dCAPS Marker Research Articles

A novel SNP within the Rsa10025320 gene is highly associated with hollowness in red-skinned radish fleshy roots.

Hollowness is a physiological disorder that frequently occurs during the growth and postharvest storage phases of fleshy radish roots, significantly diminishing quality, yield, and marketability. However, the molecular mechanism for hollowness remains elusive. To identify the QTLs and potential candidate genes for hollowness tolerance in radish, F2 and BC1 populations were constructed from hollowness-tolerant radish (C16) and hollowness-sensitive radish (C17) in the present study. Genetic analysis indicated that hollowness tolerance may be governed by two independent recessive genes. By employing bulked segregant analysis sequencing (BSA-seq), two significant candidate genomic intervals were pinpointed on chromosomes R04 (960kb, 6.48-7.44Mb) and R05 (600kb, 31.44-32.04Mb), which together harbor 107 annotated genes. Transcriptomic sequencing revealed that the downregulated differentially expressed genes (DEGs) were significantly enriched in biological processes related to cell death and the response to water stress, whereas the upregulated DEGs were significantly associated with the chitin catabolic process and the cell wall macromolecule metabolic process. A total of 46 intersecting genes were identified among these DEGs within the genomic intervals of interest. One gene with high expression (Rsa10025345) and two with low expression (Rsa10025320 and Rsa10018106) were detected in the tolerant variety C16. Furthermore, a SNP within Rsa10025320 resulting in an amino acid change (A188E) was characterized through sequence variation observed in both BSA-seq and RNA-seq data and further developed as a derived cleaved amplified polymorphic sequence (dCAPS) marker. Our study reveals potential target genes for tolerance to hollowness and paves the way for marker-assisted breeding of hollowness tolerance in red-skinned radishes.

A novel mutation Trp-2027-Gly in acetyl-CoA carboxylase confers resistance to cyhalofop-butyl in Chinese sprangletop (Leptochloa chinensis).

Chinese sprangletop [Leptochloa chinensis (L.) Nees] control is threatened by resistance to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. In this study, a L. chinensis population, HFLJ18, that survived cyhalofop-butyl [aryloxyphenoxypropionate (APP) herbicide, CyB] treatment was collected from a rice field in Lujiang County, Anhui Province, China. This study aimed to evaluate the susceptibility of HFLJ18 to herbicides with different modes-of-action and investigate the potential mechanisms of resistance to CyB. The HFLJ18 population exhibited high levels of resistance to CyB (10.92-fold) and showed resistance to the ACCase inhibitors metamifop (4.63-fold) and fenoxaprop-P-ethyl (8.39-fold), but was susceptible to clethodim, pinoxaden, florpyrauxifen-benzyl, oxadiazon and pretilachlor. Target gene sequencing revealed a novel Trp-to-Gly substitution at codon position 2027 of ACCase in the resistant plants. Molecular docking revealed that the spatial structure of ACCase changed significantly following the substitution, as indicated by reduced H-bonds. A newly derived cleaved amplified polymorphic sequence (dCAPS) marker was subsequently developed to detect the Trp-2027-Gly mutation in the ACCase of L. chinensis. Additionally, pretreatment with the cytochrome P450 (P450) inhibitor piperonyl butoxide (PBO) and the glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) did not reverse resistance to CyB, suggesting that nontarget-site resistance mechanisms were not involved in CyB resistance in the HFLJ18 population. Overall, the resistance to CyB in the HFLJ18 population derived from the mutation of ACCase gene, and to the best of our knowledge, this is the first report of the ACCase Trp-2027-Gly mutation conferring resistance to ACCase-inhibiting herbicides in grass species. © 2024 Society of Chemical Industry.

