 and CML270) genotypes. For the 62 candidate genes, 15 genes were upregulated in resistant genotypes. Among these, three (GRMZM2G028643, GRMZM2G128315, and GRMZM2G330907) and AC210003.2_FG004 were annotated as leucine-rich repeat (LRR) and peroxidase (POX) genes, respectively. These candidate genes in the QTL regions provide valuable information for further studies related to P. sorghi, P. maydis, and S. macrospora resistance.

Tetrad analysis of sake yeast and identification of an RFLP marker for the absence of phenolic off-flavour production.

Mating is a promising breeding method for industrial yeast. Although sake yeast has a low spore-formation ability, segregants exhibiting a mating type have been isolated from sake yeast K7. Here, we constructed zygotes from a cross between those segregants and a laboratory yeast strain. Because most sake and brewing yeast strains are prototrophs, we developed a PCR-based method to confirm that mating had taken place based on genome sequencing data and differences in nucleotide sequences between the two parental strains. The mated strain, termed S. cerevisiae MITOY123, showed restored spore-formation ability, unlike most sake and brewing yeast strains. By using the mated yeast strain MITOY123, it was possible to carry out tetrad analysis for the trait of the absence of off-flavour due to phenolic products such as 4-vinylguiacol (4-VG) in sake yeast K7. This tetrad analysis indicated that a single genetic region around the gene PAD1 is responsible for the absence of phenolic off-flavour in sake yeast K7. In order to aid the breeding of sake and brewing yeast strains by mating, we also identified a restriction fragment length polymorphism (RFLP) marker for the absence of phenolic off-flavour production in strains derived from sake yeast K7. Collectively, our data show that it is possible to breed new sake and brewing yeast strains by mating and to test for the absence of phenolic off-flavour production in resultant strains easily by RFLP analysis.

Quantitative trait loci (QTLs) linked with root growth in lettuce (Lactuca sativa) seedlings

In-field variation of transplanted lettuce (Lactuca sativa L.) due to variable soil and environmental conditions is one of the major restrictions in the optimization of production and yield. Marker-assisted breeding for lettuce varieties with a more rapid rooting phenotype has the potential to improve the performance of lettuce transplants. This study aimed to identify traits linked with increased primary root length, lateral root length and lateral root emergence in 14-day L. sativa seedlings from an intra-specific cross (Saladin × Iceberg). In total, 16 significant quantitative trait loci (QTLs) were associated with increased root growth traits that would allow direct introgression of the traits. Six of the QTLs were associated with increased primary root growth, accounting for 60.2% of the genetic variation for the trait. Three QTLs were associated with lateral root growth (38.6% of genetic variation); two QTLs were associated with lateral root length density (27.6% of genetic variation) and three with root number density (33.4% of genetic variation), and two QTLs were associated with mean lateral root length (21.1% of genetic variation). The statistical QTLs were located across 9 different linkage groups (LGs) representing loci on 7 of the 9 L. sativa chromosomes. A combination of restriction fragment length polymorphism (RFLPs) and Kompetitive allele specific PCR (KASPs) markers linked to these rooting traits were identified, which could allow breeders to select for a rapid establishment phenotype.

Impact of RFC1, MTHFR, and MTHFD1 polymorphism on unexplained pregnancy loss (UPL): comparative analysis of maternal and fetal components using mother–abortus paired samples

In this study, we aimed to investigate associations between polymorphisms of folate metabolic pathway genes and unexplained pregnancy loss (UPL) using matched maternal–fetal samples. A total of 113 mother–abortus and 92 mother–newborn samples were collected. Among the 113 mother–abortus samples, 50 with chromosomal abnormality and 22 with maternal cell contamination were excluded. Samples were genotyped for RFC-1 80A>G, MTHFD1 1958 G>A, MTHFR 677C>T, and MTHFR 1298A>C polymorphisms using restriction fragment length polymorphism markers. The genotypes of RFC-1 80A>G, MTHFD1 1958 G>A, MTHFR 677C>T, and MTHFR 1298A>C were not associated with UPL in maternal samples. In the fetal samples, the frequency of heterozygous genotype (GA) of MTHFD11958 G>A was significantly higher than that that of the control (OR = 2.477, 95% CI = 1.128–5.446, p = 0.037). The AA-GA genotypes of MTHFD1 1958G>A and RFC-1 80A>G were significantly higher in mother–abortus samples (p = 0.016) than in the mother–newborn samples (p = 0.029). Frequencies of allelic combinations of MTHFR 677C>T/MTHFD11958G>A and RFC-1 80A>G/MTHFR677C>T/MTHFD1 1958G>A were significantly higher in maternal samples of the UPL group. In the fetal samples, no significant differences were detected between the UPL group and the control group. This study is the first to show associations between MTHFD1 1958G>A, RFC-1 80G>A, MTHFR 677C>T, and MTHFR 1298A>C polymorphisms and UPL and to compare the effects of maternal and fetal samples on UPL using mother–abortus matched samples of Korean origin.

