Cleaved Amplified Polymorphic Sequence Markers Research Articles

Identification of genetic loci and candidate genes underlying freezing tolerance in wheat seedlings.

Ten stable loci for freezing tolerance (FT) in wheat were detected by genome-wide association analysis. The putative candidate gene TaRPM1-7BL underlying the major locus QFT.ahau-7B.2 was identified and validated. Frost damage restricts wheat growth, development, and geographical distribution. However, the genetic mechanism of freezing tolerance (FT) remains unclear. Here, we evaluated FT phenotypes of 245 wheat varieties and lines, and genotyped them using a Wheat 90K array. The association analysis showed that ten stable loci were significantly associated with FT (P < 1 × 10-4), and explained 6.45-26.33% of the phenotypic variation. In particular, the major locus QFT.ahau-7B.2 was consistently related to all nine sets of FT phenotypic data. Based on five cleaved amplified polymorphic sequence (CAPS) markers closely linked to QFT.ahau-7B.2, we narrowed down the target region to the 570.67-571.16Mb interval (0.49Mb) on chromosome 7B, in which four candidate genes were annotated. Of these, only TaRPM1-7BL exhibited consistent differential expression after low temperature treatment between freezing-tolerant and freezing-sensitive varieties. The results of cloning and whole-exome capture sequencing indicated that there were two main haplotypes for TaRPM1-7BL, including freezing-tolerant Hap1 and freezing-sensitive Hap2. Based on the representative SNP (+1956, A/G), leading to an amino acid change in the NBS domain, a CAPS marker (CAPS-TaRPM1-7BL) was developed and validated in 431 wheat varieties (including the above 245 materials) and 318 F2 lines derived from the cross of 'Annong 9267' (freezing-tolerant) × 'Yumai 9' (freezing-sensitive). Subsequently, the TaRPM1-7BL gene was silenced in 'Yumai 9' by virus-induced gene silencing (VIGS), and these silenced wheat seedlings exhibited enhanced FT phenotypes, suggesting that TaRPM1-7BL negatively regulates FT. These findings are valuable for understanding the complex genetic basis of FT in wheat.

A Novel PCR-Based Functional Marker of Rice Blast Resistance Gene Pi25

Rice blast is arguably the most devastating fungal disease of rice. Utilization of resistance genes to breed resistant cultivars is one of the most economical and environmentally friendly approaches to combat the disease. Pi25, a major resistance gene conferring broad-spectrum resistance to both leaf and neck blast, is an ideal gene resource to improve the resistance of rice varieties to blast. Recently, several allele-specific markers were developed. However, they were deficiently efficient due to either an additional process of restriction enzyme digestion for cleaved amplified polymorphic sequence (CAPS) markers or the risk of false-positive error in identifying susceptible Tetep allele (Pi25TTP) for PCR-based markers. In this study, based on a conserved single nucleotide polymorphism (SNP) between resistant and susceptible alleles, a tetra-primer amplification refractory mutation system (ARMS)-PCR marker was developed. The new marker, namely Pi25-2687R3, could effectively distinguish the resistant Gumei 2 (GM2) allele (Pi25GM2) and the susceptible allele Pi25TTP. Moreover, a perfect consistency of genotyping was exhibited between Pi25-2687R3 and published CAPS marker CAP3/Hpy 99I. A more accurate genotyping was also displayed compared to the previous PCR-based SNP marker Pi25-2566. Our finding proved that Pi25-2687R3 could achieve the same result as CAP3/Hpy 99I with less workload and cost and could promote the accuracy in the identification of genotypes superior to Pi25-2566. This study provided a quick and reliable functional marker for discriminating Pi25 alleles, which would be a valuable tool for genotypic assay and rice molecular breeding of blast resistance.

Dissecting the genetic basis of grain color and pre-harvest sprouting resistance in common wheat by association analysis

