Random Amplified Polymorphic DNA Fragments Research Articles

Molecular cloning and development of RAPD-SCAR markers for the selection of thermo-tolerant line of tropical tasar silkworm

Aim: To develop random amplified polymorphic DNA (RAPD) -sequence-characterized amplified region (SCAR) markers for selecting thermo-tolerant line of tropical tasar silkworm. Methodology: In the environmental chamber, Daba BV cocoons of A. mylitta were exposed to high temperature at 46°C/4 hr for 3 days. After the emergence, genomic DNA was extracted from thermo-tolerant moths and thermo-susceptible pupae. The genomic DNA was amplified using 30 RAPD random decamer primers. The improved RAPD fragments that can differentiate thermo-tolerant and susceptible line of A. mylitta were eluted, cloned in pJET1.2 vector and sequenced. SCAR markers were developed based on the sequence and validated in the subsequent generations. Results: Among 30 RAPD primers, OPK04, OPAJ15 and OPA17 generated polymorphic bands to differentiate thermo-tolerant and susceptible line of A. mylitta. These polymorphic bands were eluted, cloned and sequenced. Sequencing of three cloned fragments revealed that clone PB1 comprised of 1412 bp, clone PB2 comprised of 704 bp and clone PB3 comprised of 931 bp. Sequence specific stable multiplex SCAR markers TT-PB1, TT-PB2 and TT-PB3 were designed and synthesized. PCR amplification was performed using DNA templates of 10 thermo-tolerant and 10 thermo-susceptible samples. SCAR marker TT-PB1 was observed to be more specific to thermo-tolerant line of tropical tasar silkworm and validation with 25 samples each in next generation also supported the specificity of TT-PB1. Interpretation: Among three SCAR markers, TT-PB1 showed more specificity for selecting the thermo-tolerant line of A. mylitta. Therefore, the study provides an effective and precise PCR-based molecular marker system for selecting thermo-tolerant lines in tropical tasar silkworm to overcome seed crop loss due to high temperature stress in tasar rearing hotter zones. Key words: Antheraea mylitta, cloning, RAPD, SCAR m­­­arker, Thermo-tolerance

Development of Molecular Markers Associated with Resistance to Gray Mold Disease in Onion (Allium cepa L.) through RAPD-PCR and Transcriptome Analysis

Onions (Allium cepa L.) are one of the most consumed vegetable crops worldwide and are damaged by several fungal diseases in the field or during storage. Gray mold disease caused by the necrotrophic pathogens Botrytis cinerea and Botrytis squamosa is a disease that reduces the productivity and storage life in onions. However, it is difficult to control gray mold disease in onions by using physical and chemical methods. Breeding resistant onions against gray mold disease can reduce the damage caused by pathogens, reduce the labor required for control, and reduce environmental pollution caused by fungicides. However, onions have a large genome size (16Gb), making them difficult to analyze, and have a biennial cycle, resulting in a very long breeding period. Therefore, in this study, markers were developed to shorten the onion breeding period. First, random amplified polymorphic DNA (RAPD) was performed to confirm the genetic relationship between the gray mold disease-resistant and -susceptible lines through a dendrogram. In addition, the sequence characterized amplified region (SCAR)-OPAN1 marker to select resistant lines was developed using a polymorphic RAPD fragment. Second, the RNA-seq of the gray mold-resistant and -susceptible onion lines were analyzed using NGS technology. Using the RNA-seq results and DEG and GO analyses were performed, and the variants, such as SNPs and indels, were analyzed to develop a selectable marker for the resistant line. This study developed the SNP-3 HRM marker for selecting gray mold disease-resistant lines by using the SNPs present in the aldo-keto reductase (AKR) gene with high expression levels in these lines. The SCAR-OPAN1 and SNP-3 HRM markers developed in this study could be used to select gray mold disease-resistant onions in breeding programs to reduce the damage caused by gray mold disease.

