Cleaved Amplified Polymorphic Sequence Research Articles

Genetic Mapping and QTL Analysis of Stigma Color in Melon (Cucumis melo L.).

Melon is an important Cucurbitaceae crop. Field observations had shown that the green stigmas of melon are more attractive to pollinators than yellow stigmas. In this study, F2 and F2:3 populations obtained by crossing MR-1 (green stigma) and M4-7 (yellow stigma) were used for genetic analysis and mapping. A genetic map of 1,802.49 cm was constructed with 116 cleaved amplified polymorphism sequence (CAPS) markers. Two stable quantitative trait loci (QTLs) linked to the trait of stigma color were identified on chromosomes 2 (SC2.1) and 8 (SC8.1), respectively. An expanded F2 population was used to narrow down the confidence regions of SC2.1 and SC8.1. As a result, SC2.1 was further mapped to a 3.6 cm region between CAPS markers S2M3 and S2B1-3, explaining 9.40% phenotypic variation. SC8.1 was mapped to a 3.7-cm region between CAPS markers S8E7 and S8H-1, explaining 25.92% phenotypic variation. This study broadens our understanding of the mechanisms of stigma color regulation and will be of benefit to the breeding of melon.

Identification of fruit firmness QTL ff2.1 by SLAF-BSA and QTL mapping in melon

Fruit firmness is an important target of melon breeding, as it is associated with shelf life and economic value; however, the precise mechanism that determines fruit firmness during fruit ripening remains elusive. In the present study, one hundred forty-four F2 plants and F2–3 families derived from the high-firmness melon line M2–10 and the low-firmness melon line ZT091 were used to identify major quantitative trait loci by specific-locus amplified fragment sequencing with bulked segregant analysis. Simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) markers based on the resequencing of parental lines were also used to narrow the associated region to identify candidate genes. Two regions associated with fruit firmness were investigated, including a 4.87 Mb region on chr. 2 and a 28.7 Mb region on chr. 5 of the melon genome. SSR and CAPS markers were used to construct a genetic map of the associated regions: QTL ff5.1 was located between CmSSR13509 and CmSSR13423 and explained 38.44% of the observed variation, with an LOD threshold of 17.44; ff2.1 was located between CmSSR07709 and SNP22228 and explained 28.14% of the variation, with an LOD threshold of 3.8, and this region included 106 Kb and 10 candidate genes. Quantitative real-time PCR was performed to investigated the investigate candidate gene expression at 15, 20 and 25 days after pollination in the parental lines, and significant expression levels were detected for most of the genes, including four genes of unknown function and MELO3C017519, MELO3C017520, MELO3C017522, MELO3C029506, and MELO3C029520. These results revealed a new QTL, ff2.1, for melon fruit firmness-related gene identification.

Mapping of genetic loci controlling fruit linked morphological traits of melon using developed CAPS markers.

Fruit morphology traits are important commercial traits that directly affect the market value. However, studying the genetic basis of these traits in un-explored botanical groups is a fundamental objective for crop genetic improvement through marker-assisted breeding. In this study, a quantitative trait loci (QTLs) mapping strategy was used for dissecting the genomic regions of fruit linked morphological traits by single nucleotide polymorphism (SNP) based cleaved amplified polymorphism sequence (CAPS) molecular markers. Next-generation sequencing was done for the genomic sequencing of two contrasted melon lines (climacteric and non-climacteric), which revealed 97% and 96% of average coverage over the reference melon genome database, respectively. A total of 57.51% non-synonymous SNPs and 42.49% synonymous SNPs were found, which produced 149 sets of codominant markers with a 24% polymorphism rate. Total 138-F2 derived plant populations were genotyped for linkage mapping and composite interval mapping based QTL mapping exposed 6 genetic loci, positioned over distinct chromosomes (02, 04, 08, 09, and 12) between the flanking intervals of CAPS markers, whichexplained an unlinked polygenic architecture in genome. Three minor QTLs of fruit weight(FWt2.1, FWt4.1, FWt9.1), one major QTL of fruit firmness(FrFir8.1), one major QTL of fruit length(FL12.1), and one major QTL of fruit shape (FS12.1)were determined and collectively explained the phenotypic variance from 5.64 to 15.64%. Fruit phenotypic correlation exhibited the significant relationship and principal component analysis also identified the potential variability. Multiple sequence alignments also indicated the significant base-mutations in the detected genetic loci, respectively. In short, our illustrated genetic loci are expected to provide the reference insights for fine QTL mapping and candidate gene(s) mining through molecular genetic breeding approaches aimed at developing the new varieties.

