Sequence Characterized Amplified Region Research Articles

Development of SCAR markers related to heat tolerance in Kentucky bluegrass

As a high-quality cool-season grass, Kentucky bluegrass (Poa pratensis) is facing increasing threat of high temperature, so improving its heat tolerance (HT) has become an important breeding target. In this study, the HT of 84 materials was identified in the artificial climate chamber, and 15 most heat-tolerant and 15 most heat-sensitive materials were selected respectively to construct two DNA pools. There was a significant difference in high-temperature tolerance time between the plants in the two pools, which was more than 22 days. A total of 304 sequence-related amplified polymorphism (SRAP) and 88 simple sequence repeat (SSR) markers were used to screen the polymorphic bands between the two pools. Then, these bands were transformed into sequence characterized amplified region (SCAR) markers, and finally 12 SCAR dominant markers related to HT were obtained, which could detect the heat-sensitive materials efficiently. Among them, S-me8×em2 and S-me52×em5 had the best identification effect, and the consistency between the absence of these two markers and the heat-sensitive phenotype was 87%. According to the comparison with NCBI database, the sequences of 12 SCAR markers had no homology with known HT related genes. Next, we would further verify the HT identification efficiency of these SCAR markers in single plants within materials, and try to use them in molecular marker-assisted breeding.

Identification of SCAR markers linked to the Foc gene governing resistance to Fusarium oxysporum f. sp. cucumerinum in cucumber cv. SMR-18

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FOC) is an important soil-borne disease of cucumber in several parts of the world. In this study, we employed amplified fragment length polymorphic (AFLP) markers along with bulked segregant analysis (BSA), in order to identify structural markers linked to the Foc gene that governs resistance to FOC races 1, 2 and 3 in the resistant cv. SMR-18. From the four AFLP markers originally identified at the BSA level, only one (E12M50134 + T) maintained a linkage in coupling phase against the F2 mapping population. AFLP-mediated sequencing was employed in order to ensure the identification and sequencing of the truly linked AFLP fragment. This AFLP marker mapped at a distance of 6.0 cM from the Foc resistance locus and was successfully converted into two sequence-characterized amplified region (SCAR) co-dominant markers. SCAR marker SCE12M50B produced a single fragment linked to Foc and behaved as a dominant marker, while SCE12M50A amplified one strong fragment linked to Foc in parallel with a second faint fragment. The SCE12M50B fragment physically maps on chromosome 2 and lies 1.64 Mb (7,0 cM) away from the microsatellite marker SSR03084, which is closely linked to Ccu locus that controls resistance to scab, caused by Cladosporium cucumerinum in cv. SMR-18.

Phenotypic and genetic diversity of leaf rust resistance in wheat wild relatives

AbstractPuccinia triticina (Pt), the causal agent of leaf rust evolves through forming new pathotypes that adversely affect the growth and yield of wheat cultivars. Therefore, continued production of resistant varieties through exploring novel sources of resistance in wild relatives which are abundantly found in Iran and the neighbouring regions is a major task in wheat breeding programs. The aim of the present study was to explore 60 wild wheat genotypes selected from the species Triticum monococcum, Aegilops tauschii, Ae. neglecta, Ae. cylindrica, Ae. triuncialis, Ae. umbellulata, Ae. speltoides, Ae. columnaris, Ae. crassa and Ae. ventricosa for resistance to leaf rust. The cultivar ‘Boolani’ and Thatcher near‐isogenic lines were used as controls. Two‐week‐old seedlings were inoculated using 10 Pt pathotypes, and the infection types were recorded. The genotypes were also analysed for polymorphism using six sequence‐tagged sites (STS) and sequence characterized amplified region (SCAR) markers. Forty‐eight genotypes produced high infection types (3+) for two pathotypes, but the remaining genotypes produced low infection types of ‘0; =’ to ‘1+CN’ to all pathotypes. The latter included three accessions of Ae. tauschii, two accessions of each Ae. umbellulata, Ae. columnaris and Triticum monococcum, and one accession from each Ae. triuncialis, Ae. ventricosa and Ae. neglecta. Analysis for STS and SCAR markers suggested several genotypes could carry the genes Lr9, Lr10, Lr19, Lr24, Lr26 and Lr37 or their potential orthologs in addition to unknown resistance genes. In conclusion, the identified resistant genotypes could be further characterized and used in wheat breeding programs for leaf rust resistance.

