Cleaved Amplified Polymorphic Sequence Markers Research Articles

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.

Involvement of purple acid phosphatase gene into nitrogen uptake of oil palm (Elaeis guineensis)

Abstract. Maryanto SD, Tanjung ZA, Roberdi, Sudania WM, Pujianto, Hairinsyah, Utomo C, Liwang T. 2021. Involvement of purple acid phosphatase gene into nitrogen uptake of oil palm (Elaeis guineensis). Biodiversitas 22: 1385-1390. Nitrogen is the most important nutrient element in terms of plant growth. Plant purple acid phosphatases (PAPs) are known to participate in the phosphate (Pi) acquisition and utilization. Moreover, PAP gene plays an important role in nitrogen fixation. A single nucleotide polymorphism (SNP) was previously detected in the exon 7 of EgPAP3, based on SNP mining analysis of oil palm (Elaeis guineensis Jacq.). genome database. This study was aimed to obtain a Cleaved Amplified Polymorphic Sequences (CAPS) marker based on SNP within EgPAP associated with efficient nitrogen uptake oil palm progenies. Primer pairs were designed and used for PCR amplification of 3 oil palm progenies that showed low N-content, 3 progenies with moderate N-content, and 3 progenies with high N-content. The amplicon was purified prior to single-pass DNA sequencing analysis. Based on Pearson’s chi-square and odds ratio statistical analysis, the SNP has strong positive correlation with the phenotype. The SNP is located at chromosome 13 with a distance of 17.7771 cM from start codon. The sequencing analysis revealed that three progenies with high N-content samples had GG allele motif, while moderate N-content progenies had GA allele and low N-content progenies had AA allele motifs respectively. In addition, a restriction site of NIaIV was found to be adjacent to the SNP location, thus the PCR products of all samples were digested with NIaIV restriction enzyme. NIaIV was able to distinguish between high, medium and low efficient DNA samples. Whole high N-content progenies with GG allele motifs were undigested indicating a single band size of 670 bp identical to the untreated PCR product (control). Moderate N-content progenies produced a 670 bp, 550 bp, and 120 bp bands because of digested by NIaIV. Low N-content progenies also resulted in double bands of 550 bp and 120 bp due to digested by NIaIV. Furthermore, NIaIV restriction enzyme was applied to digest other 54 oil palms DNA samples with unknown genotypes. Whole GG samples were consistently shown to have single band, GA and AA samples were also consistent in producing two bands with different lengths. Based on this result, CAPS marker based on SNP in EgPAP3 was successfully developed to screen between high and low efficient N-uptake of oil palm progenies.

Development of codominant SCAR markers to detect the Pto, Tm22, I3 and Sw5 genes in tomato (Solanum lycopersicum)

AbstractMarker‐assisted selection (MAS) can speed up the plant breeding process and reduce the labour and field costs. The sequence‐characterized amplified region (SCAR) marker allows the elimination of the restriction digestion step. Hence, it is more efficient and reliable than the cleaved amplified polymorphic sequence (CAPS) marker in gel‐based screening. But we still only have CAPS marker for many tomato disease resistance genes. By comparing the coding region and its flanking sequences of the disease‐resistant genes between the resistant and the susceptible varieties, we designed primers that can amplify fragments with different sizes from these varieties for the Pto, Tm22, Sw5 and I3 genes conferring resistance to bacterial speck (BS), Tomato mosaic virus (ToMV), tomato spotted wilt virus (TSWV) and Fusarium wilt (FW) race 3, respectively in tomato. We tested these new markers using our tomato breeding lines and hybrids. All the new SCAR markers worked as expected. The fragments' sizes were amplified from the resistant variety, and the susceptible variety is 1,512 and 450 bp for Pto, 885 and 583 bp for Tm22, 521 and 451 bp for Sw5 and 673 and 480 bp for I3 genes. All the new markers are tightly‐linked to the resistant genes; the markers of Pto, Tm22, Sw5 and I3 are 3,306, 4,888, 0 and 8,310 bp from their corresponding coding sequences, respectively. These new markers may be useful for MAS in tomato breeding.

Molecular characterization of SlATG18f in response to Tomato leaf curl New Delhi virus infection in tomato and development of a CAPS marker for leaf curl disease tolerance.

