Cleaved Amplified Polymorphic Sequence Research Articles

A nonsynonymous mutation in an acetolactate synthase gene (Gh_D10G1253) is required for tolerance to imidazolinone herbicides in cotton

BackgroundHerbicide tolerance in crops enables them to survive when lethal doses of herbicides are applied to surrounding weeds. Herbicide-tolerant crops can be developed through transgenic approaches or traditional mutagenesis approaches. At present, no transgenic herbicide tolerant cotton have been commercialized in China due to the genetically-modified organism (GMO) regulation law. We aim to develop a non-transgenic herbicide-tolerant cotton through ethyl methanesulfonate (EMS) mutagenesis, offering an alternative choice for weed management.ResultsSeeds of an elite cotton cultivar Lumianyan 37 (Lu37) were treated with EMS, and a mutant Lu37-1 showed strong tolerance to imidazolinone (IMI) herbicides was identified. A novel nonsynonymous substitution mutation Ser642Asn at acetolactate synthase (ALS) (Gh_D10G1253) in Lu37-1 mutant line was found to be the potential cause to the IMI herbicides tolerance in cotton. The Ser642Asn mutation in ALS did not present among the genomes of natural Gossypium species. Cleaved amplified polymorphic sequence (CAPS) markers were developed to identify the ALS mutant allele. The Arabidopsis overexpressing the mutanted ALS also showed high tolerance to IMI herbicides.ConclusionThe nonsynonymous substitution mutation Ser642Asn of the ALS gene Gh_D10G1253 is a novel identified mutation in cotton. This substitution mutation has also been identified in the orthologous ALS genes in other crops. This mutant ALS allele can be used to develop IMI herbicide-tolerant crops via a non-transgenic or transgenic approach.Graphical abstract

Fine-Mapping Analysis of the Genes Associated with Pre-Harvest Sprouting Tolerance in Rice (Oryza sativa L.)

Pre-harvest sprouting (PHS) of rice (Oryza sativa L.) causes severe economic problems due to reduced grain quality and yield. Fine mapping was carried out to identify genes associated with PHS; the detected quantitative trait locus (QTL) was narrowed down to 50 Kbp using F3:4 populations, four polymorphic insertion and deletion (InDel) markers, and two cleaved amplified polymorphic sequence (CAPS) markers. In one region, five candidate genes were detected, and the SNP and InDel in each gene (Os01g0111400 and Os01g0111600) were confirmed to show the differences and resulting amino acid changes between parent plants. Based on haplotype, expression, and co-segregation analysis, the InDel in Os01g0111600 was confirmed to be associated with the PHS trait. The results of this study could be applied to improve the PHS tolerance of Japonica rice varieties, and they also improved our understanding of the genetic basis underlying PHS tolerance.

Molecular Markers and Their Applications in Marker-Assisted Selection (MAS) in Bread Wheat (Triticum aestivum L.)

As one of the essential cereal crops, wheat provides 20% of the calories and proteins consumed by humans. Due to population expansion, dietary shift and climate change, it is challenging for wheat breeders to develop new varieties for meeting wheat production requirements. Marker-assisted selection (MAS) has distinct advantages over conventional selection in plant breeding, such as being time-saving, cost-effective and goal-oriented. This review makes attempts to give a description of different molecular markers: sequence tagged site (STS), simple sequence repeat (SSR), genotyping by sequencing (GBS), single nucleotide polymorphism (SNP) arrays, exome capture, Kompetitive Allele Specific PCR (KASP), cleaved amplified polymorphic sequence (CAPS), semi-thermal asymmetric reverse PCR (STARP) and genotyping by target sequencing (GBTS). We also summarize some quantitative trait loci (QTL)/genes as well as their linked markers, which are potentially useful in MAS. This paper provides updated information on some markers linked to critical traits and their potential applications in wheat breeding programs.

A LuALS Mutation with High Sulfonylurea Herbicide Resistance in Linum usitatissimum L.