Development and validation of a functional molecular marker for a CaAPRR2-like gene that controls green fruit colour in pepper

Fruit colour is a crucial factor influencing both the marketability and quality of pepper (Capsicum annuum), particularly ornamental varieties. Fruit colour is a complex multigenic trait in plant species. Previously, the Arabidopsis pseudo-response regulator 2 (APRR2) gene, one of the regulators that control fruit chlorophyll content and chloroplast development, thereby influencing fruit colour at the green immature stage in tomato (Solanum lycopersicum), cucumber (Cucumis sativus) and pepper, was reported. Functional molecular markers associated with this homologous gene in pepper could be employed to discern fruit colour at the seedling stage, thus improving the efficiency of green/white-fruited pepper breeding. In this study, a derived cleaved amplified polymorphic sequences (dCAPS) marker was developed based on the mutation site in the sequence of a CaAPRR2-like gene that was cloned from the inbred line B1-2, which exhibits milky white fruit at the green immature stage. The marker was subsequently validated in a CaAPRR2-like progeny segregation population. After digestion with the restriction endonuclease TaqI, the amplification product exhibited evident polymorphic bands, enabling it to distinguish between peppers with milky white and green fruit colours. The developed molecular marker displayed remarkable stability and repeatability, thus offering a straightforward and effective tool for enhancing fruit colour in pepper breeding programs.

The Dark Side of the pollen: BSA-seq identified genomic regions linked to male sterility in globe artichoke

Globe artichoke (Cynara cardunculus var. scolymus; 2n = 2x = 34) is a food crop consumed for its immature flower heads. Traditionally, globe artichoke varietal types are vegetatively propagated. However, seed propagation makes it possible to treat the crop as annual, increasing field uniformity and reducing farmers costs, as well as pathogens diffusion. Despite globe artichoke’s significant agricultural value and the critical role of heterosis in the development of superior varieties, the production of hybrids remains challenging without a reliable system for large-scale industrial seed production. Male sterility (MS) presents a promising avenue for overcoming these challenges by simplifying the hybridization process and enabling cost-effective seed production. However, within the Cynara genus, genic male sterility has been linked to three recessive loci in globe artichoke, with no definitive genetic mechanism elucidated to date. A 250 offsprings F2 population, derived from a cross between a MS globe artichoke and a male fertile (MF) cultivated cardoon (C. cardunculus var. altilis) and fitting a monogenic segregation model (3:1), was analyzed through BSA-seq, aiming at the identification of genomic regions/genes affecting male sterility. Four QTL regions were identified on chromosomes 4, 12, and 14. By analyzing the sequence around the highest pick on chromosome 14, a cytochrome P450 (CYP703A2) was identified, carrying a deleterious substitution (R/Q) fixed in the male sterile parent. A single dCAPS marker was developed around this SNP, allowing the discrimination between MS and MF genotypes within the population, suitable for applications in plant breeding programs. A 3D model of the protein was generated by homology modeling, revealing that the mutated amino acid is part of a highly conserved motif crucial for protein folding.

Whole-genome resequencing identifies candidate genes and allelic variation in the MdNADP-ME promoter that regulate fruit malate and fructose contents in apple

Soluble sugar and organic acids are key determinants of fruit organoleptic quality and directly affect the commodity value and economic returns of fruit crops. We performed whole-genome sequencing of the apple varieties Gala and Xiahongrou, along with their F1 hybrids, to construct a high-density bin map. Our quantitative genetic analysis pinpointed 53 quantitative trait loci (QTLs) related to 11 sugar and acid traits. We identified a candidate gene, MdNADP-ME, responsible for malate degradation, in a stable QTL on linkage group 15. Sequence analysis revealed an A/C SNP in the promoter region (MEp-799) that influences binding of the MdMYB2 transcription factor, thereby affecting MdNADP-ME expression. In our study of various apple genotypes, this SNP has been demonstrated to be linked to malate and fructose levels. We also developed a dCAPS marker associated with fruit fructose content. These results substantiate the role of MdNADP-ME in maintaining the equilibrium between sugar and acid contents in apple fruits.

Genetic Insights into the Extremely Dwarf Hibiscus syriacus var. micranthus: Complete Chloroplast Genome Analysis and Development of a Novel dCAPS Marker.

This study explored the chloroplast (cp) genomes of three Hibiscus syriacus (HS) specimens endemic to Korea possessing unique ornamental and conservation values: the dwarf H. syriacus var. micranthus (HSVM), renowned for its small stature and breeding potential; HS 'Tamra', a cultivar from Korea's southernmost islands, noteworthy for its distinctive beauty; and HS Natural Monument no. 521 (N.M.521), a specimen of significant lifespan and height. Given the scarcity of evolutionary studies on these specimens, we assembled and analyzed their cp genomes. We successfully assembled genomes spanning 160,000 to 160,100 bp and identified intraspecific variants. Among these, a unique ATA 3-mer insertion in the trnL-UAA region was identified in HSVM, highlighting its value as a genetic resource. Leveraging this finding, we developed a novel InDel dCAPS marker, which was validated across 43 cultivars, enhancing our ability to distinguish HSVM and its derivatives from other HS cultivars. Phylogenetic analysis involving 23 Malvaceae species revealed that HSVM forms a clade with woody Hibiscus species, closely associating with N.M.520, which may suggest a shared ancestry or parallel evolutionary paths. This investigation advances our understanding of the genetic diversity in Korean HS and offers robust tools for accurate cultivar identification, aiding conservation and breeding efforts.