Analysis of autophagy-related genes in tomato shows the involvement of SlATG18f in leaf curl disease tolerance and a CAPS marker developed from this gene demonstrates its usefulness in marker-assisted selection. Autophagy is a highly conserved catabolic process regulating cellular homeostasis and adaptation to different biotic and abiotic stress. Several autophagy-related proteins (ATGs) are reported to be involved in autophagic processes, and considering their importance in regulating growth and stress adaptation, these proteins have been identified and characterized in several plant species. However, there is no information available on the role of autophagy-related proteins regulating the tolerance of tomatototomato leaf curl disease (ToLCD). Given this, the present genome-wide study identified thirty ATG-encoding genes (SlATG) in tomato, followed by their functional characterization. Expression profiling of the SlATG genes in contrasting tomato cultivars subjected to virus infection showed a 4.5-fold upregulation of SlATG18f in the tolerant cultivar. Further, virus-induced gene silencing of SlATG18f in the tolerant cultivar conferred disease susceptibility, which suggested the role of this gene in Tomato leaf curl New Delhi virus tolerance. Comparison of the gene sequence of both tolerant and susceptible cultivars along with the 5' upstream regions identified an SNP (A/T) at -2916 upstream of the start codon. A cleaved amplified polymorphic sequence (CAPS) marker was developed targeting this region, which showed a significant association with the tolerance characteristics in the tomato germplasm (R2 = 0.1787). Altogether, the study identified a potential gene that could be used to develop ToLCNDV tolerant tomato cultivars using transgene-based or marker-assisted breeding-based approaches.

Identification and Validation of New Stable QTLs for Grain Weight and Size by Multiple Mapping Models in Common Wheat.

Improvement of grain weight and size is an important objective for high-yield wheat breeding. In this study, 174 recombinant inbred lines (RILs) derived from the cross between Jing 411 and Hongmangchun 21 were used to construct a high-density genetic map by specific locus amplified fragment sequencing (SLAF-seq). Three mapping methods, including inclusive composite interval mapping (ICIM), genome-wide composite interval mapping (GCIM), and a mixed linear model performed with forward–backward stepwise (NWIM), were used to identify QTLs for thousand grain weight (TGW), grain width (GW), and grain length (GL). In total, we identified 30, 15, and 18 putative QTLs for TGW, GW, and GL that explain 1.1–33.9%, 3.1%–34.2%, and 1.7%–22.8% of the phenotypic variances, respectively. Among these, 19 (63.3%) QTLs for TGW, 10 (66.7%) for GW, and 7 (38.9%) for GL were consistent with those identified by genome-wide association analysis in 192 wheat varieties. Five new stable QTLs, including 3 for TGW (Qtgw.ahau-1B.1, Qtgw.ahau-4B.1, and Qtgw.ahau-4B.2) and 2 for GL (Qgl.ahau-2A.1 and Qgl.ahau-7A.2), were detected by the three aforementioned mapping methods across environments. Subsequently, five cleaved amplified polymorphic sequence (CAPS) markers corresponding to these QTLs were developed and validated in 180 Chinese mini-core wheat accessions. In addition, 19 potential candidate genes for Qtgw.ahau-4B.2 in a 0.31-Mb physical interval were further annotated, of which TraesCS4B02G376400 and TraesCS4B02G376800 encode a plasma membrane H+-ATPase and a serine/threonine-protein kinase, respectively. These new QTLs and CAPS markers will be useful for further marker-assisted selection and map-based cloning of target genes.

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene

Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low (“00”) cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new “00” varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5′-C↓TAG-3′ sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

Gametophytic self-incompatibility in Andean capuli (Prunus serotina subsp. capuli): allelic diversity at the S-RNase locus influences normal pollen-tube formation during fertilization.