Pre-harvest sprouting (PHS) adversely affects wheat quality and yield, and grain color (GC) is associated with PHS resistance. However, the genetic relationship between GC and PHS resistance remains unclear. In this study, 168 wheat varieties (lines) with significant differences in GC and PHS resistance were genotyped using an Illumina 90K iSelect SNP array. Genome-wide association study (GWAS) based on a mixed linear model showed that 67 marker-trait associations (MTAs) assigned to 29 loci, including 17 potentially novel loci, were significantly associated with GC, which explained 1.1–17.0% of the phenotypic variation. In addition, 100 MTAs belonging to 54 loci, including 31 novel loci, were significantly associated with PHS resistance, which accounted for 1.1–14.7% of the phenotypic variation. Subsequently, two cleaved amplified polymorphic sequences (CAPS) markers, 2B-448 on chromosome 2B and 5B-301 on chromosome 5B, were developed from the representative SNPs of the major common loci Qgc.ahau-2B.3/Qphs.ahau-2B.4 controlling GC/PHS resistance and PHS resistance locus Qphs.ahau-5B.4, respectively. Further validation in 171 Chinese mini-core collections confirmed significant correlations of the two CAPS markers with GC and PHS resistance phenotypes under different environments (P<0.05). Furthermore, the wheat public expression database, transcriptomic sequencing, and gene allelic variation analysis identified TraesCS5B02G545100, which encodes glutaredoxin, as a potential candidate gene linked to Qphs.ahau-5B.4. The new CAPS marker CAPS-356 was then developed based on the SNP (T/C) in the coding sequences (CDS) region of TraesCS5B02G545100. The high-density linkage map of the Jing 411/Hongmangchun 21 recombinant inbred lines (RILs) constructed based on specific locus amplified fragment sequencing markers showed that CAPS-356 collocated with a novel QTL for PHS resistance, supporting the role of TraesCS5B02G545100 as the potential candidate gene linked to Qphs.ahau-5B.4. These results provide valuable information for the map-based cloning of Qphs.ahau-5B.4 and breeding of PHS resistant white-grained varieties.

A rapid method for detection of the root-knot nematode resistance gene, Mi-1.2, in tomato cultivars.

Molecular markers have been widely used in plant breeding to improve the accuracy and efficiency of trait selection. In particular, molecular markers are powerful in facilitating the introgression of resistance genes by circumventing costly and time-consuming infection assays. To achieve their practical use, it is important to ensure the tight linkage between the markers and the traits. Here we report a new cleaved amplified polymorphic sequence (CAPS) marker, Mi1713, for the root-knot nematode resistance gene, Mi-1.2, in cultivated tomato. The Mi1713 marker is designed in the conserved region of Mi-1.2 and its homologs in tomato and other nightshade species. Combined with a single-step procedure for preparing PCR templates, the Mi1713 marker enables rapid and reliable screening for the presence of Mi-1.2. The approach described in this study is applicable in designing CAPS markers for various genes or alleles of interest in tomato and other crops.

Phenotypic and genotypic characterization to authenticate and trace a high-quality yellow tomato ecotype through the processing chain

In the last few years considerable interest is growing for high food quality, both for organoleptic and nutritional aspects. As for tomatoes, the yellow fruit varieties are conquering the consumer markets in many Mediterranean countries. Among the great variability of yellow tomatoes, one landrace of “Pomodorino giallo del Vesuvio”, named GiaGiù (E40), differs from the traditional “Pomodorino del Piennolo” for the fruit color and stands out for its quality traits, such as high glutamic acid, pectin content and titratable acidity of fruit, thus increasing its demand for both fresh consumption and cooking purposes. The aim of this work was to phenotypically and genotypically distinguish the GiaGiù landrace through morphological descriptors and molecular markers, in order to provide an effective tool to authenticate this product as fresh and processed tomatoes. Morphological characterization evidenced that the distinctive traits of GiaGiù landrace were the potato leaf morphology and the pyriform shape with a pointed apex of the yellow fruits. The genotypic distinction of E40 was performed by using two Cleaved Amplified Polymorphic Sequence (CAPS) markers designed on a Single Nucleotide Polymorphism (SNP) of the Phytoene synthase 1 (psy1) gene that confers the yellow color to tomato fruit and already known as specific of GiaGiù genotype. Additional CAPS markers were designed on two private mutations of E40 genes derived from data retrieved from a Genotyping-By-Sequencing (GBS) dataset, already available. These findings were confirmed by comparing E40 private mutations with the 360 accessions of the BGI tomato 360 genomes resequencing project. The designed markers allowed to distinguish GiaGiù genotype in all fresh and processed fruit tomato matrices, thus representing a molecular tool able to prevent food fraud and authenticate GiaGiù products.

Fine Mapping the Soybean Mosaic Virus Resistance Gene in Soybean Cultivar Heinong 84 and Development of CAPS Markers for Rapid Identification.