Genetic diversity of grain yield traits and identification of a grain weight gene SiTGW6 in foxtail millet.

Agronomic traits were evaluated in 1250 foxtail millet accessions, and a crucial gene SiTGW6 governing grain yield was identified. Elite haplotypes and dCAPS markers developed for SiTGW6 facilitate molecular breeding. A comprehensive evaluation of phenotypic characteristics and genetic diversity in germplasm resources are important for gene discovery and breeding improvements. In this study, we conducted a comprehensive evaluation of 1250 foxtail millet varieties, assessing seven grain yield-related traits and fourteen common agronomic traits over two years. Principal component analysis, correlation analysis, and cluster analysis revealed a strong positive correlation between 1000-grain weight and grain width with grain yield, emphasizing their importance in foxtail millet breeding. Additionally, we found that panicle weight positively correlated with 1000-grain weight but negatively correlated with branch and tiller numbers, indicating selection factors during domestication and breeding. Using this information, we identified 27 germplasm resources suitable for high-yield foxtail millet breeding. Furthermore, through an integration of haplotype variations and phenotype association analysis, we pinpointed a crucial gene, SiTGW6, responsible for governing grain yield in foxtail millet. SiTGW6 encodes an IAA-glucose hydrolase, primarily localized in the cytoplasm and predominantly expressed in flowering panicles. Employing RNAseq analysis, we identified 1439 differentially expressed genes across various SiTGW6 haplotypes. Functional enrichment analysis indicating that SiTGW6 regulates grain yield through the orchestration of auxin and glucan metabolism, as well as plant hormone signaling pathways. Additionally, we have identified elite haplotypes and developed dCAPS markers for SiTGW6, providing valuable technical tools to facilitate molecular breeding efforts in foxtail millet.

A Novel Allele in the Promoter of Wx Decreases Gene Expression and Confers Lower Apparent Amylose Contents in Japonica Rice (Oryza sativa L.).

Wx is the key gene that controls amylose content (AC), and various alleles have been found in rice populations. Wxb is the major allele in japonica and produces moderate AC (15~18%). It was recently found that editing the promoter of Wx could produce a series of alleles that have different Wx activities. Although some studies have edited the promoter, few studies have focused on the natural variations in Wx. Here, we used the Rice3K database to investigate variations in the Wx promoter and found that the allele Wx1764178 (A/G) has a higher LD (linkage disequilibrium) with the two key SNPs (1765751, T/G; 1768006, A/C), which could produce different Wx alleles and influence AC, as reported previously. Further study showed that the Wx1764178 allele (A/G) is functional and influences the expression of Wx positively. Editing the A allele using CRISPR‒Cas9 produced 36 and 3 bp deletions and caused a decrease in the expression of Wx. The apparent amylose content (AAC) in the edited lines was decreased by 7.09% and 11.50% compared with that of the wild type, which was the japonica variety Nipponbare with Wxb and the A allele at 1764178, while a complementary line with the G allele showed a lower AAC than the A allele with no effect on other agronomic traits. The AAC of the edited lines showed a higher increase than that of the wild type (Nipponbare, Wxb) in low-nitrogen conditions relative to high-nitrogen conditions. We also developed a dCAPS marker to identify the allele and found that the G allele has widely been used (82.95%) in japonica-bred varieties from Jiangsu Province, China. Overall, we found a functional allele (Wx1764178, A/G) in the Wx promoter that could affect AAC in japonica cultivars and be developed as markers for quality improvement in rice breeding programs.

Fine mapping of ClLOX, a QTL for powdery mildew resistance in watermelon (Citrullus lanatus L.).