Capuli (Prunus serotina subsp. capuli) is a tree species that is widely distributed in the northern Andes. In Prunus, fruit set and productivity appears to be limited by gametophytic self-incompatibility (GSI) which is controlled by the S-Locus. For the first time, this research reveals the molecular structure of the capuli S-RNase (a proxy for S-Locus diversity) and documents how S-Locus diversity influences GSI in the species. To this end, the capuli S-RNase gene was amplified and sequenced in order to design a CAPS (Cleaved Amplified Polymorphic Sequence) marker system that could unequivocally detect S-alleles by targeting the highly polymorphic C2–C3 S-RNase intra-genic region. The devised system proved highly effective. When used to assess S-Locus diversity in 15 P. serotina accessions, it could identify 18 S-alleles; 7 more than when using standard methodologies for the identification of S-alleles in Prunus species. CAPS marker information was subsequently used to formulate experimental crosses between compatible and incompatible individuals (as defined by their S-allelic identity). Crosses between heterozygote individuals with contrasting S-alleles resulted in normal pollen tube formation and growth. In crosses between individuals with exactly similar S-allele identities, pollen tubes often showed morphological alterations and arrested development, but for some (suspected) incompatible crosses, pollen tubes could reach the ovary. The latter indicates the possibility of a genotype-specific breakdown of GSI in the species. Overall, this supports the notion that S-Locus diversity influences the reproductive patterns of Andean capuli and that it should be considered in the design of orchards and the production of basic propagation materials.

PCR-Based InDel Marker Associated with Powdery Mildew-Resistant MR-1

Powdery mildew (PM) is a fungal disease occurring in both field and greenhouse conditions worldwide. It infects many plant species and reduces both the productivity and quality of crops. Melon (Cucumis melo L.) is an economically important crop. In order to develop a molecular marker that can be used more conveniently in the development of PM-resistant melon using MR-1 melon resources, the previously reported cleaved amplified polymorphic sequence (CAPS) marker was improved with a length polymorphism PCR marker. Two cleaved CAPS markers—BSA12-LI3ECORI and BSA12-LI4HINFI—associated with BPm12.1, a major quantitative trait locus (QTL) corresponding to the PM resistance of MR-1, have been reported. In this study, we found that in the BSA12-LI3ECORI CAPS marker specifically, a 41 bp deletion was present in the PCR DNA region of the MR-1 melon genome. A new marker capable of distinguishing polymerase chain reaction (PCR) length polymorphism was produced using insertion-deletion (InDel) information in this region. This PCR-based InDel marker distinguished the genotypes of PM-resistant MR-1, PM-susceptible Top Mark, and their F1 progeny. These results suggest that this InDel marker could be used to develop PM-resistant melon varieties based on MR-1.

The Variability of Puroindoline-Encoding Alleles and Their Influence on Grain Hardness in Modern Wheat Cultivars Cultivated in Poland, Breeding Lines and Polish Old Landraces (Triticum aestivum L.)

Wheat (Triticum aestivum L.) grain hardness is determined mainly by variations in puroindoline genes (Pina-D1 and Pinb-D1), which are located on the short arm of chromosome 5D. This trait has a direct effect on the technological properties of the flour and the final product quality. The objective of the study was to analyze the mutation frequency in both Pin genes and their influence on grain hardness in 118 modern bread wheat cultivars and breeding lines cultivated in Poland, and 80 landraces from Poland. The PCR products containing the Pin gene coding sequences were sequenced by the Sanger method. Based on detected the SNPs (single-nucleotide polymorphisms) we designed CAPS (cleaved amplified polymorphic sequence) markers for the fast screening of Pinb alleles in a large number of genotypes. All analyzed cultivars, breeding lines, and landraces possess the wild-type Pina-D1a allele. Allelic variation was observed within the Pinb gene. The most frequently occurring allele in modern wheat cultivars and breeding lines (over 50%) was Pinb-D1b. The contribution of the remaining alleles (Pinb-D1a, Pinb-D1c, and Pinb-D1d) was much less (approx. 15% each). In landraces, the most frequent allele was Pinb-D1a (over 70%), followed by Pinb-D1b (21% frequency). Pinb-D1c and Pinb-D1g were found in individual varieties. SKCS (single-kernel characterization system) analysis revealed that grain hardness was strictly connected with Pinb gene allelic variation in most tested cultivars. The mean grain hardness values were significantly greater in cultivars with mutant Pinb variants as compared to those with the wild-type Pinb-D1a allele. Based on grain hardness measured by SKCS, we classified the analyzed cultivars and lines into different classes according to a previously proposed classification system.