ISSR and RAPD primers selection for assessing genetic diversity of Enhalusacoroides (L.f.) Royle

Inter Simple Sequence Repeat (ISSR) and Random Amplified Polymorphic DNA (RAPD) are common PCR-based molecular markers used to study genetic diversity both between and within species. One of important steps in ISSR and RAPD analyses is primer selection to obtain bright PCR products. The aim of this study was to select ISSR and RAPD primers that produced scorable fragments of PCR product for genetic diversity evaluation of seagrass Enhalusacoroides (L.f.) Royle. The study also aimed to compare ISSR and RAPD technique in producing informative fragments. Enhalusacoroides is one seagrass species widely distributed in Indonesia. Seagrass samples were collected from Sanur Coastal Water and from Nusa Dua Coastal Water. DNA was extracted using CTAB method. Ten ISSR primers and eight RAPD primers were tested. Results showed that RAPD was more informative than ISSR. Among ten ISSR primers, only four primers resulted in scorable fragments in all samples, and only one among those primers gave polymorphic pattern. As many as 15 fragments have resulted from three ISSR primers and only 1 fragment was polymorphic. All RAPD primers produced scorable products, and three primers resulted in polymorphic fragments. A total of 42 RAPD fragments were produced and 4 from them were polymorphic fragments

Molecular characterization of oil seed Brassica using RAPD markers

Twelve varieties of oil seed Brassica spp. were characterized at molecular level using Random Amplified Polymorphic DNA (RAPD) primers to explore genetic diversity and to find out relationship among them. Twelve random primers used in the study generated 94 RAPD fragments and 53 (56.38%) of them were considered as polymorphic indicating high level of polymorphism within the materials. The size of amplified fragments ranged between 300-3000 bp. The values of pair-wise genetic distance ranged from 0.1613 to 0.5543. To find out phylogenetic relationships among the varieties, dendrogram based on Nei’s genetic distance was constructed using Unweighted Pair Group Method of Arithmetic Means (UPGMA) separating the 12 Brassica spp into two major clusters C1 and C2. This result will be useful for designing future breeding programmes for improvement of Brassica varieties.
 Bangladesh J. Sci. Ind. Res.55(1), 1-8, 2020

Sequence-characterized amplified region markers and multiplex-polymerase chain reaction assays for kiwifruit cultivar identification

A simple and reliable multiplex polymerase chain reaction (PCR) method was developed using sequence-characterized amplified region (SCAR) markers to identify kiwifruit (Actinidia spp.) cultivars. We observed 183 different polymorphic random amplified polymorphic DNA (RAPD) markers using 23 arbitrary primers in 30 kiwifruit cultivars. The number of polymorphic bands per primer varied from five (OPM-15, OPN-05, and OPN-19) to 12 (OPG-01), with an average of eight. Fifty-eight cultivar-specific polymorphic RAPD fragments were converted into 16 SCAR markers after validating their specificity. Among them, 15 SCAR markers retained the dominant behavior of the original RAPD fragment, whereas the KA11_774 SCAR marker showed a co-dominant polymorphism. Based on RAPD and SCAR data sets, cluster analyses were performed with the unweighted pair-group method of arithmetic averages. The mean genetic similarities were estimated as 0.703 and 0.677 using the RAPD and SCAR markers, respectively. Thirty kiwifruit cultivars were divided into three clusters for RAPD analysis and into two clusters for SCAR analysis, based on a similarity value of 0.67. Overall, the kiwifruit cultivars with a similar breeding background tended to cluster together. Therefore, RAPD and SCAR markers could be successfully applied to assay genetic relationships for kiwifruit cultivar identification. Cluster analysis of combined RAPD and SCAR markers showed very similar results when compared to the RAPD analysis in dendrogram form. The kiwifruit cultivars tested in this study could be identified using only 10 different SCAR markers (KB08_182, KK08_287, KG19_295, KN15_316, KG01_411, KF07_433, KG09_468, KG09_477, KL01_764, and KA11_774). Furthermore, we optimized SCAR markers for simultaneous identification of the 30 kiwifruit cultivars by multiplex PCR, using two sets of five SCAR primer pairs. The developed SCAR markers and multiplex PCR approach facilitate efficient identification of kiwifruit cultivars.