The cultivation of herbicide-resistant crops is an effective tool for weed management in agriculture. Weed control in flax (Linum usitatissimum L.) remains challenging due to the lack of available herbicide-resistant cultivars. In this study, a mutant resistant to acetolactate synthase (ALS)-inhibiting herbicides was obtained by ethyl methanesulphonate (EMS) mutagenesis using an elite cultivar, Longya10. Whole-plant dose-response assays revealed that, compared to Longya10, the mutant was 11.57-fold more resistant to tribenuron-methyl (TBM) and slightly resistant to imazethapyr (resistance index (mutant/Longya10) < 3). In vitro acetolactate synthase assays showed that the relative resistance of the mutant was 12.63 times more than that of Longya10. A biochemical analysis indicated that there was a Pro197Ser (relative to the Arabidopsis thaliana ALS sequence) substitution within the LuALS1, conferring high resistance to sulfonylurea herbicides in the mutant. Additionally, two cleaved amplified polymorphic sequence (CAPS) markers, BsaI-LuALS1 and EcoO109I-LuALS1, were developed based on the mutation site for marker assistant selection in breeding. Moreover, the mutant did not cause losses in natural field conditions. We find a mutant with ALS-inhibiting herbicide resistance chemically induced by EMS mutagenesis, providing a valuable germplasm for breeding herbicide-resistant flax varieties.

Primary mapping of quantitative trait loci regulating multivariate horticultural phenotypes of watermelon (Citrullus lanatus L.).

Watermelon fruits exhibit a remarkable diversity of important horticultural phenotypes. In this study, we initiated a primary quantitative trait loci (QTL) mapping to identify the candidate regions controlling the ovary, fruit, and seed phenotypes. Whole genome sequencing (WGS) was carried out for two differentiated watermelon lines, and 350 Mb (96%) and 354 Mb (97%) of re-sequenced reads covered the reference de novo genome assembly, individually. A total of 45.53% non-synonymous single nucleotide polymorphism (nsSNPs) and 54.47% synonymous SNPs (sSNPs) were spotted, which produced 210 sets of novel SNP-based cleaved amplified polymorphism sequence (CAPS) markers by depicting 46.25% co-dominant polymorphism among parent lines and offspring. A biparental F2:3 mapping population comprised of 100 families was used for trait phenotyping and CAPS genotyping, respectively. The constructed genetic map spanned a total of 2,398.40 centimorgans (cM) in length and averaged 11.42 cM, with 95.99% genome collinearity. A total of 33 QTLs were identified at different genetic positions across the eight chromosomes of watermelon (Chr-01, Chr-02, Chr-04, Chr-05, Chr-06, Chr-07, Chr-10, and Chr-11); among them, eight QTLs of the ovary, sixteen QTLs of the fruit, and nine QTLs of the seed related phenotypes were classified with 5.32-25.99% phenotypic variance explained (PVE). However, twenty-four QTLs were identified as major-effect and nine QTLs were mapped as minor-effect QTLs across the flanking regions of CAPS markers. Some QTLs were exhibited as tightly localized across the nearby genetic regions and explained the pleiotropic effects of multigenic nature. The flanking QTL markers also depicted significant allele specific contributions and accountable genes were predicted for respective traits. Gene Ontology (GO) functional enrichment was categorized in molecular function (MF), cellular components (CC), and biological process (BP); however, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were classified into three main classes of metabolism, genetic information processing, and brite hierarchies. The principal component analysis (PCA) of multivariate phenotypes widely demonstrated the major variability, consistent with the identified QTL regions. In short, we assumed that our identified QTL regions provide valuable genetic insights regarding the watermelon phenotypes and fine genetic mapping could be used to confirm them.

Embryo rescue of inter-specific hybrid of Solanum lycopersicum × S. neorickii

The tomato wild accession LA2325 (Solanum neorickkii) shows strong resistance to early blight (EB) caused by Alternaria solani. To study genetics of the resistance and to transfer the resistance to cultivated tomato, crosses were made between accessionLA2325 and highly susceptible genotype Hawaii 3998. The crosses yielded no viable seeds. It is well known that fertilization barriers like incompatibility and embryo abortion come in the way of development of inter-specific hybrid. This hampers introgression of resistance to cultivated tomato. In the subsequent season, pollen from LA2325was used to pollinate the susceptible cultivar and pro-embryos were collected from developing fruits 39-49 days after pollination. Rescue of immature embryos was done on Murashige and Skoog medium (MS-medium). Pro-embryos from fruits of 42 - 44 days after pollination gave good response. A total of ten plants were regenerated. Developed seedlings were hardened and shifted to green house. The hybridity of the rescued plants was confirmed by comparing the morphological characters of the interspecific hybrid and parents. Further, cleaved amplified polymorphic sequences (CAPS) marker was also used for verifying the hybridity. The embryo rescued inter-specific hybrid is being used as a resource for generating different genetic populations to study the genetics of the EB resistance and to transfer the resistance to cultivated tomato.