A major and stable QTL controlling wheat thousand grain weight: identification, characterization, and CAPS marker development

Thousand grain weight (TGW) is one of the three crucial determinants of wheat (Triticum aestivum) grain yield. The identification of functional genes and development of closely linked markers are urgently needed for wheat yield improvements. In this study, quantitative trait locus (QTL) mapping for TGW was carried out using one RIL population in four environments. A total of 8 QTL controlling TGW were detected across four chromosomes. Of them, QGW4B.4-17 was confirmed as a major and stable QTL for TGW increases of 2.19–3.06 g and high PVE of 22.5–36.3% in three environments. It was located in an interval (Rht1-TaGW-4B) of 2 Mb in physical distance that contains the Rht1 locus. Three high-confidence (HC) genes were predicted as potential candidates according to their annotated functions and expression profiles. Furthermore, a cleaved amplified polymorphic sequence (CAPS) marker, TaGW-4B, was developed and verified in a panel of 205 wheat cultivars and showed a highly significant correlation with TGW, demonstrating high value for efficient marker-assisted selection. The study provided a basis for cloning the functional genes underlying the QTL and a practical and accurate marker for molecular breeding.

Genetic localization of the SPC gene controlling pod coiling direction in Medicago truncatula.

Handedness in plants introduced by helical growth of organs is frequently observed, and it has fascinated plant scientists for decades. However, the genetic control of natural handedness has not been revealed. In the model legume Medicago truncatula, pods can be coiled in a clockwise or anti-clockwise manner, providing a model for genetic analysis of plant handedness. We aimed to localize the Sense of Pod Coiling (SPC) gene controlling pod coiling direction in M. truncatula. Linkage analysis was used with a biparental population for fine mapping of the SPC gene. The genome sequence of M. truncatula Mt4.0 was used for marker identification and physical mapping. Single nucleotide polymorphisms (SNPs) between the parental lines were converted to CAPS (cleaved amplified polymorphic sequences) markers. Genetic map was constructed using the software JoinMap version 3.0. Gene predication and annotation provided by the M. truncatula genome database (http://www.medicagogenome.org) was confirmed with the programs of FGENESH and Pfam 32.0, respectively. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze the relative expression levels of candidate genes. The genetic analysis indicated that the anti-clockwise coiling is dominant to clockwise and is controlled by the single gene, SPC. The SPC gene was delimited to a 250 kb-region on Chromosome 7. Total of 15 protein-coding genes were identified in the SPC locus through gene annotation and sequence analysis. Of those, two genes, potentially encoding a receptor-like kinase and a vacuolar cation/proton exchanger respectively, were selected as candidates for the SPC gene. The result presented here lay a foundation for gene cloning of SPC, which will help us to understand the molecular mechanisms underlying helical growth in plant organs.

A New SNP in Rice Gene Encoding Pyruvate Phosphate Dikinase (PPDK) Associated with Floury Endosperm.

Rice varieties with suitable flour-making qualities are required to promote the rice processed-food industry and to boost rice consumption. A rice mutation, Namil(SA)-flo1, produces grains with floury endosperm. Overall, grains with low grain hardness, low starch damage, and fine particle size are more suitable for use in flour processing grains with waxy, dull endosperm with normal grain hardness and a high amylose content. In this study, fine mapping found a C to T single nucleotide polymorphism (SNP) in exon 2 of the gene encoding cytosolic pyruvate phosphate dikinase (cyOsPPDK). The SNP resulted in a change of serine to phenylalanine acid at amino acid position 101. The gene was named FLOURY ENDOSPERM 4-5 (FLO4-5). Co-segregation analysis with the developed cleaved amplified polymorphic sequence (CAPS) markers revealed co-segregation between the floury phenotype and the flo4-5. This CAPS marker could be applied directly for marker-assisted selection. Real-time RT-PCR experiments revealed that PPDK was expressed at considerably higher levels in the flo4-5 mutant than in the wild type during the grain filling stage. Plastid ADP-glucose pyrophosphorylase small subunit (AGPS2a and AGPS2b) and soluble starch synthase (SSIIb and SSIIc) also exhibited enhanced expression in the flo4-5 mutant.