Genetic authentication of Eclipta prostrate (Asteraceae) from Penthorum chinense (Penthoraceae) by Sequence Characterized Amplified Region (SCAR) markers

Introduction: For the rapid and accurate genetic identification and authentication of living organisms, improved random amplified polymorphic DNA (RAPD) fragment based development of sequence-characterized amplified region (SCAR) markers is an important genetic technique. Objective: This study aimed to develop SCAR markers for perennial herb Eclipta prostrate (E. prostrate). Methods: Here the RAPD fragments by improved RAPD amplification with primers A11 and N-7 for E. prostrate were cloned into pGEX-T vector, and PCR amplification identified the positive clones. After the enzymatic digestion, they were sequenced with Sanger sequencing. Results: Two SCAR markers were developed, which were very specific to E. prostrate, not found in Penthorum chinense Pursh (P. chinense). The nucleotide sequence search by BLAST GenBank database showed that they are novel in E. prostrate, therefore they were deposited in Genbank with accession number KX671034, KX671035. The markers did not show any identity to other species. Conclusions: Thus, in this study two specific SCAR markers were developed for genetically distinguishing and identifying the plant species E. prostrate from herb P. chinense and others.

Molecular characterization of the two postharvest biological control agents Aureobasidium pullulans L1 and L8

L1 and L8 are Aureobasidium pullulans strains previously shown to effectively act as biocontrol agents (BCA) against fruit postharvest pathogens. Here, L1 and L8 strains were tested in vivo against Monilinia laxa of peaches showing the ability to reduce the brown rot incidence by 95% and by 80% respectively. To develop future bioformulation and allow commercial use of these biocontrol agents (BCAs) it is necessary to develop specific identification tools. Since A. pullulans strains share very similar morphological features and are not distinguishable by visual analysis, the RAPD (Random Amplified Polymorphic DNA) technique was applied to genomic DNA of L1 and L8 and of other 40 different A. pullulans strains, and SCAR markers were developed from a L1 and L8 specific RAPD fragment of 984 bp. These primers amplify a fragment of 137 bp exclusively in L1 and L8, and can be used to distinguish the two yeasts strains from other A. pullulans strains with high specificity. The droplet digital PCR, used to determine the precise limit of detection (LOD), showed that these markers allow to detect up to 43 and 215 cells of L1 and L8 respectively.

Genetic DiversityAssessment in Ten Aromatic Rice Varieties of Bangladesh

The availability of molecular marker systems allowed estimating the relationships among various taxa. This study was aimed at assessing the genetic diversity among ten aromatic rice (Oryza sativa L.) pools from Bangladesh by means of randomly amplified polymorphic DNA (RAPD) markers. These varieties were evaluated for polymorphisms after amplification with 10 decamer primers. A total of 60 RAPD fragments were generated among the assessed varieties with a polymorphism percentage of 80. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that these 10 varieties could be placed into two groups with a similarity ranging from 65 to 86% depicting adjacent association between Rajbhog and Kalijira‐12, whereas Maloti belongs to a separate group maintaining maximum distance from rest of the varieties. The analysis revealed that the intervarietal genetic relationship of several varieties is related to their center of origin. As expected, most of the varieties have a narrow genetic base. The present results could be used for the selection of possible parents to generate a mapping population and utilized by the breeders for assessing the genetic diversity of rice genotypes.Plant Tissue Cult. & Biotech. 27(2): 217-225, 2017 (December)

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

Mei Z, Khan MA, Zhang X, Fu J. 2017. Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers. Biodiversitas 18: 1243-1249. For the genetic identification and authentication of living organisms, development of Sequence-Characterized Amplified Region (SCAR) markers from Random Amplified Polymorphic DNA (RAPD) fragments is a valuable molecular approach. By using SCAR markers, molecular analysis is simplified to a Polymerase Chain Reaction (PCR) analysis using PCR primers designed from specific sequences of the RAPD amplicons. In this study, RAPD fragments from improved RAPD amplification of a perennial herb Penthorum chinense Pursh from China were cloned into a T-vector, and positive clones were identified by PCR amplification, and sequenced with the Sanger sequencing method for the SCAR marker development. Five SCAR markers were developed that were very specific to P. chinense, and deposited in GenBank (accession numbers: KX671029, KX671030, KX671031, KX671032 and KX671033). BLAST searches of these five nucleotide sequences in the GenBank database showed no identity with markers from other species. This study was developing five specific SCAR markers, has enabled reliable genetic identification of the herbal plant species Penthorum chinense Pursh, useful for authenticating future samples of this important medicinal herb.