Use of molecular markers SSR and SCAR for identification of olive accessions

Morphological and agronomical describers are traditionally used in plant characterization. However, the usage of these describers have some limitations such as susceptibility to abiotic and biotic stress and environmental factors. Furthermore, the describers are not stable over time and many can only be evaluated during the adult phase of the plants, which requires time and physical space. Molecular markers offer numerous advantages compared to the conventional alternatives based on phenotype: they are stable and detectable in all vegetable tissues, and are independent of the environment and development phase. One of the main advantages of the use of molecular markers is the time reduction in the identification of genetic diversity among the studied subjects, as the genotypes may even be described for the seed or seedling phase. Many countries have already adopted molecular markers to identify olive cultivars more accurately. The aim of this study was to evaluate the genetic identity of eight olive accessions supposedly belonging to cultivar Arbequina by using microsatellite (SSR) and Sequence Characterized Amplified Region (SCAR) markers. One accession corresponding to the cultivar was also incorporated into the analysis as a reference genotype. The molecular marker data were analyzed on the software GENALEX6. The markers generated an accumulated PI and PE of 1.26 x 10-6 and 0.949, respectively. The results supported the hypothesis that all accessions belong to the cultivar Arbequina, and the markers can therefore be applied to other varieties of olive species.

The application of DNA molecular markers in the study of Codonopsis species genetic variation, a review

Codonopsis genus is comprised of species that are perennial plants primarily distributed across all east, southeast, and Central Asia. The most famous species of Codonopsis are C. tangshen, C. lanceolate, and C. pilosula. The records showed that they have a long story usage as traditional Chinese medicines, as they were alleged to be able to intensify the spleen and the lung as well as enriching blood and engendering liquid. Certain species have a culinary value in southern China and Southeast Asia, where they are considered as tea, wine, soup, plaster, and porridge. Codonopsis species were shown to be of great importance in medicine, due to their broad biological activity. Therefore, a clear understanding of their genetic diversity is needed. Adequate distinctions and descriptions of those species are necessary to preserve plant reservoir, investigations of genes associated with desirable traits, and understanding of evolutionary relationships. Subsequently, various molecular marker techniques such as Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSR), and Inter Simple Sequence Repeat (ISSR), Single Nucleotide Polymorphism (SNP), internal transcribed spacer (ITS), and Sequence-Characterized Amplified Region (SCAR) have been improved to provide detailed informations about genomes, that historically were not possible to obtain based on only phenotypic methods. This review represents the usage of DNA molecular markers for molecular diversity analysis of medically important species belonging to the genus Codonopsis.

Detection of Powdery Mildew Resistance Gene in Melon Cultivar Meloni Based on SCAR Markers

Powdery mildew is one of the diseases caused by fungal infections that can reduce the production of melon fruit worldwide including in Indonesia. A powdery mildew-resistant cultivar of melon is needed to increase melon yield crops. This study aimed to detect resistance gene linked to powdery mildew using a sequence characterized amplified region (SCAR) markers. The melon cultivar Meloni was used in this study. SL-3, PI 371795, and Aramis cultivar were used to compare. Amplification of the marker was performed employing a pair of primers. The result showed that Meloni had a powdery mildew resistance gene by the presence of a DNA target band at 1058 base pair (bp). Based on this result, it could be concluded that Meloni was an excellent melon cultivar because of its ability to overcome the powdery mildew infections naturally. SCAR markers have been used for various purposes, especially to detect resistance genes to plant diseases. The present study had provided information for plant breeders about Meloni as the new melon cultivar that was genetically resistant against powdery mildew infections. Furthermore, Meloni could be proposed as an alternative to native Indonesian superior melon seeds.