ClLOX, is located on chromosome 2 and encodes a lipoxygenase gene, which induced watermelon powdery mildew resistance by inhibiting pathogen spread. Powdery mildew is one of the most severe fungal diseases reducing yield and quality of watermelon (Citrullus lanatus L.) and other cucurbit crops. Genes responsible for powdery mildew resistance in watermelon are highly valuable. In this study, we first identified the QTL pm-lox for powdery mildew resistance in watermelon, located within a 0.93Mb interval of chromosome 2, via XP-GWAS method using two F2 populations. The F2:3 families from one of the F2 populations were then used for fine-mapping the pm-lox locus into a 9,883bp physical region between 29,581,906 and 29,591,789, containing only two annotated genes. Of these, only ClG42_02g0161300 showed a significant differential expression between the resistant and susceptible lines after powdery mildew inoculation based on RNA sequencing (RNA-seq) and qRT-PCR analysis, and is designated ClLOX. Derived Cleaved Amplified Polymorphic Sequence (dCAPs) markers were developed and validated. In addition, our tests showed that the resistance was anti-spread rather than anti-infection of the pathogen. This study identified a new resistance gene (ClLOX), provided insights into the mechanism of powdery mildew resistance, and developed a molecular marker for watermelon breeding.

Fine mapping of leaf delayed virescence gene dv4 in Triticum aestivum

Wheat (Triticum aestivum L.) is one of the most important crops worldwide, and its yield affects national food security. Wheat leaves are key photosynthetic organs where carbohydrates are synthesized for grain yield. Leaf colour mutants are ideal germplasm resources for molecular genetic studies of wheat chloroplast development, chlorophyll synthesis and photosynthesis. We obtained a wheat mutant delayed virescence 4 (dv4) from cultivar Guomai 301. The leaves of mutant dv4 were pale yellow at the seedling stage, golden yellow at the turning green stage, and they started to turn green at the jointing stage. Genetic analysis demonstrated that the yellow-leaf phenotype was controlled by a single recessive gene named as dv4. Gene dv4 was fine mapped in a 1.46 Mb region on chromosome 7DS by SSR and dCAPS marker assays. Three putative candidate genes were identified in this region. Because no leaf colour genes have been reported on wheat chromosome arm 7DS previously, dv4 is a novel leaf colour gene. The result facilitates map-based cloning of dv4 and provides information for the construction of a high-photosynthetic efficiency ideotype for improving wheat yield.

Determination of the Allelic Composition of the sdw1/denso (HvGA20ox2), uzu1 (HvBRI1) and ari-e (HvDep1) Genes in Spring Barley Accessions from the VIR Collection

The lodging of barley significantly limits its potential yield, leads to the deterioration of grain quality, and complicates mechanized harvesting. More than 30 dwarfness and semi-dwarfness genes and loci are known for barley, and their involvement in breeding can solve the problem of lodging. The most common dwarfing alleles are of the genes sdw1/denso (HvGA20ox2), uzu1 (HvBRI1), and ari-e (HvDep1). The aim of this study was the design of dCAPS markers for the sdw1.c and ari-e.GP alleles and the molecular screening of barley accessions from the VIR collection for identifying these and other dwarfing alleles commonly used in breeding. Two dCAPS markers have been developed to identify the sdw1.c allele of the HvGA20ox2 gene and ari-e.GP of HvDep1. These dCAPS markers and two known from the literature CAPS and dCAPS markers of the alleles sdw1.a/sdw1.e, sdw1.c, sdw1.d, and uzu1.a were used in the molecular screening of 32 height-contrasting barley accessions. This made it possible to identify the accessions with alleles sdw1.a/sdw1.e, sdw1.c, and sdw1.d of the HvGA20ox2 gene, as well as accessions with a combination of sdw1.c and uzu1.a alleles of the genes HvGA20ox2 and HvBRI1. A comparison of the results of genotyping and phenotyping showed that the presence of dwarfing alleles in all genotypes determines high or medium lodging resistance regardless of the influence of weather conditions. Twelve accessions were found to contain the new allele sdw1.ins of the HvGA20ox2 gene, which differs from the known allele sdw1.c by a larger size of PCR products. It is characterized by the Thalos_2 transposon insertion; the subsequent GTTA insertion, common with the sdw1.c allele; and by a single-nucleotide G→A substitution at the 165 position.

THB1, a putative transmembrane protein that causes hybrid breakdown in rice.