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the Xfm rpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Identification of resistance sources in common bean (

Context Among biotic factors, bean anthracnose caused by Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara is one of the most destructive diseases of common bean (Phaseolus vulgaris L.). Resistance in common bean genotypes is controlled by anthracnose resistance loci (designated Co). Aims The objective of this study was to evaluate the presence of Co resistance genes in common bean genotypes from Turkey and to examine promising genotypes as candidates for parent plants in breeding studies. Methods The presence of Co resistance genes in 693 common bean genotypes from 35 provinces of Turkey was determined by 12 SCAR (sequence characterised amplified region), RAPD (random amplified polymorphic DNA), STS (sequence-tagged site) and CAPS (cleaved amplified polymorphic sequence) markers. The disease reaction of 40 agronomically promising genotypes to different pathogen isolates was evaluated in a pot study. Key results The results indicated the presence of Co-1, Co-14, Co-15, Co-2, Co-33, Co-4, Co-42, Co-6, Co-10, Co-11 and Co-13 resistance genes, alone or in combination; Co-5 was not detected in any of the genotypes. Among the genotypes, Co-15 was the most frequent resistance source, followed by Co-4, Co-6 and Co-11, respectively. Common bean genotypes carrying Co-14, Co-2, Co-33, Co-11 and Co-13 have been identified for the first time in Turkish germplasm. Five genotypes carrying the various combinations of Co-1, Co-15, Co-4, Co-6, Co-10, Co-11 and Co-13 were resistant to all isolates in pathogenicity tests. Conclusions This study is the most comprehensive to date on Co genes linked to the resistance of common bean genotypes throughout Turkey and provides an important gene pool for breeding studies into bean anthracnose. Resistant genotypes identified will be useful as parent plants in resistance breeding studies. Implications Resistance genotypes carrying different Co genes to bean anthracnose will contribute significantly to the disease management strategy.

Purine alkaloids in tea plants: component, biosynthetic mechanism and genetic variation

The purine alkaloids, including caffeine, theobromine, and theacrine, are one of the most important quality and functional components of tea plants and commercial teas. In this paper, we review the component, biosynthetic mechanism and genetic variation of purine alkaloids in tea plants. The content of caffeine and theobromine in 403 accessions Chinese tea germplasms core collection preserved in the National Germplasm Hangzhou Tea Repository were analyzed using High Performance Liquid Chromatography (HPLC). The purine alkaloid profiles of different tea varieties, germplasm types, geographical origin, seasons, were highlighted. Some naturally low caffeine or caffeine-free, high theobromine or high theacrine germplasms were identified and the possible biosynthetic mechanism of hypernormal purine alkaloid content in tea plants were partially revealed. Some Cleaved Amplified Polymorphic Sequences (CAPS) DNA markers were developed for the identification of purine alkaloid content. The potential application of genetic engineering and DNA markers developed based on the low caffeine <italic>TCS1</italic> (tea caffeine synthesis gene) alleles for marker assisted selection (MAS), and cocoa tea and kucha in low caffeine/high theacrine tea cultivar breeding programs were also reviewed.

Identification of starch candidate genes using SLAF-seq and BSA strategies and development of related SNP-CAPS markers in tetraploid potato.