South American species <i>Solanum alandiae</i> Card. and <i>S. okadae</i> Hawkes et Hjerting as potential sources of genes for potato late blight resistance

For several decades, wild species of Solanum L. section Petota Dumort. have been involved in potato cultivar breeding for robust resistance to pests and diseases. Potato late blight (LB) is caused by oomycete Phytophthora infestans (Mont.) de Bary, and the genes for race-specific resistance to P. infestans (Rpi genes) have been introgressed into cultivated potatoes by remote crosses and trans- or cisgenesis, first from S. demissum Buk. and, more recently, from other wild species, such as S. bulbocastanum Dun., S. stoloniferum Schlechtd. et Bché, and S. venturii Hawkes et Hjerting (according to the nomenclature by Hawkes, 1990). Most wild species already involved in breeding for LB resistance came from North and Central Americas: series Bulbocastana (Rydb.) Hawkes, Demissa Buk. and Longipedicellata Buk., and some Rpi genes of these species have been already characterized in much detail. Rpi genes of South American species, including the series Tuberosa (Rydb.) Hawkes, have not been sufficiently investigated. Among the latter, this study focuses on the Rpi genes of S. alandiae Card. and S. okadae Hawkes et Hjerting. Four accessions of S. alandiae, one accession of S. okadae and 11 clones of interspecific potato hybrids comprising S. alandiae germplasm from the VIR collection were PCR-screened using specific SCAR (Sequence Characterized Amplified Region) markers for eight Rpi genes. SCAR amplicons of five Rpi genes registered in this study were validated by comparing their sequences with those of prototype genes deposited in the NCBI Genbank. Among the structural homologues of Rpi genes found in S. alandiae and S. okadae, of special interest are homologues of CC-NB-LRR resistance genes with broad specificity towards P. infestans races, in particular R2=Rpi-blb3, R8, R9a, Rpi-vnt1 and Rpi-blb2 (94–99, 94–99, 86–89, 92–98 and 91% identity with the prototype genes, respectively). Our data may help to better understand the process of Rpi gene divergence along with the evolution of tuberbearing Solanum species, particularly in the series Tuberosa.

Development of the sequence-characterized amplified region (SCAR) marker for distinction of intergeneric hybrids between Argyranthemum frutescens (L.) Sch. Bip. and Rhodanthemum gayanum (Cross. & Durieu) B.H. Wilcox, K. Bremer & Humphries.

Distinction of intergeneric hybrids between Argyranthemum frutescens (L.) Sch. Bip. and Rhodanthemum gayanum (Cross. & Durieu) B.H. Wilcox, K. Bremer & Humphries is important for the development of new intergeneric hybrids. The aim of this study was to develop a simple and cost-effective DNA marker to distinguish between a large number of putative hybrids of A. frutescens × R. gayanum during primary selection. We designed sequence-characterized amplified region (SCAR) markers based on the genetic sequences of the internal transcribed spacer (ITS) region for A. frutescens and R. gayanum. Specific SCAR markers for A. frutescens amplified bands in A. frutescens and hybrids but not in R. gayanum. In contrast, specific SCAR markers for R. gayanum amplified bands in R. gayanum and hybrids but not in A. frutescens. Additionally, we confirmed the reproducibility of the SCAR marker using 31 cultivars. The findings demonstrated that these SCAR markers could be used to distinguish putative hybrids between A. frutescens and R. gayanum. This is the first report of the development of a SCAR marker to facilitate distinction of these hybrids.

The development and validation of new DNA markers linked to the thousand-grain weight QTL in bread wheat (Triticum aestivum L.)

Thousand-grain weight (TGW) is an important trait affecting wheat production. We previously identified a major quantitative trait loci (QTL) controlling the TGW on the 2D chromosome of wheat using a recombinant inbred line (RIL) population constructed by the cross between Tibetan semi-wild wheat Q1028 (Q1028) and Zhengmai 9023 (ZM9023). The positive allele at this QTL is from ZM9023. To further characterise this QTL, here, we try to develop and validate the high-resolution melting (HRM) and sequence-characterised amplified region (SCAR) markers. One HRM marker (0C98-411) and two SCAR markers (E301-700 and B0BB-10470) were developed and integrated into the genetic map. All of these three markers were validated in three populations with different genetic backgrounds. 0C98-411 is the most closely linked marker that could trace QTgw.sau-2D in molecular marker assisted breeding.