A Convenient Co-Dominant Marker for Height-Reducing Ddw1 Allele Useful for Marker-Assisted Selection

Reducing plant height improves lodging resistance and helps to obtain high grain yield under various environmental conditions. So far, the introduction and maintenance of the dwarfing allele of the Ddw1 gene is the most effective height-reducing genetic approach in rye and triticale breeding programs. However, the dominance of the dwarfing Ddw1 allele makes it difficult to select against heterozygous lines for further breeding based on plant phenotype. To assist breeders in the identification of the allele status of the Ddw1 gene, we developed a cleaved amplified polymorphic sequence (CAPS) marker that requires basic equipment and can be easily applied. The CAPS marker was tested on two F2 segregating populations of triticale, and the test showed the association of the Ddw1 genotype with plant height. The application of the marker for marker-assisted selection (MAS) for rye and triticale is discussed in detail.

Genome-wide association studies for inflorescence type and remontancy in Hydrangea macrophylla

Inflorescence type and remontancy are two valuable traits in bigleaf hydrangea (Hydrangea macrophylla L.) and both are recessively inherited. Molecular marker-assisted selection (MAS) can greatly reduce the time necessary to breed cultivars with desired traits. In this study, a genome-wide association study (GWAS) using 5803 single-nucleotide polymorphisms (SNPs) was performed using a panel of 82 bigleaf hydrangea cultivars. One SNP locus (Hy_CAPS_Inflo) associated with inflorescence type was identified with general linear model (GLM) and mixed linear model (MLM) methods that explained 65.5% and 36.1% of the phenotypic variations, respectively. Twenty-three SNPs associated with remontancy were detected in GLM whereas no SNP was detected in MLM. The SNP locus (Hy_CAPS_Inflo) was converted to a cleaved amplified polymorphic sequence (CAPS) marker that showed absolute identification accuracy (100%) of inflorescence type in a validation panel consisting of eighteen H. macrophylla cultivars. The SNP was investigated in 341 F1 progenies using genotyping by sequencing (GBS) and co-segregated with inflorescence type (χ2 = 0.12; P = 0.73). The SNP was subsequently used for breeding selection using kompetitive allele specific PCR (KASP) technology. Future directions for the use of genomics and MAS in hydrangea breeding improvement are discussed. The results presented in this study provide insights for further research on understanding genetic mechanisms behind inflorescence type and remontancy in H. macrophylla. The CAPS and KASP markers developed here will be immediately useful for applying MAS to accelerate breeding improvement in hydrangea.

Construction of a High-Density Genetic Map and Analysis of Seed-Related Traits Using Specific Length Amplified Fragment Sequencing for Cucurbita maxima.

Seed traits are agronomically important for Cucurbita breeding, but the genes controlling seed size, seed weight and seed number have not been mapped in Cucurbita maxima (C. maxima). In this study, 100 F2 individual derived from two parental lines, “2013-12” and “9-6”, were applied to construct a 3,376.87-cM genetic map containing 20 linkage groups (LGs) with an average genetic distance of 0.47 cM using a total of 8,406 specific length amplified fragment (SLAF) markers in C. maxima. Ten quantitative trait loci (QTLs) of seed width (SW), seed length (SL) and hundred-seed weight (HSW) were identified using the composite interval mapping (CIM) method. The QTLs affecting SW, SL and HSW explained a maximum of 38.6%, 28.9% and 17.2% of the phenotypic variation and were detected in LG6, LG6 and LG17, respectively. To validate these results, an additional 150 F2 individuals were used for QTL mapping of SW and SL with cleaved amplified polymorphic sequence (CAPS) markers. We found that two major QTLs, SL6-1 and SW6-1, could be detected in both SLAF-seq and CAPS markers in an overlapped region. Based on gene annotation and non-synonymous single-nucleotide polymorphisms (SNPs) in the major SWand SL-associated regions, we found that two genes encoding a VQ motif and an E3 ubiquitin-protein ligase may be candidate genes influencing SL, while an F-box and leucinerich repeat (LRR) domain-containing protein is the potential regulator for SW in C. maxima. This study provides the first high-density linkage map of C. maxima using SNPs developed by SLAF-seq technology, which is a powerful tool for associated mapping of important agronomic traits, map-based gene cloning and marker-assisted selection (MAS)-based breeding in C. maxima.