Potato starch is an essential nutrient for humans and is widely used worldwide. Locating relevant genomic regions, mining stable genes and developing candidate gene markers can promote the breeding of new high-starch potato varieties. A total of 106 F1 individuals and their parents (YSP-4 × MIN-021) were used as test materials, from which 20 plants with high starch content and 20 with low starch content were selected to construct DNA pools for site-specific amplified fragment sequencing (SLAF-seq) and bulked segregation analysis (BSA). A genomic region related to the starch traits was first identified in the 0–5.62 Mb of chromosome 2 in tetraploid potato. In this section, a total of 41 non-synonymous genes, which were considered as candidate genes related to the starch trait, were annotated through a basic local alignment search tool (BLAST) search of multiple databases. Six candidate genes for starch (PGSC0003DMG400017793, PGSC0003DMG400035245, PGSC0003DMG400036713, PGSC0003DMG400040452, PGSC0003DMG400006636 and PGSC0003DMG400044547) were further explored. In addition, cleaved amplified polymorphic sequence (CAPS) markers were developed based on single nucleotide polymorphism (SNP) sites associated with the starch candidate genes. SNP-CAPS markers chr2-CAPS6 and chr2-CAPS21 were successfully developed and validated with the F2 population and 24 tetraploid potato varieties (lines). Functional analysis and cloning of the candidate genes associated with potato starch will be performed in further research, and the SNP-CAPS markers chr2-CAPS6 and chr2-CAPS21 can be further used in marker-assisted selection breeding of tetraploid potato varieties with high starch content.

Whole-Genome Resequencing of Near-Isogenic Lines Reveals a Genomic Region Associated with High Trans-Lycopene Contents in Watermelon.

Trans-lycopene is a functional phytochemical abundant in red-fleshed watermelons, and its contents vary among cultivars. In this study, the genetic basis of high trans-lycopene contents in scarlet red flesh was evaluated. Three near-isogenic lines (NILs) with high trans-lycopene contents were derived from the scarlet red-fleshed donor parent DRD and three coral red-fleshed (low trans-lycopene contents) recurrent parents. The lycopene contents of DRD (589.4 ± 71.8 µg/g) were two times higher than that of the recurrent parents, and values for NILs were intermediate between those of the parents. Coral red-fleshed lines and F1 cultivars showed low trans-lycopene contents (135.7 ± 18.0 µg/g to 213.7 ± 39.5 µg/g). Whole-genome resequencing of two NILs and their parents and an analysis of genome-wide single-nucleotide polymorphisms revealed three common introgressed regions (CIRs) on chromosomes 6, 9, and 10. Twenty-eight gene-based cleaved amplified polymorphic sequence (CAPS) markers were developed from the CIRs. The CAPS markers derived from CIR6 on chromosome 6, spanning approximately 1 Mb, were associated (R2 = 0.45–0.72) with the trans-lycopene contents, particularly CIR6-M1 and CIR6-M4. Our results imply that CIR6 is a major genomic region associated with variation in the trans-lycopene contents in red-fleshed watermelon, and CIR6-M1 and CIR6-M4 may be useful for marker-assisted selection.

QTL analysis of flowering‐related traits by specific length amplified fragment sequencing in melon

AbstractMelon (Cucumis melo L., 2n = 24) belongs to the family Cucurbitaceae and is an important vegetable crop. This study aimed to investigate the quantitative trait loci (QTL) of flowering‐related traits to understand the genetic inheritance and candidate gene of flowering‐related traits. The specific length amplified fragment sequencing (SLAF‐seq) technology was used to construct a genetic map of 115 individual plants in the melon F2 population. A total of 82.6 Gb of data were generated using SLAF‐seq. A genetic map was constructed with 10,596 specific length amplified fragment markers in 12 linkage groups, which spanned 1,383.88 cM with an average distance of 0.13 cM between markers. Ten QTL of flowering‐related traits were detected, which explained 2.16–19.34% of the phenotypic variance. The logarithm of the odds (LOD) values ranged from 3.5 to 24.9. Ninety‐four F2:3 families were used for mapping mft and fft in candidate regions by simple sequence repeat (SSR) and cleaved amplified polymorphic sequences (CAPS) markers. The mapping results showed that mft2.1 and fft2.1 were located in the same region, between CmSSR07071 and CmSSR07102. By gene annotation, 36 candidate genes were detected in this region, 26 of which were annotated. Quantitative reverse transcription–polymerase chain reaction (PCR) showed that candidate genes, including WRKY transcription factor and AP2/ERF and B3 domain‐containing transcription factor, were differentially expressed in female and male flowers in the flower budding stages. These genes may provide the theoretical foundation for gene cloning and genetic transformation in melons.