Genetic variation of Prochilodus lineatus (Valenciennes, 1836) from Paraná, Baía, Miranda, and Corumbá rivers, Brazil.

The curimbatá (Prochilodus lineatus) is one of the migratory species in the Paraná River Basin impacted by the construction of dams. Mitochondrial DNA sequences and random amplified polymorphic DNA (RAPD) fragments were used to investigate genetic variability and geographic structure of five populations of curimbatá from the Paraná River Basin. A total of 1815 bp from seven polymerase chain reaction-amplified fragments representing five protein-coding mitochondrial genes were sequenced from 12 individuals. Estimates of nucleotide sequence divergence ranged from 0.00 to 0.95%. A total of 86 RAPD markers from 58 individuals were detected. Results from the Fisher exact test indicated that P. lineatus is not genetically subdivided, although significant differences in the frequencies of a few RAPD fragments were observed. This study provides useful information for stocking and management programs for resource planning of P. lineatus.

Development of two novel specific SCAR markers by cloning improved RAPD fragments from the medicinal mushroom Ganoderma lucidium (Leysser: Fr) Karst.

Development of sequence-characterized amplified region (SCAR) markers from random-amplified polymorphic DNA (RAPD) fragments is a valuable molecular approach for the genetic identification of different species. By using SCAR markers, molecular analysis is reduced to a simple polymerase chain reaction (PCR) analysis using primers designed from the amplicon sequence of RAPD. In this study, the DNA fragments from an improved RAPD amplification of Ganoderma species were cloned into a pGM-T vector; positive clones were identified by PCR amplification and enzymatic digestion, and finally, DNA fragments were sequenced using the Sanger sequencing method for developing the SCAR markers. Two SCAR markers, named LZ4-1 with 534 nucleotides, and LZ5-2 with 337 nucleotides were identified, which are specific to Ganoderma lucidium (Leysser: Fr) Karst species. BLAST of these two nucleotide sequences in the GenBank database showed no identity to other species. We deposited these sequences into the GenBank database (LZ4-1 accession No. KM391933, LZ5-2 accession No. KM391934). PCR assays confirmed them as novel molecular markers for G. lucidium (Leysser: Fr) Karst, which might be used for genetic authentication of adulterant samples. Thus, our study developed two specific SCAR markers for identifying and distinguishing the medicinal mushroom G. lucidium (Leysser: Fr) Karst from other Ganoderma species.

RAPD-SCAR Markers: An Interface Tool for Authentication of Traits

The versatility of the PCR technique is that several kinds of primers can be explored for genome analysis depending on the purpose of study. The easy to access and low cost PCR-based markers include Random Amplified Polymorphic DNA (RAPD). The RAPD markers are easy to develop but lack of reproducibility makes it less reliable and obstacles to their further use in authentication of traits. In addition, other PCR and non PCR based markers like Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeat (SSR) and Restriction Fragment Length Polymorphism (RFLP) are also employed in authentication of traits with certain restrictions vis-a-vis use of radioactive materials, high cost and requirement of sequence information etc. Therefore, this problem can be overcome by converting RAPD markers to more robust sequence characterized amplified regions i.e. SCAR markers. SCARs are locus specific, co-dominant in nature and amplified by PCR using specific 15 - 30 bp DNA fragments. For developing SCAR markers, primers are designed from the nucleotide sequences of a cloned RAPD fragments linked to a trait of interest. SCAR markers are easy to develop and reliable tools for DNA fingerprinting. This mini review is an attempt to summarize efficacy of RAPD-SCAR interface in authentication of traits.