Development of a Species Identification Method for the Egg and Fry of the Three Korean Bitterling Fishes (Pisces: Acheilognathinae) using RFLP (Restriction Fragment Length Polymorphism) Markers

Development of a Species Identification Method for the Egg and Fry of the Three Korean Bitterling Fishes (Pisces: Acheilognathinae) using RFLP (Restriction Fragment Length Polymorphism) Markers

Mapping of easy to screen SSR markers for selection of RFLP markers-bracketed downy mildew resistance QTLs in pearl millet

Downy mildew (DM) of pearl millet, caused by fungus Sclerospora graminicola, is the most destructive disease of pearl millet which can cause grain yield losses up to 60%. Also referred to as ‘green ear’, this disease is widely distributed in temperate and tropical areas of the world and is especially important in India and Africa. In an earlier study, quantitative trait loci (QTL) for resistance to Sclerospora graminicola have been reported on linkage groups (LG) 1 and 4 using a F2 population derived from the cross of H 77/833–2 and ICMP 451. However, these QTLs were bracketed with labour-intensive restriction fragment length polymorphism (RFLP) markers, thus hampering their use in marker-assisted selection (MAS). The current study reported confirmation of these QTLs using the same cross and phenotypic data generated using seven different pathogen isolates obtained from Africa and India and saturation of the identified genomic regions with easy to screen simple sequence repeat (SSR) markers. The inclusion of SSR markers resulted in a map covering 101.1 cM and 91.4 cM on LG 1 and LG 4 respectively. QTL likelihood maps developed for each of seven Sclerospora graminicola isolates revealed that for most of the isolates, QTLs for DM resistance (DMR) were located either on LG 1 or LG 4, while Niger isolate had QTLs on both the linkage groups and no QTL was located for Mysore isolate. The SSRs can be efficiently utilized in MAS programmes targeting improvement of elite cultivars of pearl millet against this disease.

Relative Contribution of Seed Tuber- and Soilborne Inoculum to Potato Disease Development and Changes in the Population Genetic Structure of Rhizoctonia solani AG 3-PT under Field Conditions in South Africa.

Understanding the contribution of seed tuber- and soilborne inocula of Rhizoctonia solani AG 3-PT in causing potato disease epidemics is an important step in implementing effective management strategies for the pathogen. A 2-year study was conducted to evaluate the contribution of each source of inoculum using an integrative experimental approach combining field trials and molecular techniques. Two distinct sets of genetically marked isolates were used as seed tuberborne and soilborne inocula in a mark-release-recapture experiment. Disease assessments were done during tuber initiation and at tuber harvest. Both inoculum sources were found to be equally important in causing black scurf disease, whereas soilborne inocula appeared to be more important for root and stolon infection, and seedborne inocula contributed more to stem canker. However, seed tuber-transmitted genotypes accounted for 60% of the total recovered isolates when genotyped using three polymerase chain reaction restriction fragment length polymorphism markers. The changes in population structure of the experimental R. solani population over the course of the growing season and across two growing seasons were investigated using eight microsatellite markers. The populations at different sampling times were somewhat genetically differentiated, as indicated by Nei's gene diversity (0.24 to 0.27) and the fixation index (FST). The proportion of isolates with genotypes that differed from the inoculants ranged from 13 to 16% in 2013 and 2014, respectively, suggesting the possibility of emergence of new genotypes in the field. Because both soilborne and tuberborne inocula are critical, it is important to ensure the use of pathogen-free seed tubers to eliminate seed tuberborne inoculum and the introduction of new genotypes of R. solani for sustainable potato production in South Africa.