Selection of the Salt Tolerance Gene GmSALT3 During Six Decades of Soybean Breeding in China.

Salt tolerance is an important trait that affects the growth and yield of plants growing in saline environments. The salt tolerance gene GmSALT3 was cloned from the Chinese soybean cultivar Tiefeng 8, and its variation evaluated in Chinese wild soybeans and landraces. However, the potential role of GmSALT3 in cultivation, and its genetic variation throughout the history of Chinese soybean breeding, remains unknown. Here we identified five haplotypes of GmSALT3 in 279 Chinese soybean landraces using a whole genome resequencing dataset. Additionally, we developed five PCR-based functional markers: three indels and two cleaved amplified polymorphic sequences (CAPS) markers. A total of 706 Chinese soybean cultivars (released 1956–2012), and 536 modern Chinese breeding lines, were genotyped with these markers. The Chinese landraces exhibited relatively high frequencies of the haplotypes H1, H4, and H5. H1 was the predominant haplotype in both the northern region (NR) and Huanghuai region (HHR), and H5 and H4 were the major haplotypes present within the southern region (SR). In the 706 cultivars, H1, H2, and H5 were the common haplotypes, while H3 and H4 were poorly represented. Historically, H1 gradually decreased in frequency in the NR but increased in the HHR; while the salt-sensitive haplotype, H2, increased in frequency in the NR during six decades of soybean breeding. In the 536 modern breeding lines, H2 has become the most common haplotype in the NR, while H1 has remained the highest frequency haplotype in the HHR, and H5 and H1 were highest in the SR. Frequency changes resulting in geographically favored haplotypes indicates that strong selection has occurred over six decades of soybean breeding. Our molecular markers could precisely identify salt tolerant (98.9%) and sensitive (100%) accessions and could accurately trace the salt tolerance gene in soybean pedigrees. Our study, therefore, not only identified effective molecular markers for use in soybean, but also demonstrated how these markers can distinguish GmSALT3 alleles in targeted breeding strategies for specific ecoregions.

Evaluation of Anthracnose Resistance in Pepper (Capsicum spp.) Genetic Resources

Anthracnose (Colletotrichum spp.), is one of the major yield losing fungal disease in both pre- and post-harvest stage of pepper (Capsicum spp.) production worldwide. Among the Colletotrichum spp., C. acutatum has strong pathogenicity, which infects both immature and mature pepper fruit leads to severe economic losses in pepper production. Inheritance of anthracnose disease resistance was evaluated with 3738 pepper genetic resources which was collected from different countries and conserved at Korean genebank. The resistance analysis against pepper anthracnose (C. acutatum) was performed on detached mature green and red fruits under laboratory conditions by spray (non-wounding) and microinjection (wounding) inoculation methods. In the primary screening, about 261 accessions were appeared to be resistant against C. acutatum in spray inoculation. The resistant accessions were further evaluated with microinjection (wounding) inoculation method using the fungal (C. acutatum) isolate of pepper anthracnose. There were highly significant differences in the disease severity and distribution of disease rating scale, considering all the sources has significant genetic variation. Finally, the anthracnose resistant pepper accessions have been validated with cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers in which, the CAPS and HRM marker analysis showed four types of genotypes such as resistant (R), susceptible (S), heterozygous (H) and Unidentified type (UT) or not detection. The Capsicum accessions showing high level of resistance to the pathogen could be used as source material in breeding programs for resistance to anthracnose disease.

Investigation and rapid detection of fungal pathogens causing mulberry sclerotial disease in south‐west China