Hybrid breakdown is a post-zygotic reproductive isolation that hinders genetic exchange between species or populations in both animals and plants. Two complementary recessive genes, temperature sensitive hybrid breakdown1 (thb1) and thb2, cause hybrid breakdown in rice (Oryza sativa). The present study delimited the THB1 locus to a 9.1-kb sequence, containing a single gene encoding a putative transmembrane protein with unknown functions. Haplotype analysis of THB1 in the two core collections of 119 accessions revealed that these accessions were divided into 22 haplotypes. A test cross with thb2 carrier showed that haplotype2 (H2) was assigned to thb1 and was restricted to temperate japonica. A nonsynonymous nucleotide polymorphism (SNP) specific to H2 was identified as a causal mutation in thb1. A test cross with thb1 carrier indicated that six accessions, including temperate japonica, tropical japonica, and indica, carried thb2. These results suggest that thb1 has recently evolved in temperate japonica, whereas thb2 arose in an ancient japonica and introgressed into the present three subgroups. Furthermore, we developed a derived cleaved amplified polymorphic sequence (dCAPS) marker to detect causal SNP in THB1. Our findings provide new insights into reproductive isolation and may benefit rice breeding.

Flavonoid Biosynthesis Pathway May Indirectly Affect Outcrossing Rate of Cytoplasmic Male-Sterile Lines of Soybean.

(1) Background: Cytoplasmic male sterility (CMS) is important for exploiting heterosis. Soybean (Glycine max L.) has a low outcrossing rate that is detrimental for breeding sterile lines and producing hybrid seeds. Therefore, the molecular mechanism controlling the outcrossing rate should be elucidated to increase the outcrossing rate of soybean CMS lines; (2) Methods: The male-sterile soybean lines JLCMS313A (with a high outcrossing rate; HL) and JLCMS226A (with a low outcrossing rate; LL) were used for a combined analysis of the transcriptome (RNA-seq) and the targeted phenol metabolome; (3) Results: The comparison between HL and LL detected 5946 differentially expressed genes (DEGs) and 81 phenolic metabolites. The analysis of the DEGs and differentially abundant phenolic metabolites identified only one common KEGG pathway related to flavonoid biosynthesis. The qRT-PCR expression for eight DEGs was almost consistent with the transcriptome data. The comparison of the cloned coding sequence (CDS) regions of the SUS, FLS, UGT, and F3H genes between HL and LL revealed seven single nucleotide polymorphisms (SNPs) only in the F3H CDS. Moreover, five significant differentially abundant phenolic metabolites between HL and LL were associated with flavonoid metabolic pathways. Finally, on the basis of the SNPs in the F3H CDS, one derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to distinguish between HL and LL soybean lines; (4) Conclusions: The flavonoid biosynthesis pathway may indirectly affect the outcrossing rate of CMS sterile lines in soybean.

A Frameshift Mutation in the Mg-Chelatase I Subunit Gene OsCHLI Is Associated with a Lethal Chlorophyll-Deficient, Yellow Seedling Phenotype in Rice.

Chlorophyll biosynthesis is a crucial biological process in plants, and chlorophyll content is one of the most important traits in rice breeding programs. In this study, we identified a lethal, chlorophyll-deficient, yellow seedling (YS) phenotype segregating in progeny of CR5055-21, an F2 plant derived from a backcross between Korean japonica variety 'Hwaseong' (Oryza sativa) and CR5029, which is mostly Hwaseong with a small amount of Oryza grandiglumis chromosome segments. The segregation of the mutant phenotype was consistent with a single gene recessive mutation. Light microscopy of YS leaf cross-sections revealed loosely arranged mesophyll cells and sparse parenchyma in contrast to wildtype. In addition, transmission electron microscopy showed that chloroplasts did not develop in the mesophyll cells of the YS mutant. Quantitative trait loci (QTL)-seq analysis did not detect any significant QTL, however, examination of the individual delta-SNP index identified a 2-bp deletion (AG) in the OsCHLI gene, a magnesium (Mg)-chelatase subunit. A dCAPs marker was designed and genotyping of a segregating population (n = 275) showed that the mutant phenotype co-segregated with the marker. The 2-bp deletion was predicted to result in a frameshift mutation generating a premature termination. The truncated protein likely affects formation and function of Mg-chelatase, which consists of three different subunits that together catalyze the first committed step of chlorophyll biosynthesis. Transcriptome analysis showed that photosynthesis and carbohydrate metabolism pathways were significantly altered although expression of OsCHLI was not. Chlorophyll- and carotenoid-related genes were also differentially expressed in the YS mutant. Our findings demonstrated that OsCHLI plays an important role in leaf pigment biosynthesis and leaf structure development in rice.