Development of molecular markers for Ty-2 and Ty-3 selection in tomato breeding

Tomato yellow leaf curl virus (TYLCV) is a disease causing serious yield reduction of tomato in the world. An effective control strategy for this virus is required for stable tomato production. Six resistant genes, Ty-1 to Ty-6 were previously identified in several wild species. In this study, we investigated novel sequence variations associated with Ty-2 and Ty-3 resistance to develop gene-based or functional markers suitable for improving TYLCV resistance in tomato breeding programs. Two resistant and susceptible inbred varieties were used for sequence analysis of each gene. For a Ty-2 candidate gene, we found a total of 39 single nucleotide polymorphisms (SNPs) and seven insertion/deletions (InDels). Of these, three SNPs were derived from coding sequences but were synonymous mutations. The SNPs and InDels from intron sequences were not responsible for alternative splicing in this gene. In addition, three large InDels of 50 bp, 42 bp, and 15 bp were found in upstream sequences, which are located at 688 bp, 611 bp, and 499 bp from 5’ UTR. Using the 50 bp and 42 bp InDels, a molecular marker was developed for Ty-2 selection. Nine non-synonymous SNPs and a 12 bp InDel were detected on coding sequences of the Ty-3 gene. Three of these (two SNPs and an InDel) were used to develop cleaved amplified polymorphic sequence (CAPS) markers. The resulting markers for Ty-2 and Ty-3 selection were validated using 18 commercial F1 cultivars, nine inbred lines, and three wild species with known phenotypes against TYLCV. Furthermore, these markers were used for genetic analysis of Ty-2 and Ty-3 resistance in a collection of 171 germplasm. The InDel and CAPS markers will be a useful resource to facilitate marker-assisted selection for pyramiding Ty-2 and Ty-3 in elite tomato varieties.

QTL mapping of pericarp and fruit-related traits in melon (Cucumis melo L.) using SNP-derived CAPS markers

As an important fruit vegetable crop, the quality of melon affects consumer preferences and the market value. Most quantitative trait loci (QTLs) related to fruit quality are controlled by polygenes and influenced by environmental factors. The objective of the present study was to identify QTLs for fruit quality traits in the F2 population derived from thick-skin line Elizabeth (M4-5) and thin-skin line M1-15 melons using single-nucleotide polymorphism (SNP)-derived cleaved amplified polymorphic sequence (CAPS) markers. We sequenced the melon lines M4-5 and M1-15 using next-generation sequencing (NGS) to identify CAPS loci across the assembled genome and constructed a linkage map with 195 CAPS markers to map the QTLs associated with fruit quality traits. Forty QTLs for fruit quality traits were identified with the new CAPS-based linkage map. Among these, 12 QTLs were associated with pericarp-related traits (exocarp type and thickness, pericarp thickness and firmness, and cracking) and 28 QTLs were associated with fruit-related traits (fruit length, width, and weight, and brix content). Our results suggest that the genetic components for fruit quality traits are complex and additive. The majority of QTLs for fruit quality traits were clustered around LG6 and LG9, among which two QTLs showed pleiotropic effects. The location of these QTLs with narrow genomic intervals could facilitate marker-assisted selection (MAS) application of the underlying genes in breeding programs. The findings of the present study provide the fundamentals for pyramiding the favored allele at multiple QTL locations into elite material using highly efficient molecular breeding tools.

Mikan Genome Database (MiGD): integrated database of genome annotation, genomic diversity, and CAPS marker information for mandarin molecular breeding.

Citrus species are some of the most valuable and widely consumed fruits globally. The genome sequences of representative citrus (e.g., Citrus clementina, C. sinensis, C. grandis) species have been released but the research base for mandarin molecular breeding is still poor. We assembled the genomes of Citrus unshiu and Poncirus trifoliata, two important species for citrus industry in Japan, using hybrid de novo assembly of Illumina and PacBio sequence data, and developed the Mikan Genome Database (MiGD). The assembled genome sizes of C. unshiu and P. trifoliata are 346 and 292 Mb, respectively, similar to those of citrus species in public databases; they are predicted to possess 41,489 and 34,333 protein-coding genes in their draft genome sequences, with 9,642 and 8,377 specific genes when compared to C. clementina, respectively. MiGD is an integrated database of genome annotation, genetic diversity, and Cleaved Amplified Polymorphic Sequence (CAPS) marker information, with these contents being mutually linked by genes. MiGD facilitates access to genome sequences of interest from previously reported linkage maps through CAPS markers and obtains polymorphism information through the multiple genome browser TASUKE. The genomic resources in MiGD (https://mikan.dna.affrc.go.jp) could provide valuable information for mandarin molecular breeding in Japan.