AbstractMulberry sclerotial disease is a destructive disease that afflicts mulberry fruits throughout the world. In recent years, the impact of mulberry sclerotial disease has increased with the development of the mulberry fruit industry. Ciboria carunculoides, C. shiraiana, and Scleromitrula shiraiana are all pathogens causing mulberry sclerotial disease, but the dominant pathogen may be different in different mulberry planting areas. In this study, we investigated the causal agents of mulberry sclerotial disease in Chongqing and Sichuan, the main mulberry planting areas in south‐west China. The results showed that C. carunculoides was the dominant pathogen in the investigated areas, and the disease incidence rate on some widely cultivated mulberry varieties exceeded 90%. Based on the differences in internal transcriber spacer sequences of different pathogens, we developed a method using cleaved amplified polymorphic sequences (CAPS) from digestion with the three restriction enzymes HindIII, EcoRI, and MluI for rapid detection of pathogens on mulberry fruits. Use of this method confirmed that the diseased fruits of some mulberry varieties were infected by a mixture of C. carunculoides and S. shiraiana. Long‐fruit mulberry, which was considered to be resistant to sclerotial disease in the past, was also infected by C. carunculoides. Diseased fruit infected by C. shiraiana or by S. shiraiana alone were not detected. Our results suggest that C. carunculoides is probably the dominant pathogen causing mulberry sclerotial disease in south‐west China. The CAPS method for rapid detection of pathogens could assist diagnosis, epidemiology, and disease monitoring.

Marker-Assisted Evaluation of Two Powdery Mildew Resistance Candidate Genes in Korean Cucumber Inbred Lines

Two genes, CsLRR-RPK2 (CsGy5G015660) and CsaMLO8 (Csa5G623470), have been considered as powdery mildew (PM) resistance genes in cucumbers. In this study, we evaluated the involvement of the alleles of these two genes in PM resistance in 100 commercial Korean cucumber inbred lines. To achieve this, we developed cleaved amplified polymorphic sequences (CAPS) and InDel markers from CsLRR-RPK2 and CsaMLO8. Genotyping analysis indicated that the CsLRR-RPK2-CAPS marker showed a stronger correlation with the PM-resistant phenotype, with an 84% consistency compared to the CsaMLO8-InDel marker. The use of the CsaMLO8-InDel marker showed a 70% consistency between phenotype and genotype results. It was proposed that the CsLRR-RPK2-CAPS marker successfully eliminated PM-susceptible inbred lines, since both genotype and phenotype results were 100% identical. Furthermore, the present study revealed that the introduction of one of these alleles is probably enough to confer PM resistance in cucumbers. However, seven PM-resistant inbred lines harbored either CsaMLO8 or CsLRR-RPK2 alleles, indicating that there is another PM-resistant resource(s) besides CsaMLO8- and CsLRR-RPK2–originated resistance in the commercial Korean inbred lines. Our results provide reliable evidence confirming two PM-resistant candidate genes for the detection of PM resistance resources in cucumber inbred lines.

Loss of chromatin remodeler DDM1 causes segregation distortion in Arabidopsis thaliana.

In ddm1 mutants, the DNA methylation is primarily affected in the heterochromatic region of the chromosomes, which is associated with the segregation distortion of SNPs in the F2 progenies. Segregation distortion (SD) is common in most genetic mapping experiments and a valuable resource to determine how gene loci induce deviation. Meiotic DNA crossing over and SD are under the control of several types of epigenetic modifications. DNA methylation is an important regulatory epigenetic modification that is inherited across generations. In the present study, we investigated the relationship between SD and DNA methylation. The ecotypes Col-0/C24 and chromatin remodeler mutants ddm1-10/Col and ddm1-15/C24 were reciprocally crossed to obtain F2 generations. A total of 300 plants for each reciprocally crossed plant in the F2 generations were subjected to next-generation sequencing to detect the single-nucleotide polymorphisms (SNPs) as DNA markers. All SNPs were analyzed using the Chi-square test method to determine their segregation ratio in F2 generations. Through the segregation ratio, whole-genome SNPs were classified into 16 classes. In class 10, the SNPs in the reciprocal crosses of wild type showed the expected Mendelian ratio of 1:2:1, while those in the reciprocal crosses of ddm1 mutants showed distortion. In contrast, all SNPs in class 16 displayed a normal 1:2:1 ratio, and class 1 showed SD, regardless of wild type or mutants, as assessed using CAPS (cleaved amplified polymorphic sequences) marker analysis to confirm the next-generation sequencing. In ddm1 mutants, the DNA methylation is highly reduced throughout the whole genome and more significantly in the heterochromatic regions of chromosomes. Our results showed that the ddm1 mutants exhibit low levels of DNA methylation, which facilitates the SD of SNPs primarily located in the heterochromatic region of chromosomes by reducing the heterozygous ratio. The present study will provide a strong base for future research focusing on the impact of DNA methylation on trait segregation and plant evolution.