Homolog of Pea SGR Controls Stay-Green in Faba Bean (Vicia faba L.).

Faba bean is an important legume crop consumed as a vegetable or snack food, and its green cotyledons could present an attractive color for consumers. A mutation in SGR causes stay-green in plants. In this study, vfsgr was identified from a green-cotyledon-mutant faba bean, SNB7, by homologous blast between the SGR of pea and the transcriptome of faba bean. Sequence analysis revealed that a SNP at position 513 of the CDS of VfSGR caused a pre-stop codon, resulting in a shorter protein in the green-cotyledon faba bean SNB7. A dCaps marker was developed according to the SNP that caused the pre-stop, and this marker was completely associated with the color of the cotyledon of faba bean. SNB7 stayed green during dark treatment, while the expression level of VfSGR increased during dark-induced senescence in the yellow-cotyledon faba bean HST. Transient expression of VfSGR in Nicotiana. benthamiana leaves resulted in chlorophyll degradation. These results indicate that vfsgr is the gene responsible for the stay-green of faba bean, and the dCaps marker developed in this study provides a molecular tool for the breeding of green-cotyledon faba beans.

Genome-wide association study of leaf-related traits in tea plant in Guizhou based on genotyping-by-sequencing

BackgroundStudying the genetic characteristics of tea plant (Camellia spp.) leaf traits is essential for improving yield and quality through breeding and selection. Guizhou Plateau, an important part of the original center of tea plants, has rich genetic resources. However, few studies have explored the associations between tea plant leaf traits and single nucleotide polymorphism (SNP) markers in Guizhou.ResultsIn this study, we used the genotyping-by-sequencing (GBS) method to identify 100,829 SNP markers from 338 accessions of tea germplasm in Guizhou Plateau, a region with rich genetic resources. We assessed population structure based on high-quality SNPs, constructed phylogenetic relationships, and performed genome-wide association studies (GWASs). Four inferred pure groups (G-I, G-II, G-III, and G-IV) and one inferred admixture group (G-V), were identified by a population structure analysis, and verified by principal component analyses and phylogenetic analyses. Through GWAS, we identified six candidate genes associated with four leaf traits, including mature leaf size, texture, color and shape. Specifically, two candidate genes, located on chromosomes 1 and 9, were significantly associated with mature leaf size, while two genes, located on chromosomes 8 and 11, were significantly associated with mature leaf texture. Additionally, two candidate genes, located on chromosomes 1 and 2 were identified as being associated with mature leaf color and mature leaf shape, respectively. We verified the expression level of two candidate genes was verified using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and designed a derived cleaved amplified polymorphism (dCAPS) marker that co-segregated with mature leaf size, which could be used for marker-assisted selection (MAS) breeding in Camellia sinensis.ConclusionsIn the present study, by using GWAS approaches with the 338 tea accessions population in Guizhou, we revealed a list of SNPs markers and candidate genes that were significantly associated with four leaf traits. This work provides theoretical and practical basis for the genetic breeding of related traits in tea plant leaves.

Development of short tandem repeat (STR) and derived cleaved amplified polymorphic (dCAPS) markers for distinguishing species and varieties of the genus Panax in Vietnam

In this study, we developed a protocol for the authentication of Panax vietnamensis var. vietnamensis (Ngoc Linh ginseng) by combining two molecular markers: short tandem repeat (STR) and derived cleaved amplified polymorphic sequences (dCAPS). STR markers: Pvm30 and Pvm31 were found in the chloroplast genome of P. vietnamensis var. vietnamensis. These markers were able to accurately identify Panax stipuleanatus, Panax vietnamensis var. fuscidiscus, and Panax ginseng. Panax vietnamensis var. vietnamensis and Panax vietnamensis var. langbianensis had a high similarity of chloroplast genomic sequence (99.96%) leading to STR markers could not distinguish these two ginseng varieties. Therefore, dCAPS marker: PvmdCAPS was applied to compensate for the defect of the STR markers. From the alignment result of the matK coding sequences of these two varieties, PvmdCAPS primers were designed at the position of single nucleotide polymorphisms (SNP) at the 248th nucleotide and had the ability to discriminate between these two Panax varieties. In summary, the combination of STR and dCAPS was used to distinct Panax species in Vietnam, especially P. vietnamensis var. vietnamensis.