Heinong 84 is one of the major soybean varieties growing in Northeast China, and is resistant to the infection of all strains of soybean mosaic virus (SMV) in the region including the most prevalent strain, N3. However, the resistance gene(s) in Heinong 84 and the resistant mechanism are still elusive. In this study, genetic and next-generation sequencing (NGS)-based bulk segregation analysis (BSA) were performed to map the resistance gene using a segregation population from the cross of Heinong 84 and a susceptible cultivar to strain N3, Zhonghuang 13. Results show that the resistance of Heinong 84 is controlled by a dominant gene on chromosome 13. Further analyses suggest that the resistance gene in Heinong 84 is probably an allele of Rsv1. Finally, two pairs of single-nucleotide-polymorphism (SNP)-based primers that are tightly cosegregated with the resistance gene were designed for rapidly identifying resistant progenies in breeding via the cleaved amplified polymorphic sequence (CAPS) assay.

Validation of CAPS marker WR003 for the leaf rust resistance gene Lr1 and the molecular evolution of Lr1 in wheat

The wheat leaf rust resistance gene Lr1 encodes a typical coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) of resistance protein containing 1 344 amino acids. WR003, a cleaved amplified polymorphic sequence (CAPS) marker is derived from the LRR regions of Lr1. In this study, a worldwide collection of 120 Aegilops tauschii accessions and 282 hexaploid wheat varieties was screened for Lr1 alleles using WR003, and the specificity of WR003 for Lr1 was confirmed by pathogenicity tests and genotype analysis. The sequence alignment and phylogenetic tree analysis of 38 Lr1 haplotypes provided a further view of the molecular evolution of Lr1. The results showed that there were very few polymorphisms between the Lr1 alleles from Ae. tauschii and hexaploid wheat with the same resistance phenotype. The polymorphisms of the Lr1 haplotypes were mainly between the different resistance lines, rather than between the different ploidy levels. These results indicate that Lr1 originated from Ae. tauschii and differentiated into resistant and susceptible genotypes before its introgression into hexaploid wheat. Therefore, it is likely that wheat Lr1 has at least two major variants for disease resistance and susceptibility, and except for certain point mutations, few gene conversions and genetic re-combinations occurred during the hexaploid wheat domestication.

Genome-wide association study reveals a NAC transcription factor TaNAC074 linked to pre-harvest sprouting tolerance in wheat.

Twelve QTL associated with pre-harvest sprouting tolerance were identified using association analysis in wheat. Two markers were validated and a candidate gene TaNAC074 for Qgpf.cas-3B.2 was verified using Agrobacterium-mediated transformation. Pre-harvest sprouting (PHS) is a considerable global threat to wheat yield and quality. Due to this threat, breeders must identify quantitative trait loci (QTL) and genes conferring PHS-tolerance (PHST) to reduce the negative effects of PHS caused by low seed dormancy. In this study, we evaluated a panel of 302 diverse wheat genotypes for PHST in four environments and genotyped the panel with a high-density wheat 660K SNP array. By using a genome-wide association study (GWAS), we identified 12 stable loci significantly associated with PHST (P < 0.0001), explaining 3.34 - 9.88% of the phenotypic variances. Seven of these loci co-located with QTL and genes reported previously. Five loci (Qgpf.cas-3B.2, Qgpf.cas-3B.3, Qgpf.cas-3B.4, Qgpf.cas-7B.2, and Qgpf.cas-7B.3), located in genomic regions with no known PHST QTL or genes, are likely to be new QTL conferring PHST. Additionally, two molecular markers were developed for Qgpf.cas-3A and Qgpf.cas-7B.3, and validated using a different set of 233 wheat accessions. Finally, the PHST-related function of candidate gene TaNAC074 for Qgpf.cas-3B.2 was confirmed by CAPS (cleaved amplified polymorphic sequences) marker association analysis in 233 wheat accessions and by expression and phenotypic analysis of transgenic wheat. Overexpression of TaNAC074 significantly reduced seed dormancy in wheat. This study contributes to broaden the genetic basis and molecular marker-assisted breeding of PHST.

Intergeneric hybridization of marguerite (Argyranthemum frutescens (L.) Sch. Bip.) and Roman chamomile (Chamaemelum nobile (L.) All.) using ovule culture and confirmation of hybridity.