Development of Sequence-characterized Amplified Region Markers for the Identification of Grapevine Cultivars

Grapevine cultivars have traditionally been identified based on the morphological characteristics, but the identification of closely related cultivars has been difficult because of their similar pedigree backgrounds. In this study, we developed DNA markers for genetic fingerprinting in 37 grapevine cultivars, including 20 cultivars bred in Korea. A total of 180 randomly amplified polymorphic DNA (RAPD) markers were obtained using 30 different primers. The number of polymorphic bands ranged from three (OPG-08 and OPU-19) to nine (OPV-01 and UBC116), with an average of six. RAPD markers were used in cluster analysis performed with the unweighted pair-group method of arithmetic averages (UPGMA). The average similarity value was 0.69 and the dendrogram clustered the 37 grapevine cultivars into five clusters. The relationships among the grapevine cultivars were consistent with the known pedigrees of the cultivars. The 50 RAPD fragments selected were sequenced for the development of sequence-characterized amplified region (SCAR) markers. As a result, 16 of 50 fragments were successfully converted into SCAR markers. A single polymorphic band, the same size as the RAPD fragments or smaller, was amplified depending on the primer combinations in the 14 SCAR markers, and codominant polymorphisms were detected using the SCAR markers G119_412 and GB17_732. Among these markers, combination of 11 SCAR markers, GG05_281, G116_319, G146_365, G119_412, GW04_463, G169_515, G116_539, GV04_618, GV01_678, GG05_689, and GB17_732, provided sufficient polymorphisms to distinguish the grapevine cultivars investigated in this study. These newly developed markers could be a fast and reliable tool for identifying grapevine cultivars.

English

Stemphylium solani isolates infect two varieties of eggplant, Solanum aethiopicum and Solanum melongena grown in Senegal (West Africa) and are morphologically quite similar. Using the random amplified polymorphic DNA (RAPD) procedure with arbitrary 10-mer primers, we were able to differentiate these S. solani isolates into two groups directly related to their plant host origin. A RAPD product of approximately 480 bases pairs obtained with OPF-20 primer were polymorphic between the two groups. Four new primers (F20F1, F20F2, F20R1 and F20R2) based on nucleotide sequence analysis of this 480 bases pairs RAPD fragment were developed. Such primers used pairwise amplified a single fragment from the DNA of S. solani isolates whatever their host origin. However, DNA extracted from Fusarium oxysporum (f. sp. vasinfectum, f. sp. elaeidis), Verticillium dahliae and Phyllosticta sp. isolates did not amplify using these primers. Our results indicate that these primers sets were good tools for specific identification of these two eggplants S. solani isolates by polymerase chain reaction (PCR). Key words: Stemphylium solani isolates, Solanum aethiopicum, Solanum melongena, random amplified polymorphic DNA (RAPD) markers, identification.

Development and significance of RAPD-SCAR markers for the identification of Litchi chinensis Sonn. by improved RAPD amplification and molecular cloning

article i nfo Background: Analysis of genetic diversity is important for the authentication of a species. Litchi (Litchi chinensis Sonn.) is a subtropical evergreen tree. Recently, L. chinensis has been characterized by an improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis. The goal of this study was to develop sequence-characterized amplified region (SCAR) markers from the improved RAPD fragments for the genetic analysis of L. chinensis. Results: The improved RAPD fragments from L. chinensis were cloned, sequenced and converted into stable SCAR markers. Sequencing of three cloned RAPD fragments revealed that the clone L7-16 consisted of 222 nucleotides (GenBank accession number KM235222), clone L9-6 consisted of 648 nucleotides (GenBank accession number KM235223), and clone L11-26 consisted of 369 nucleotides (GenBank accession number KM235224). Then, specific primers for SCAR markers L7-16, L9-6, and L11-26 were designed and synthesized. PCR amplification was performed using DNA templates from 24 different samples, including 6 samples of L. chinensis and other plants. The SCAR marker L9-6 was specific for all of the L. chinensis samples, the SCAR marker L11-26 specific for five L. chinensis samples, and the SCAR marker L7-16 only specific for the samples from Luzhou. Conclusions: This study developed stable SCAR markers for the identification of L. chinensis by the cloning of the improved RAPD fragments. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species.