Variations in Grain Traits among Local Rice Varieties Collected More Than Half-Century Ago in Indochinese Countries

More than half-century ago, local rice varieties were collected from Indochinese countries (Cambodia, Thailand, Laos, and Vietnam). Of these, 162 local varieties were examined for 7 grain-size traits: seed length/width/thickness, brown rice length/width/thickness, and 100-seed weight. Since these traits varied considerably, a survey of functional mutations was performed in the genes related to these traits. In total, 19 markers (12 InDel and 7 dCAPS markers) were used to investigate the mutations at 14 grain-size loci of GW2, GS2, qLGY3, GS3, GL3.1, TGW3, GS5, GW5, GS6, TGW6, GW6a, GLW7, GL7, and GW8. Significant allele effects were observed with six markers detecting base substitution mutations at GW2 and GS3 and insertion/deletion mutations at GS5, GW5, and GW6a, suggesting that these mutations might have affected the grain trait and caused variation among local varieties in the Indochinese countries. In addition to grain size, the hull color, grain color, and glutinosity were also examined using a survey of loss-of-function mutations at major responsible loci. Most phenotypes were reflected based on functional mutations at these loci. Since the local varieties have wide genetic variation, they are important genetic resources for future rice breeding.

Fine mapping and identification of candidate genes for fruit color in pepper (Capsicum chinense)

The quality of pepper fruits is determined by numerous characteristics such as, flavor, color, and nutritional value. Through little is known about it, fruit color impacts breeding preferences in Capsicum chinense in a substantial way. In the present study, two C. chinense germplasms, HNUCC16 (dark green immature and yellow ripe fruit) and HNUCC22 (light green immature and red ripe fruit) were used to produce an F2 population. Based on genotyping-by-sequencing, we constructed a high-density genetic map of the F2 population with 1,347 bin markers and covered 1,206.37 cM in twelve pepper linkage groups. The trait segregation analysis of the F2 population show that the color of immature and mature fruits is controlled by a single locu . Bin markers 19 (chromosome 1) and 849 (Chromosome 6) were closely associated with the locus that regulates the color of immature and ripe fruits, respectively. The transformation of the two bin markers into dCAPS markers indicate that inactive shikimate kinase-like 2 (BC332_00870) may be a strong candidate gene for regulating immature fruit color, and the premature termination mutation of capsanthin–capsorubin synthase (BC332_17237) gene may possibly cause the yellow color in HNUCC16 pepper fruits. Overall, the findings of this study offer fundamental knowledge concerning the development of color and offer a new resource for pepper fruit color genetic improvement and molecular breeding.

Association study of SNP locus for color related traits in herbaceous peony (Paeonia lactiflora Pall.) using SLAF-seq.

Paeonia lactiflora Pall. (P. lactiflora) is a famous ornamental plant with showy and colorful flowers that has been domesticated in China for 4,000 years. However, the genetic basis of phenotypic variation and genealogical relationships in P. lactiflora population is poorly understood due to limited genetic information, which brings about bottlenecks in the application of effective and efficient breeding strategies. Understanding the genetic basis of color-related traits is essential for improving flower color by marker-assisted selection (MAS). In this study, a high throughput sequencing of 99 diploid P. lactiflora accessions via specific-locus amplified fragment sequencing (SLAF-seq) technology was performed. In total, 4,383,645 SLAF tags were developed from 99 P. lactiflora accessions with an average sequencing depth of 20.81 for each SLAF tag. A total of 2,954,574 single nucleotide polymorphisms (SNPs) were identified from all SLAF tags. The population structure and phylogenetic analysis showed that P. lactiflora population used in this study could be divided into six divergent groups. Through association study using Mixed linear model (MLM), we further identified 40 SNPs that were significantly positively associated with petal color. Moreover, a derived cleaved amplified polymorphism (dCAPS) marker that was designed based on the SLAF tag 270512F co-segregated with flower colors in P. lactiflora population. Taken together, our results provide valuable insights into the application of MAS in P. lactiflora breeding programs.