Assessment of genetic purity of two sunflower (Helianthus annuus L.) hybrids using sequence characterized amplified region (SCAR) markers

Random amplified polymorphic DNA (RAPD) technique is easier to identify true hybrids. However, it hasinherent disadvantage of less reproducibility and that can be overcome by conversion of RAPD markers into sequence characterized amplified region (SCAR) markers. In the present investigation, RAPD analysis was carried out with the two hybrids, KBSH 41 and KBSH 42 along with their parents, polymorphic markers were identified and those markers were converted into SCAR markers. These markers would be of immense potential during routine genetic purity assessment of the two sunflower hybrids.

Rapid and Simple Species Identification of Cicada Exuviae Using COI-Based SCAR Assay.

Cicadidae periostracum (CP), the medicinal name of cicada exuviae, is well-known insect-derived traditional medicine with various pharmacological effects, e.g., anticonvulsive, anti-inflammatory, antitussive, and anticancer effects; it is also beneficial for the treatment of Parkinson’s disease. For appropriate CP application, accurate species identification is essential. The Korean pharmacopoeia and the pharmacopoeia of the People’s Republic of China define Cryptotympana atrata as the only authentic source of CP. Species identification of commercially distributed CP based on morphological features, however, is difficult because of the combined packaging of many cicada exuviae in markets, damage during distribution, and processing into powder form. DNA-based molecular markers are an excellent alternative to morphological detection. In this study, the mitochondrial cytochrome c oxidase subunit I sequences of C. atrata, Meimuna opalifera, Platypleura kaempferi, and Hyalessa maculaticollis were analyzed. On the basis of sequence alignments, we developed sequence-characterized amplified-region (SCAR) markers for efficient species identification. These markers successfully discriminated C. atrata from the three other cicada species, and detected the adulteration of market CP samples. This SCAR assay is a rapid, simple, cheap, reliable, and reproducible method for species identification, regardless of sample form and status, and contributes to CP quality control.

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.

Identification of a molecular marker associated with lignotuber in Eucalyptus ssp

About 95% of Eucalyptus species present an organ known as a lignotuber, a basal woody swelling that holds a large number of dormant buds in a protected position along with carbohydrates and other nutrients. The importance of this trait in Eucalyptus species relates to its regenerative capacity, particularly in the context of coppicing practices and survival in regions of high abiotic stress, especially fire. In this study, we identified and characterized a genomic region associated with the lignotuber trait in commercially important Eucalyptus species by developing a polymorphic marker that co-segregates with lignotuber presence. The marker was then converted into a SCAR (Sequence Characterized Amplified Region) marker, validated in four other Eucalyptus species and hybrids and analyzed in silico. Our investigation presents a marker (ELig) that is effective in identifying individuals with lignotuber. In silico and Southern blot analyses show that the marker is present in a single copy region and is related to auxilin/cyclin-G associated kinase, containing a DnaJ domain. The ELig marker is an important tool that can be used to manage crosses in Eucalyptus breeding programs and inform studies involving lignotuber development and genetics.

Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of Viola serpens Wall. ex Ging.

An efficient protocol of plant regeneration through indirect organogenesis in Viola serpens was developed in the present study. Culture of leaf explants on MS (Murashige and Skoog) medium supplemented with 2.0 mg/L 6-benzyladenine and 0.13 mg/L 2,4-dichloro phenoxy acetic acid. Adventitious shoot formation was observed when calli were transferred on to MS medium containing 0.5 mg/L α-naphthalene acetic acid and 2.25 mg/L kinetin, which showed the maximum 86% shoot regeneration frequency. The highest root frequency (80.92%) with the 5.6 roots per explant and 1.87 cm root length was observed on MS medium supplemented with 2 mg/L indole-3-butyric acid. The plantlets were transferred to the mixture of sand, coffee husk and soil in the ratio of 1:2:1 in a pot, and placed under 80% shade net for one month. It was then transferred to 30% shade net for another one month, prior to transplantation in the field. These plantlets successfully acclimatized under field conditions. A Sequence Characterized Amplified Region (SCAR) marker was also developed using a 1135 bp amplicon that was obtained from RAPD (Random Amplification of Polymorphic DNA) analysis of six accessions of V. serpens. Testing of several market samples of V. serpens using the SCAR marker revealed successful identification of the genuine samples of V. serpens. This study, therefore, provides a proficient in vitro propagation protocol of V. serpens using leaf explants and a SCAR marker for the authentic identification of V. serpens. This study will be helpful for conservation of authentic V. serpens.