Development of RAPD-SCAR markers for Lonicera japonica (Caprifolicaceae) variety authentication by improved RAPD and DNA cloning.

Genetic diversity within a species is a common feature, which plays a vital role in its survival and adaptability, and is important for the identification and authentication of a species. Lonicera japonica is a traditionally used medicinal plant, which have been recently genetically characterized by an improved ran- dom amplified polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific L. japonica variety. The DNAs were extracted from fresh young leaves of different samples of L. japonica collected from Shenzhen, Yichang, Leshan, Emei and Loudi, China. The DNA materials were amplified using improved RAPD PCR. Different RAPD bands were excised, cloned and developed for stable sequence-characterized amplified region (SCAR) markers with differ- ent species. Two SCAR markers, JYH3-3 and JYH4-3, have been successfully cloned from improved.RAPD fragments. The SCAR marker JYH3-3 was found specific for all of the L. japonica samples collected from the different regions, and another marker JYH 4-3 was strictly specific to the Shenzhen sample from Guangdong province, which is geographically distant from Hubei, Sichuan and Hunan Provinces (source of other L. japonica samples). The marker JYH3-3 was found as specific molecular marker for the identification of L. japonica, while JYH4-3 was found as molecular marker strictly specific for the Shenzhen sample. The developed SCAR markers might serve as more specific molecular markers for L. japonica variety authentication. The combination of improved RAPD analysis and SCAR marker development have resulted useful tools to study the genetic variety of any organism, which we have successfully applied here in L. japonica.

Genetic Variability in Bangladeshi Aromatic Rice through RAPD Analysis

Genetic polymorphism and relationships among 30 commercial varieties of Bangladeshi aromatic rice (Oryza sativa L.) were established using random amplified polymorphic DNA (RAPD) primers. Out of fifty 10-mer RAPD primers screened initially, four were chosen and used in a comparative analysis of different varieties of indigenous Bangladeshi aromatic rice. Of the 33 total RAPD fragments amplified, 7 (21.21%) were found to be shared by individuals of all eight varieties. The remaining 26 fragments were found to be polymorphic (78.79%). Pair-wise estimates of similarity ranged from 0.101 to 0.911. Highest genetic diversity was determined between Radhunipagol and Dubsail varieties (0.911). The amount of genetic diversity within aromatic rice germplasm was quite high as determined by the genetic similarity coefficients between varieties. Genetic similarities obtained from RAPD data were also used to create a cluster diagram. Cluster analysis using an un-weighted pair-group method with arithmetic averages (UPGMA) was used to group the varieties and the 30 aromatic rice varieties were grouped into 6 clusters where cluster I includes the maximum number of varieties (9). Cluster VI includes minimum number of varieties (2). This Study offered a rapid and reliable method for the estimation of variability between different varieties which could be utilized by the breeders for further improvement of the local aromatic rice varieties.

Genetic Diversity Assessment of Acid Lime (Citrus Aurantifolia Swingle) Landraces of Eastern Nepal Using RAPD Markers