To introduce useful characteristics such as fragrance into Argyranthemum frutescens (L.) and to expand the variation, we conducted crosses using A. frutescens as the seed parent and Chamaemelum nobile (L.) All. as the pollen parent. All the tested cross combinations between the three strains of A. frutescens and one strain of C. nobile produced embryos, and healthy plants were obtained by ovule culture. The obtained plantlets had a white ray floret, and the leaf shape was intermediate to those of the parents. The individuals obtained from this cross were subjected to two methods to determine hybridity: flow cytometry analyses and cleaved amplified polymorphic sequence (CAPS) markers. For the CAPS marker, we selected the internal transcribed spacer (ITS) region, which is highly variable among the genera, as the region to be amplified. We selected restriction enzymes BmgT120 I and Afl II, which selectively cut common sequences in the genus Argyranthemum, based on the sequence analysis of one parent strain each of A. frutescens and C. nobile and alignment with known sequences of related species. Flow cytometry analyses and CAPS markers revealed that the individuals obtained from the cross between A. frutescens and C. nobile are intergeneric hybrids. In addition, these established methods were capable of quickly and reliably identifying hybrids between A. frutescens and C. nobile. This report shows for the first time that crossbreeding between A. frutescens (seed parent) and C. nobile (pollen parent) is possible, and further development of Argyranthemum breeding, such as the expansion of variation by intergeneric crosses, is expected.

Cultivar-specific markers, mutations, and chimerisim of Cavendish banana somaclonal variants resistant to Fusarium oxysporum f. sp. cubense tropical race 4

BackgroundThe selection of tissue culture–derived somaclonal variants of Giant Cavendish banana (Musa spp., Cavendish sub-group AAA) by the Taiwan Banana Research Institute (TBRI) has resulted in several cultivars resistant to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a destructive fungus threatening global banana production. However, the mutations in these somaclonal variants have not yet been determined. We performed an RNA-sequencing (RNA-seq) analysis of three TBRI Foc TR4–resistant cultivars: ‘Tai-Chiao No. 5’ (TC5), ‘Tai-Chiao No. 7’ (TC7), and ‘Formosana’ (FM), as well as their susceptible progenitor ‘Pei-Chiao’ (PC), to investigate the sequence variations among them and develop cultivar-specific markers.ResultsA group of single-nucleotide variants (SNVs) specific to one cultivar were identified from the analysis of RNA-seq data and validated using Sanger sequencing from genomic DNA. Several SNVs were further converted into cleaved amplified polymorphic sequence (CAPS) markers or derived CAPS markers that could identify the three Foc TR4–resistant cultivars among 6 local and 5 international Cavendish cultivars. Compared with PC, the three resistant cultivars showed a loss or alteration of heterozygosity in some chromosomal regions, which appears to be a consequence of single-copy chromosomal deletions. Notably, TC7 and FM shared a common deletion region on chromosome 5; however, different TC7 tissues displayed varying degrees of allele ratios in this region, suggesting the presence of chimerism in TC7.ConclusionsThis work demonstrates that reliable SNV markers of tissue culture–derived and propagated banana cultivars with a triploid genome can be developed through RNA-seq data analysis. Moreover, the analysis of sequence heterozygosity can uncover chromosomal deletions and chimerism in banana somaclonal variants. The markers obtained from this study will assist with the identification of TBRI Cavendish somaclonal variants for the quality control of tissue culture propagation, and the protection of breeders’ rights.

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.

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. &amp; Magnus) Briosi &amp; 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.

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.

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.

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.

A novel rice dull gene, LowAC1, encodes an RNA recognition motif protein affecting Waxyb pre-mRNA splicing

A new dull grain rice mutant with low amylose content, designated lowac1, has been isolated and characterized. To identify the causal mutation site, resequencing of the whole genome and analysis of a cleaved amplified polymorphic sequence (CAPS) marker were performed. Genotypes using the CAPS marker of the identified LowAC1 gene encoding an RNA recognition motif (RRM) protein were entirely consistent with low amylose phenotypes in BC1F2 progeny. Moreover, the segregation of BC1F2 population indicated that the low amylose phenotype was controlled by a single recessive gene. lowac1 involves a single-nucleotide polymorphism from G to A within the gene, resulting in the stop codon generation. The RRM protein deletion in the mutant seed specifically affected the splicing efficiency of Waxyb (Wxb) in the 5′ splice site of intron 1, resulting in decreased protein levels of granule-bound starch synthase I (GBSSI) encoded by Wxb. Whereas, the RRM protein did not affect amylose content in Wxa of indica variety. Also, the mutation induced a little variation in the expression levels of some genes involved in starch biosynthesis. Particularly, expression levels of SBEIIb, PUL, and AGPL2 mRNAs in lowac1 mutant were approximately two times higher compared to the corresponding wild type (WT) genes. Aside from low amylose content, lowac1 seeds included an amylopectin structure reducing short chains compared to that of WT seeds. Overall, our data suggest that LowAC1 is a novel regulatory factor for starch synthesis in rice.