Exaiptasia diaphana from the great barrier reef: a valuable resource for coral symbiosis research

The sea anemone, Exaiptasia diaphana, previously known as Exaiptasia pallida or Aiptasia pallida, has become increasingly popular as a model for cnidarian-microbiome symbiosis studies due to its relatively rapid growth, ability to reproduce sexually and asexually, and symbiosis with diverse prokaryotes and the same microalgal symbionts (family Symbiodiniaceae) as its coral relatives. Clonal E. diaphana strains from Hawaii, the Atlantic Ocean, and Red Sea are now established for use in research. Here, we introduce Great Barrier Reef (GBR)-sourced E. diaphana strains as additions to the model repertoire. Sequencing of the 18S rRNA gene confirmed the anemones to be E. diaphana while genome-wide single nucleotide polymorphism analysis revealed four distinct genotypes. Based on Exaiptasia-specific inter-simple sequence repeat (ISSR)-derived sequence characterized amplified region (SCAR) marker and gene loci data, these four E. diaphana genotypes are distributed across several divergent phylogenetic clades with no clear phylogeographical pattern. The GBR E. diaphana genotypes comprised three females and one male, which all host Breviolum minutum as their homologous Symbiodiniaceae endosymbiont. When acclimating to an increase in light levels from 12 to 28 μmol photons m−2 s−1, the genotypes exhibited significant variation in maximum quantum yield of Symbiodiniaceae photosystem II and Symbiodiniaceae cell density. The comparatively high levels of physiological and genetic variability among GBR anemone genotypes make these animals representative of global E. diaphana diversity and thus excellent model organisms. The addition of these GBR strains to the worldwide E. diaphana collection will contribute to cnidarian symbiosis research, particularly in relation to the climate resilience of coral reefs.

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.

Identification, Characterization, and Evaluation of Novel Stripe Rust-Resistant Wheat-Thinopyrum intermedium Chromosome Translocation Lines.

Stripe rust is an important disease in wheat, and development of genetic resistance in cultivars is an effective approach to control the disease. Wild species of wheat, such as Thinopyrum intermedium, are an excellent gene source for wheat improvement. In this study, two stripe rust-resistant wheat-Th. intermedium chromosome translocation lines, CH4131 and CH4132, were characterized by cytogenetic and pathological methods. The introgressed chromosome fragment was tagged using amplified fragment-length polymorphism-derived sequence-characterized amplified region (SCAR) markers and intron targeting markers, indicating that CH4131 and CH4132 both possess a homologous group 3 chromatin of Th. intermedium. Genomic in situ hybridization results suggested that a very small Th. intermedium chromosome segment was translocated to the terminal region of wheat 1BS for both lines, forming a configuration of T3Ai-1BS.1BL. The two translocation lines were resistant to stripe rust, and the resistance gene, temporarily designated YrCH-1BS, was likely derived from Th. intermedium. The translocated chromosome fragments have no genetic linkage drag to agronomic performance. The grain quality indexes of these two translocations were higher than local wheat varieties. Therefore, CH4131 and CH4132 could be used as potential gene sources in wheat improvement programs. The SCAR markers are useful to select stripe rust resistance from Th. intermedium.

Stable Genome Incorporation of Sperm-derived DNA Fragments in Gynogenetic Clone of Gibel Carp.