Novel phytoplasma strains of X‐disease group unveil genetic markers that distinguish North American and South American geographic lineages within subgroups 16SrIII‐J and 16SrIII‐U

AbstractPhytoplasmas in the X‐disease group (16SrIII) are highly diverse in terms of geographic distributions, vectorship and plant host specificity. Such biological and ecological diversity is often correlated with distinctive genetic markers present in evolutionarily conserved genes. Based on restriction fragment length polymorphism (RFLP) markers in the 16S rRNA gene sequences, 29 subgroups have been delineated, with most of them being found in the Americas. However, it has been unknown whether distinct geographic lineages are present within a given subgroup. Prior to this study, phytoplasmas belonging to subgroups 16SrIII‐J and 16SrIII‐U were reported only in countries located in South America. In the present study, we identified new phytoplasmas strains closely related to the reference strains of the two subgroups in Mexico, a North American country. These newly identified Mexican strains possess unique RFLP, single nucleotide polymorphism, and fragmental deletion markers in 16S rRNA‐ and/or ribosomal protein‐encoding genes. Since these markers consistently distinguished the Mexican strains from their South American counterparts, they may represent emerging or previously unknown North American geographic lineages of the subgroups 16SrIII‐J and 16SrIII‐U.

Analysis of bulked segregants to identify molecular markers linked with cocoon weight and cocoon shell weight in the silkworm Bombyx mori L

Two silkworm strains viz, B20 Λ (high cocoon shell ratio) and C. Nichi (low cocoon shell ratio) were sib mated for 10 generations to determine the homozygosis. Both bulked segregant analysis (BSA) and near isogenic lines (NIL) studies were done to identify the RFLP markers closely linked to cocoon shell parameters. Three hundred and fifty-two random clones were identified as the low copy number sequence and used for identification of Restriction Fragment Length Polymorphic (RFLP) marker linked to cocoon weight and cocoon shell character. In the bulk segregant analysis>DNA from the parents (B20 Λ C. Nichi), F1 and F2 progeny of high shell ratio (HSR) and low shell ratio (LSR) were screened for hybridization with the random clones. Polymorphic banding pattern achieved through southern hybridization with different probes indicated the probable correlation of polymorphism with high and low cocoon shell character which are possible landmarks in identifying the putative marker(s) for the cocoon shell character. Out of the 100 probes tried with parents, F1, F2 and their bulks, 10 probes were found to be closely linked to cocoon shell characters.

Diagnosis of Swine Toxoplasmosis by PCR and Genotyping of Toxoplasma gondii from pigs in Henan, Central China

BackgroundToxoplasma gondii, a widely prevalent protozoan parasite, causes serious toxoplasmosis infections in humans and other animals. Among livestock, pigs are susceptible to T. gondii infection. Despite Henan being one of the biggest pig-raising provinces in China, little information exists on the epidemiology of toxoplasmosis in this location. Therefore, we molecularly characterized DNA samples from pigs in Henan. A total of 1647 samples, including 952 from dead piglets, 478 from seriously sick fattening pigs and 217 from abortion sows, were collected from different animal hospitals or pig farms from 10 different cities in Henan (2006–2008). Each pig corresponded to a separate pig farm. DNA was extracted from 3 to 5 g of the most severely affected pig tissue (liver, spleen, lung, hilar lymph nodes and amniotic fluid) after postmortem examination. The presence of the T. gondii B1 gene was detected using nested polymerase chain reactions (PCR). Genotyping was performed directly on DNA from the PCR-positive tissue samples using 11 PCR restriction fragment length polymorphism markers (SAG1, 5′- and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22–8, c29–2, and Apico).ResultsOf all samples, thirty-four were positive for the T. gondii B1 gene (2.06%, 95% CI: 1.86%–2.26%) from four cities, including 31 from NanYang city, one (PgXY 1) from Xinyang City, one (PgZZ 1) from Zhengzhou City and one (PgZK1) from Zhoukou City. The prevalence was found to be highest in piglets than in fattening pigs and sows. And the difference was statistically significant (P<0.01). The following 32 samples were genotyped with complete data: 13 hilar lymph node tissue samples, seven liver tissue samples, seven lung tissue samples, four spleen tissue samples, and one amniotic fluid sample. Only one genotype, belonging to ToxoDB Genotype #9, was identified.ConclusionsThis is the first large-scale survey molecularly characterizing T. gondii from pigs in Henan. The results of the present study revealed that T. gondii infection is present in swine in Henan and is a potential source of foodborne toxoplasmosis in the investigated areas. Implementation of effective control measures for T. gondii to reduce the chance of zoonotic toxoplasmosis spreading from pig farms may be warranted. The results show that the ToxoDB #9 genotype may be the dominant T. gondii lineage in mainland China. These findings strengthen the limited Chinese T. gondii epidemiology database.