Uncovering candidate genes involved in postharvest ripening of tomato using the Solanum pennellii introgression line population by integrating phenotypic data, RNA-Seq, and SNP analyses

Fruit shelf life in tomato (Solanum lycopersicum) and many other fleshy fruits is determined by several factors including cell wall structure, turgor, and cuticle properties. In this study, an introgression line (IL) population derived from a cross between Solanum pennellii (LA0716) and the S. lycopersicum cultivar M82 was used to identify candidate genes (CGs) that might be responsible for controlling tomato shelf life. IL4-2 and IL4-3-2 were identified as soft lines, IL5-1 and IL9-1-3 as firm lines based on their Br7 and Br14 firmness and shelf-life performance. To gain deeper insights into the genes that might play a regulatory key role in tomato shelf-life, transcriptome profiling was performed for the four ILs and M82 as the reference genome. A total of 5,580 Differentially Expressed Genes (DEGs) with 2,333 up-regulated and 3,247 down-regulated genes, were differentially expressed in IL4-2 vs M82, IL4-3-2 vs M82, IL5-1vs M82, and IL9-1-3 vs M82 combinations. Forty-two DEGs were selected as putative ripening related genes. The expression of nine genes potentially involved in ripening/softening process out of 42 DEGs was verified by qPCR. The RNA-Seq data was also used to discover SNPs between the ILs and the reference genome. The SNPs on CGs were converted to Cleaved Amplified Polymorphic Sequence (CAPS) markers which could be used in breeding programs in the future.

VCF2CAPS-A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset.

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS–a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters–selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software–VCF2CAPS–fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

Study for salt tolerance using CAPS markers in key DNA regulation se-quences in durum wheat (Triticum turgidum L. var. durum) as new alternative

In arid and semi-arid regions, particularly around the Mediterranean basin, soil salinization is one of the abiotic factors that reduces the agricultural yield of several crops and especially the cereal species. Molecular genetics, as an alternative method, makes it possible to select eligible plants for desi-rable traits at an earlier age. Selection based on markers such as CAPS (cleaved amplified polymorphic sequence) can lead to increased accuracy and selection responses in plants. A simple method for detecting polymor-phism of the WRKY candidate gene was performed on nine varieties of du-rum wheat (Triticum turgidum L. var. durum) for the identification of tolerant varieties. The DNA was amplified with a pair of primers, and digested with a restriction endonuclease that recognizes the SNP sequence. The electropho-retic analysis revealed the amplification of 7 bands of different sizes per pro-file in all individuals tested. The analysis of profiles after digestion shows that out of a total of seven bands, five bands do not change size. While, the two fast bands of 130 and 144 bp disappear and two other bands of 122 and 118 bp appear only after digestion, in all individuals studied. In our work, we were unable to demonstrate restriction variation in the DNA binding domain sequences of the transcription factor WRKY1, On the other hand, we show that the CAPS technique can be an easily used alternative, for the detection of variation in key DNA sequences, such as those of the transcription factor binding domains or their cis-acting elements sequences.

Phenotypic and molecular analyses in rice weevil, Sitophilus oryzae (Linneaus) (Coleoptera: Curculionidae): identification of a super kdr mutation, T929I, conferring resistance to deltamethrin.

The rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) is a cosmopolitan pest of stored cereal grains and other commodities globally. Infestations caused by S. oryzae makes grains unsuitable for consumption, processing, and export. Deltamethrin, a synthetic pyrethroid insecticide, is widely used in major grain storages in India as a prophylactic treatment to control this pest. However, recurrent use of this insecticide had led to genetic resistance in S. oryzae, questioning its ongoing use at the current recommended concentration. Dose response analysis of resistant (Delta-R) and susceptible (Lab-S) strains of S. oryzae collected from grain storages across southern India, revealed that Delta-R was 134-fold more resistant than the Lab-S at median lethal concentration (LC50 ). A concentration of 180 ppm over 48 h effectively discriminated 16 resistant field populations from Lab-S with per cent resistance ranging from 8.72% to 75.86%. Exposing all the resistant populations to 1000 ppm over 48 h identified 12 populations with strongly resistant individuals and confirmed the existence of two distinct resistance phenotypes, 'weak' and 'strong' in S. oryzae. Furthermore, sequence analysis of the voltage-gated sodium channel (vgsc) gene in Delta-R identified a single target site mutation, T929I conferring resistance in S. oryzae. CAPS (Cleaved Amplified Polymorphic Sequence) marker analysis of this allele confirmed that frequency of resistance is high (up to 0.96) supporting the results of phenotypic analysis. Both phenotype and molecular marker analyses clearly demonstrated that deltamethrin at 180 and 1000 ppm can be used to discriminate weakly and strongly resistant populations in S. oryzae, respectively. Resistance diagnostics based on the mutation, T929I, supports our phenotypic data and indicates that resistance to deltamethrin in S. oryzae is prevalent in southern parts of India, stressing the need to identify a synergist or suitable alternatives. © 2021 Society of Chemical Industry.

Discovery and fine mapping of Rph28: a new gene conferring resistance to Puccinia hordei from wild barley.

A new gene Rph28 conferring resistance to barley leaf rust was discovered and fine-mapped on chromosome 5H from wild barley. Leaf rust is a highly destructive disease of barley caused by the fungal pathogen Puccinia hordei. Genetic resistance is considered to be the most effective, economical and eco-friendly approach to minimize losses caused by this disease. A study was undertaken to characterize and fine map a seedling resistance gene identified in a Hordeum vulgare ssp. spontaneum-derived barley line, HEB-04-101, that is broadly effective against a diverse set of Australian P. hordei pathotypes. Genetic analysis of an F3 population derived from a cross between HEB-04-101 and the H. vulgare cultivar Flagship (seedling susceptible) confirmed the presence of a single dominant gene for resistance in HEB-04-101. Selective genotyping was performed on representative plants from non-segregating homozygous resistant and homozygous susceptible F3 families using the targeted genotyping-by-sequencing (tGBS) assay. Putatively linked SNP markers with complete fixation were identified on the long arm of chromosome 5H spanning a physical interval between 622 and 669Mb based on the 2017 Morex barley reference genome assembly. Several CAPS (cleaved amplified polymorphic sequences) markers were designed from the pseudomolecule sequence of the Morex assembly (v1.0 and v2.0), and 16 polymorphic markers were able to delineate the RphHEB locus to a 0.05cM genetic interval spanning 98.6kb. Based on its effectiveness and wild origin, RphHEB is distinct from all other designated Rph genes located on chromosome 5H and therefore the new locus symbol Rph28 is recommended for RphHEB in accordance with the rules and cataloguing system of barley gene nomenclature.

In planta Genome Editing in Commercial Wheat Varieties.

Limitations for the application of genome editing technologies on elite wheat (Triticum aestivum L.) varieties are mainly due to the dependency on in vitro culture and regeneration capabilities. Recently, we developed an in planta particle bombardment (iPB) method which has increased process efficiency since no culture steps are required to create stably genome-edited wheat plants. Here, we report the application of the iPB method to commercially relevant Japanese elite wheat varieties. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaQsd1 were bombarded into the embryos of imbibed seeds with their shoot apical meristem (SAM) exposed. Mutations in the target gene were subsequently analyzed within flag leaf tissue by using cleaved amplified polymorphic sequence (CAPS) analysis. A total of 9/358 (2.51%) of the bombarded plants (cv. “Haruyokoi,” spring type) carried mutant alleles in the tissue. Due to the chimeric nature of the T0 plants, only six of them were inherited to the next (T1) generation. Genotypic analysis of the T2 plants revealed a single triple-recessive homozygous mutant of the TaQsd1 gene. Compared to wild type, the homozygous mutant exhibited a 7 days delay in the time required for 50% seed germination. The iPB method was also applied to two elite winter cultivars, “Yumechikara” and “Kitanokaori,” which resulted in successful genome editing at slightly lower efficiencies as compared to “Haruyokoi.” Taken together, this report demonstrates that the in planta genome editing method through SAM bombardment can be applicable to elite wheat varieties that are otherwise reluctant to callus culture.