How unisexual animals eliminate deleterious mutations to avoid dead ends is one of the most interesting puzzles in evolutionary genetics. Incorporation of microchromosomes derived from exogenous sperm had been observed in gynogenetic animals, but little is known about their detailed process and hereditary fate. Here, we show a stable genome incorporation case in an artificial clone F of gynogenetic gibel carp (Carassius gibelio). A total of 12 exogenous DNA fragments were screened through a read depth-dependent comparison strategy and confirmed to be specific to the clone F and the paternal blunt snout bream (Megalobrama amblycephala Yin) by SCAR (sequence characterized amplified regions) marker detection. Moreover, these sperm-derived DNA fragments were not detected in some samples in early gynogenetic generations, but they were found to exist in all examined individuals through artificial gynogenetic selections of 13 generations, implying that they might have stably incorporated into the genome of clone F. Furthermore, chromosome localization and sequence characterization indicate that the largest fragment CgA22_34 is derived from blunt snout bream non-LTR retrotransposon and durably incorporated into only one of three homologous chromosomes of gibel carp clone F. Our results suggest that the incorporated sperm-derived DNA fragments by allogynogenesis should increase genetic diversity and introduce new traits into unisexual animals which will benefit genetic breeding of gibel carp. During the process, transposable elements (TEs) may play significant roles in shaping the genome structures. Simultaneously, the incorporated DNA fragments are able to be used as genetic markers to perform selective breeding programs in aquaculture practices of gibel carp.

RAPD and ISSR derived SCAR marker(s) for Aphid tolerance in Brassica juncea Czern. And Coss

In this present study, eight genotypes of Brassica juncea comprising four tolerant (IC-399802, IC-491089, IC-312545, IC-312553) and four susceptible (IC-385686, IC-264131, IC-426392, Laxmi) genotypes for aphid tolerance were used to generate Sequence Characterized Amplified Region (SCAR) markers through dominant PCR based markers (51 RAPD and 12 ISSR markers). 13 RAPD (Random Amplified Polymorphic DNA) and 8 ISSR (Inter Simple Sequence Repeat) markers were found to be polymorphic but only 3 primers OPE16 (RAPD), UBC 839 (ISSR) and UBC 864 (ISSR) were obtained which could distinguish tolerant genotypes from susceptible ones. OPE16 (RAPD) obtained unique band of size approx. 600 bp in susceptible genotypes while it was absent in the tolerant genotypes. Similarly, UBC 839 (ISSR) yielded ~800 bp unique band in bulk tolerant while UBC 864 (ISSR) yielded three bands of ~1200 bp, ~1000 bp, and ~500 bp in tolerant genotypes which was absent in the susceptible genotypes. These unique bands were excised to generate five sets of SCAR markers. Among the five sets of SCAR marker, only BJSCAR F1 and BJSCAR R1 set yielded the promising result in all for 4 susceptible genotypes as well as bulk susceptible and was absent in all the tolerant genotypes and Brassica fruticulosa (highly tolerant to aphid, used as a control). So, this SCAR marker developed could be successfully used in screening of B.juncea genotypes in future breeding programs.

Positional cloning and characterization of the papaya diminutive mutant reveal a truncating mutation in the CpMMS19 gene.

The papaya diminutive mutant exhibits miniature stature, retarded growth and reduced fertility. This undesirable mutation appeared in the variety 'Sunset', the progenitor of the transgenic line 'SunUp', and was accidentally carried forward into breeding populations. The diminutive mutation was mapped to chromosome 2 and fine mapped to scaffold 25. Sequencing of a bacterial artificial chromosome in the fine mapped region led to the identification of the target gene responsible for the diminutive mutant, a gene orthologous to MMS19 with a 36.8kb deletion co-segregating with the diminutive mutant. The genomic sequence of CpMMS19 is 62kb, consisting of 20 exons and 19 introns. It encodes a protein of 1143 amino acids while the diminutive allele encodes a truncated protein of 287 amino acids. Expression of the full-length CpMMS19 was able to complement the thermosensitive growth of the yeast mms19 deletion mutant while expression of the diminutive allele resulted in increased thermosensitivity. Over-expression of the diminutive allele in Arabidopsis met18 mutant results in a high frequency of seed abortion. The papaya diminutive phenotype is caused by an alteration in gene function rather than a loss-of-function mutation. SCAR (sequence characterized amplified region) markers were developed for rapid detection of the diminutive allele in breeding populations.