Identification of an A2 population of Phythophthora andina attacking tree tomato in Peru indicates a risk of sexual reproduction in this pathosystem

Tree tomato, Solanum betaceum, is an Andean fruit crop previously shown to be attacked by Phytophthora andina in Ecuador and Colombia. Blight‐like symptoms were discovered on tree tomato plants in the central highlands of Peru in 2003 and shown to be caused by P. andina. Isolates of P. andina, collected from three different plantations in Peru over a 6‐year time span (2003–2008), were compared genetically with P. andina isolates from Colombia and Ecuador to test whether the pathogen population is geographically structured in the Andes. Restriction fragment length polymorphism (RFLP), mitochondrial DNA and simple sequence repeat (SSR) genetic markers, and mating type behaviour indicated that the Peruvian P. andina population from tree tomato is genetically distinct from populations infecting tree tomato in Colombia (CO‐1) and Ecuador (EC‐3, Ia, A1), but is more similar to the population infecting solanaceous hosts of the Anarrhichomenum complex (EC‐2, Ic, A2). Such geographic substructuring within this pathogen species could result from spatial isolation. Most strikingly, in contrast to the Ecuadorian and Colombian P. andina isolates from tree tomato, the Peruvian isolates have the A2 mating type. The presence of both mating types in the Andean population of P. andina attacking tree tomato indicates a risk of sexual reproduction and the presence of long‐lasting oospores in this pathosystem.

Development of a high-resolution melting marker for selecting Fusarium crown and root rot resistance in tomato.

Fusarium crown and root rot is a severe fungal disease of tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In this study, the genomic location of the FORL-resistance locus was determined using a set of molecular markers on chromosome 9 and an F2 population derived from FORL-resistant inbred 'AV107-4' (Solanum lycopersicum) × susceptible 'L3708' (Solanum pimpinellifolium). Bioassay performed using Korean FORL strain KACC 40031 showed single dominant inheritance of FORL resistance in the F2 population. In all, 13 polymerase chain reaction-based markers encompassing approximately 3.6-72.0 Mb of chromosome 9 were developed based on the Tomato-EXPEN 2000 map and SolCAP Tomato single nucleotide polymorphism array analysis. These markers were genotyped on 345 F2 plants, and the FORL-resistance locus was found to be present on a pericentromeric region of suppressed chromosomal recombination in chromosome 9. The location of the FORL-resistance locus was further confirmed by testing these markers against diverse commercial tomato and stock cultivars resistant to FORL. A restriction fragment length polymorphism marker, PNU-D4, located at approximately 6.1 Mb of chromosome 9 showed the highest match with the resistance locus and was used for conducting high-resolution melting analysis for marker-assisted selection of FORL resistance.

PCR-RFLP analysis of cpDNA in Gigantochloa scortechinii (Poaceae: Bambuseae) in Peninsular Malaysia and implications for the use of cpDNA markers in systematic studies

Abstract Gigantochloa is a paleotropical woody bamboo genus that has been widely cultivated in SE Asia because of its usefulness. Recent studies have shown that species of this genus enter into an introgression complex with other genera of the same subtribe Bambusinae. Within G. scortechinii, a common species indigenous to Malay Peninsula and common in Peninsular Malaysia, two distinct chloroplast DNA (cpDNA) lineages, the Gombak- and Langat-type, were recovered. We report the development of a PCR-based restriction fragment length polymorphism (RFLP) marker for depicting the genetic differentiation in G. scortechinii based on cpDNA. We determined a cpDNA region and its corresponding restriction enzyme which can produce different RFLP profiles for the two cpDNA lineages. Our design was verified with empirical studies. The Gombak-type was the dominant cpDNA genotype for G. scortechinii in Peninsular Malaysia. Implications for the continued use of cpDNA markers in systematic studies are discussed.