Development and Complex Application of Methods for the Identification of Mutations in the FAD3A and FAD3B Genes Resulting in the Reduced Content of Linolenic Acid in Flax Oil.

Flax is grown worldwide for seed and fiber production. Linseed varieties differ in their oil composition and are used in pharmaceutical, food, feed, and industrial production. The field of application primarily depends on the content of linolenic (LIN) and linoleic (LIO) fatty acids. Inactivating mutations in the FAD3A and FAD3B genes lead to a decrease in the LIN content and an increase in the LIO content. For the identification of the three most common low-LIN mutations in flax varieties (G-to-A in exon 1 of FAD3A substituting tryptophan with a stop codon, C-to-T in exon 5 of FAD3A leading to arginine to a stop codon substitution, and C-to-T in exon 2 of FAD3B resulting in histidine to tyrosine substitution), three approaches were proposed: (1) targeted deep sequencing, (2) high resolution melting (HRM) analysis, (3) cleaved amplified polymorphic sequences (CAPS) markers. They were tested on more than a thousand flax samples of various types and showed promising results. The proposed approaches can be used in marker-assisted selection to choose parent pairs for crosses, separate heterogeneous varieties into biotypes, and select genotypes with desired homozygous alleles of the FAD3A and FAD3B genes at the early stages of breeding for the effective development of varieties with a particular LIN and LIO content, as well as in basic studies of the molecular mechanisms of fatty acid synthesis in flax seeds to select genotypes adequate to the tasks.

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.

QTL Analysis and CAPS Marker Development Linked with Russet in Pear (Pyrus spp.).

The fruit skin types of pear (Pyrus spp.) are divided into russet, smooth, and intermediate. One of the important traits in pear breeding programs is russet on pear fruit skin because it affects the commercial value. In the present study, a high-density genetic linkage map of ‘Whangkeumbae’ (smooth) × ‘Minibae’ (russet) was constructed. In addition, quantitative trait loci (QTL) analysis was performed to identify russet related QTL and develop a cleaved amplified polymorphism sequence (CAPS) marker. Together with SNPs derived from Axiom Pear 70K Genotyping Array and genotyping-by-sequencing derived SNPs and SSRs generated in previous study, an integrated genetic linkage map of ‘Whangkeumbae’ × ‘Minibae’ was constructed. A total of 1263 markers were anchored in 17 linkage groups (LGs) with a total genetic distance of 1894.02 cM and an average marker density of 1.48 cM. The chromosome coverage of ‘Whangkeumbae’ × ‘Minibae’ map was improved because the SNPs derived from Axiom Pear 70K Genotyping Array were anchored. QTL analysis was performed using previous russet phenotype data evaluated with russet coverage and Hunter a. As a result of QTL analysis, russet coverage- and Hunter a-related QTLs were identified in LG8 of the ‘Whangkeumbae’ × ‘Minibae’ map, and SNPs located in the QTL region were heterozygous in the ‘Minibae’. Although the russet coverage- and Hunter a-related QTLs were commonly detected in LG8, the logarithm of odds values of SNPs in the QTL region were higher in QTL related to russet coverage than to Hunter a. The CAPS marker (CBp08ca01) was developed using an array SNP located in the russet coverage related QTL, and the genotype of CBp08ca01 showed a 1:1 ratio in ‘Whangkeumbae’ × ‘Minibae’ (χ2 = 0.65, p > 0.05). ‘Whangkeumbae’ and ‘Minibae’ were thought to have rr and Rr genotypes, respectively, and the genetic factors controlling the russet formation might be located in chromosome 8. The CBp08ca01 was able to select F1 individuals with less than 30% russet coverage. Thus, it will be a useful tool for marker-assisted selection in pears.

Genome-wide association study and genetic mapping of BhWAX conferring mature fruit cuticular wax in wax gourd