Acid lime (Citrus aurantifolia Swingle) is an important commercial fruit crop, cultivated from terai to high hill landscapes of Nepal. However, production and productivity is very low due to various reasons including infestations by various diseases and pests, lack of diseases and pests resistant and high yielding varieties. In this context, determination of genetic variation at molecular level is fundamental to citrus breeders for the development of elite cultivars with desirable traits. In the present study, Random Amplified Polymorphic DNA (RAPD) marker technique has been employed to assess genetic diversity in 60 acid lime landraces representing different agro-ecological zones of eastern Nepal. Nine selected arbitrary primers generated 79 RAPD fragments of which 75 were polymorphic (94.94%). Phenogram was constructed by NTSYSPC ver. 2.21i using UPGMA cluster analysis based on Jaccard’s similarity coefficient to deduce overall genetic diversity and relationships of the acidlime genotypes under study. Sixty acid lime landraces formed seven clusters and similarity value ranged from 38% to 98% with an average of 72%. Genetic variation at different agro-ecological zones was assessed using Popgene ver. 1.32 and found 47% to 69.6% polymorphism. Shannon’s index and Nei’s gene diversity showed highest level of acid lime diversity in Terai zone (PPB, 69.62%; H, 0.213; I, 0.325) followed by mid-hill zone (PPB, 67.09%; H, 0.208; I, 0.317). The results obtained will be useful to citrus breeders for elite cultivar development. The RAPD-PCR technique is found to be the rapid and effective tool for genetic diversity assessment in acid lime landraces of Nepal. DOI: http://dx.doi.org/10.3126/ijasbt.v2i3.10950 Int J Appl Sci Biotechnol, Vol. 2(3): 315-327

Development of a SCAR Marker for Sex Identification in Asparagus

A sex-linked random amplified polymorphic DNA (RAPD) marker was identified from Asparagus officinalis L. and was converted into a sequence-characterized amplified regions (SCAR) marker for the large-scale screening of male and female plants. A total of 100 arbitrary decamer oligonucleotide primers were used for the RAPD analysis. Among them, the primer UBC347 amplified one female-specific 400 base pair DNA. Subsequently, the amplified RAPD fragment was cloned and sequenced. The fragment was abundant in AT and shared sequence homology with retrotransposon elements. On the basis of the sequence obtained, a pair of SCAR primer was designed. The amplification product, named F400, was the same size as the respective RAPD fragment from which it was derived. The F400 SCAR marker resulted to be female-specific in the three asparagus varieties tested in this study. This SCAR marker can be used for an early and rapid identification of female and male plants during breeding programs of asparagus.

Fertility restoration in three CMS systems of eggplant by the Rf genes of each other's systems and their SCAR marker

Three pollen non-formation type cytoplasmic male sterility (CMS) systems of eggplant (Solanum melongena L.) were developed in our previous study, through repeated backcrossing, by using wild Solanum species Solanum aethiopicum L. Aculeatum Group, Solanum anguivi Lam. and Solanum grandifolium C. V. Morton as the cytoplasm donor and eggplant ‘Uttara’ as the recurrent pollen parent. Each CMS system has male fertile (MF) line which carries the fertility restorer (Rf) genes for the pollen non-formation type sterility. In the present study, fertility restoration ability in three CMS systems of eggplant by the Rf genes of each other's systems was evaluated. The male sterile (MS) line of each CMS system was pollinated with the pollen of MF lines of other CMS systems as well as its own CMS system. Among the nine cross combinations, six groups were obtained from crossing MS lines with MF lines of other CMS systems and three groups were obtained from crossing MS lines with MF lines of their own CMS system. The segregation of MF and MS plants was fitted well with a 3:1 ratio in almost all cross combinations. The segregation patterns revealed that two independent dominant Rf genes restore fertility in each CMS system. Pollen stainability and in vitro pollen germination rate of the progenies obtained from different cross combinations were varied from 61.0 to 89.8% and 0.7 to 8.0%, respectively. Each Rf gene of three CMS systems restores fertility of other CMS systems as well as its own CMS system. Moreover, fertility recovery action in these three kinds of CMS is similar. DNA markers linked to Rf genes were screened by bulked segregant analysis (BSA) using random amplified polymorphic DNA (RAPD). A total of 420 primers were used, and more than 65% of the primers yielded a considerable number of RAPD fragments in both MF and MS bulks. Only one Operon primer OPAB10 gave a reproducible polymorphic band of 1.9kbp (OPAB101900) between bulks and was identified as linked to the Rf genes. Additionally, the RAPD marker OPAB101900 was converted to sequenced characterized amplified region (SCAR) marker designated as SCAB101900 by nucleotide sequencing. Findings of the present study may give us a potential way to explore CMS and the corresponding Rf genes from wild Solanum resources for the breeding program of eggplant.