BackgroundWax gourd [Benincasa hispida (Thunb) Cogn. (2n = 2x = 24)] is an economically important vegetable crop of genus Benincasa in the Cucurbitaceae family. Fruit is the main consumption organ of wax gourd. The mature fruit cuticular wax (MFCW) is an important trait in breeding programs, which is also of evolutionary significance in wax gourd. However, the genetic architecture of this valuable trait remains unrevealed.ResultsIn this study, genetic analysis revealed that the inheritance of MFCW was controlled by a single gene, with MFCW dominant over non-MFCW, and the gene was primarily named as BhWAX. Genome-wide association study (GWAS) highlighted a 1.1 Mb interval on chromosome 9 associated with MFCW in wax gourd germplasm resources. Traditional fine genetic mapping delimited BhWAX to a 0.5 Mb region containing 12 genes. Based on the gene annotation, expression analysis and co-segregation analysis, Bhi09G001428 that encodes a membrane bound O-acyltransferase (MBOAT) was proposed as the candidate gene for BhWAX. Moreover, it was demonstrated that the efficiency of a cleaved amplified polymorphic sequences (CAPS) marker in the determination of MFCW in wax gourd reached 80%.ConclusionsIn closing, the study identified the candidate gene controlling MFCW and provided an efficient molecular marker for the trait in wax gourd for the first time, which will not only be beneficial for functional validation of the gene and marker-assisted breeding of wax gourd, but also lay a foundation for analysis of its evolutionary meaning among cucurbits.

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.

Branding and tracing seaweed: Development of a high-resolution genetic kit to identify the geographic provenance of Alaria esculenta

Global seaweed production increased rapidly in the past 50 years, mostly in Asia. Recently seaweed aquaculture has been attracting more attention worldwide because of its potential as a form of sustainable agriculture that provides food, feed, fertiliser, bio-stimulants and other valuable products. Quality characteristics of seaweeds are species-dependent, influenced by the genetic makeup of the individual, and by the environmental conditions where the seaweeds are grown. Those two factors largely depend on their place of origin. Traceability in terms of geographic provenance would therefore add commercial value to seaweed products . Here we describe the development of a method to identify/certify the place of origin of the blown alga Alaria esculenta . We identified Single Nucleotide Polymorphisms (SNPs) on the nuclear, plastid and mitochondrial genomes by analysing whole-genome sequences of individuals originated from Ireland, Northern Ireland, Faroe Islands, Greenland, Norway and Atlantic Canada. Multi-dimensional scaling (MDS) plots of the nuclear SNPs, but not of the organellar SNPs, showed a clear separation of individual samples by the country of origin. We then identified country-specific nuclear SNPs and designed a set of cleaved amplified polymorphic sequence (CAPS) assays to detect two or three of specific SNPs per country. The SNPs were tested on individuals, including those whose genome sequence had not been determined, and were shown to successfully identify individual's country of origin. This method allows accurate determination of the geographic provenance of Alaria esculenta . This tool could form the base for branding seaweed products with certified origin, and for the development of mass identification kits using metabarcoding . It also has the potential to be used to monitor seaweed farms for invasion of non-native varieties of a seaweed species, and, more broadly, to monitor effects of climate change on the diversity of seaweed populations. • Nuclear SNPs distinguish the geographical location of Alaria esculenta strains. • Organellar SNPs failed to distinguish geographical location of the strains. • Country-specific nuclear SNPs allowed developing CAPS assays. • The method can be deployed for both ecological and industrial applications.

A preliminary mapping of QTL qsg5.1 controlling seed germination in melon (Cucumis melo L.).

Melon (Cucumis melo L.) seed germination significantly affects its economic value. Cultivation of melon varieties with high germination ability and seedling vigor is beneficial in large-scale melon propagation. In this study, two melon genotypes differing in their germination ability, P5 with low and P10 with high germination ability, were used to identify the optimal seed germination conditions by evaluating different water immersion times and germination temperatures. The germination rate of the P5 and P10 parental genotypes and their segregating population, consisting of 358 F2:3 families, were evaluated for 2 years to identify their genetic basis. QTL analysis was performed on a high-density genetic map constructed using specific-locus amplified fragment sequencing (SLAF-seq). The germination rate of F1 and F2 populations treated with water immersion for 8 h at 28°C and measured at 48 h showed a normal distribution Genetic mapping carried out using the high-density genetic map revealed eight QTLs in chromosomes 2, 4, 5, 6, and 8 that control melon seed germination, of which 2020/2021-qsg5.1 was consistently significant in both years of experimentation. qsg5.1 explained 15.13% of the phenotypic variance with a LOD of 4.1. To fine map the candidate region of qsg5.1, eight cleaved amplified polymorphism sequence (CAPS) markers were used to construct a genetic map with another 421 F2 individual fruits. The major QTL qsg5.1 was located between SNP53 and SNP54 within a 55.96 Kb interval containing four genes. qRT-PCR gene expression analysis of the candidate genes showed that MELO3C031219.2 (Phosphorus transporter PHO-5) exhibited a significant difference in gene expression between the parental lines at 24, 32, and 48 h after germination, potentially being the underlying gene controlling melon seed germination. These results provide a theoretical basis for the molecular mechanisms controlling melon seed germination and can practically contribute to further improving germination to increase the propagation efficiency of commercial melon cultivars.

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.

Occurrence of Bensulfuron-Methyl Resistance and Target-Site Resistance Mechanisms in Ammannia auriculata Biotypes from Paddy Fields.

Ammanniaauriculata is a troublesome broadleaf weed, widely distributed in the paddy fields of southern China. In this study, 10 biotypes of A. auriculata were sampled from Yangzhou City, China, where the paddy fields were seriously infested with A. auriculata, and their resistance levels to acetolactate synthase (ALS) inhibitor bensulfuron-methyl were determined. The whole-plant response assays showed that nine A. auriculata biotypes were highly resistant (from 16.4- to 183.1-fold) to bensulfuron-methyl in comparison with a susceptible YZ-S biotype, and only one YZ-6 biotype was susceptible. ALS gene sequencing revealed that three ALS gene copies existed in A. auriculata, and four different amino acid substitutions (Pro197-Leu, -Ala, -Ser, and -His) at site 197 in the AaALS1 or 2 genes were found in eight resistant biotypes. In addition, no amino acid mutations in three ALS genes were found in the YZ-3 biotype. These results suggested that target-site mutations or non-target-site resistance mechanisms were involved in tested resistant A. auriculata biotypes. Finally, a cleaved amplified polymorphic sequence (CAPS) marker was identified to rapidly detect the Pro197 mutations in A. auriculata.

BoCAPS: Rapid screening of chemical races in Botryococcus braunii with direct PCR-CAPS

The colony-forming microalga Botryococcus braunii produces petroleum-like hydrocarbons and is a promising source for biofuel production. B. braunii is classified into three chemical races (A, B, L) plus a tentative class (S), based on the structure of its hydrocarbons. The determination of hydrocarbon structure in a newly isolated strain requires several months for culturing and analysis. As potential for biofuel production differs among races, a rapid method for estimating race is needed for the effective bioprospecting. This study aimed to develop a DNA-based rapid estimation method (BoCAPS) of the chemical races of B. braunii and to test the feasibility of direct PCR from a single colony. A total of 247 wild strains were isolated from 29 ponds in Japan and Indonesia, their 18S rRNA sequences determined, and a phylogenetic tree constructed based on the sequences of standard strains with known chemical races. Polymorphisms between clades of the phylogenetic tree corresponding to chemical races were mapped, and a cleaved amplified polymorphic sequence (CAPS) marker was developed for race discrimination. Direct PCR was tested on a single colony as a template. We demonstrated that HaeIII restriction of the PCR product enables race discrimination on agarose gels, and that direct PCR amplifies the 18S rRNA gene from a single colony without any pre-treatment. With BoCAPS, we investigated race compositions of the isolated strains and determined that the B-race was dominant (74 %). Neither the A-race nor S-race strains were isolated from tropical regions. In addition, we observed a few ponds in which minor races (L or A) were dominant and some ponds with a mixture of different races. The BoCAPS allows us to estimate chemical race without the time-consuming and expensive analysis of hydrocarbons and will be useful for high-throughput screening and ecological studies of wild populations.

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.

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.

Cleaved Amplified Polymorphic Sequences (CAPS) Markers for Characterization of the LuFAD3A Gene from Various Flax (Linum usitatissimum L.) Cultivars

Depending on the variety, flax (Linum usitatissimum L.) provides an oil rich in omega-3 acids (especially 50% α-linolenic acid) with proven health properties, and is used as a raw industrial material. Alpha-linolenic acid is a polyunsaturated fatty acid easily subject to oxidative transformation. The auto-oxidation of α-linolenic acid is the main process contributing to off-flavor, color loss, and change in the nutritional value of flax oil. We used six flax genotypes differing in fatty acid content in our research. For all the DNA samples extracted from the leaf tissue of the studied flax, the new, cleaved amplified polymorphic sequences (CAPS) markers specific to the LuFAD3A desaturase gene were applied. A specific PCR product from the LuFAD3A flax gene comprising fragments of exon 5, exon 6, and the intron between these exons was digested using six various restriction enzymes. These experiments could differentiate between some of the studied varieties of flax. We also proved that the LuFAD3A gene mutation previously detected by other authors in the low-linolenic forms of flax (solin line 593–708) was absent in the low-linolenic forms of flax that we studied (Linola KLA and Linola KLB).