dCAPS Marker Research Articles

Genetic identification of the pleiotropic gene Tasg-D1/2 affecting wheat grain shape by regulating brassinolide metabolism

Improving yield potential is a major goal of wheat breeding that depends on identifying key genetic loci. In this study, two residual heterozygous line RHL351- and RHL78-derived populations were employed for genetic linkage map construction and QTL detection. Two genetic populations indicated a robust grain-size QTL between Marker6 and Marker10. It covered a 95.54–99.38 Mb physical interval and was named Qpleio.nwafu.3D, containing the candidate gene Tasg (TraesCS3D02G137200). Intriguingly, RNA-seq analysis and sequencing revealed two different allelic variants in Tasg, named Tasg-D1 (G>A) and Tasg-D2 (C>G), respectively. Although the relationship between Tasg-D1 and grain size had been demonstrated previously, here we provided the first genetic evidence that C/G allelic variation in Tasg-D2 was associated with grain shape and size through a newly developed dCAPS marker. In addition, transcriptome comparison indicated that Tasg-D1/2 might primarily contribute to significant expression differences in brassinolide (BR) metabolism-related genes rather than those related to BR responses in developing grains and spikes. Our study provided new evidence and a breeder-friendly dCAPS marker for improving grain size through the selection of Tasg, as well as a basis to understand Tasg function in the future.

Comparative Analysis of Chloroplast Genome in Saccharum spp. and Related Members of 'Saccharum Complex'.

High ploids of the sugarcane nuclear genome limit its genomic studies, whereas its chloroplast genome is small and conserved, which is suitable for phylogenetic studies and molecular marker development. Here, we applied whole genome sequencing technology to sequence and assemble chloroplast genomes of eight species of the ‘Saccharum Complex’, and elucidated their sequence variations. In total, 19 accessions were sequenced, and 23 chloroplast genomes were assembled, including 6 species of Saccharum (among them, S. robustum, S. sinense, and S. barberi firstly reported in this study) and 2 sugarcane relative species, Tripidium arundinaceum and Narenga porphyrocoma. The plastid phylogenetic signal demonstrated that S. officinarum and S. robustum shared a common ancestor, and that the cytoplasmic origins of S. sinense and S. barberi were much more ancient than the S. offcinarum/S. robustum linage. Overall, 14 markers were developed, including 9 InDel markers for distinguishing Saccharum from its relative species, 4 dCAPS markers for distinguishing S. officinarum from S. robustum, and 1 dCAPS marker for distinguishing S. sinense and S. barberi from other species. The results obtained from our studies will contribute to the understanding of the classification and plastome evolution of Saccharinae, and the molecular markers developed have demonstrated their highly discriminatory power in Saccharum and relative species.

Development and validation of gene-specific dCAPS markers for a temperature-sensitive male sterile gene TMS5 in rice

Development and validation of gene-specific dCAPS markers for a temperature-sensitive male sterile gene TMS5 in rice

Identification of a Candidate restorer-of-fertility Gene Rf3 Encoding a Pentatricopeptide Repeat Protein for the Cytoplasmic Male Sterility in Soybean.

The cytoplasmic male sterility/restorer-of-fertility (CMS/Rf) system plays a vital role in high-efficiency hybrid seed production in crops, including soybean (Glycine max (L.) Merr.). The markers linked to fertility restoration and the restorer-of-fertility (Rf) genes are essential because they can facilitate the breeding of new CMS lines and production of commercial hybrid soybean seeds. To date, several soybean Rf genes have been mapped to various genetic loci in diverse genetic populations. However, the mapping range of restorer genes remains narrow, with relatively limited practical applicability. Therefore, in the present study, F2 and F3 segregating populations derived from the CMS line JLCMS5A crossed with the restorer line JLR2 were developed and used for Rf3 gene fine mapping. Genetic investigation indicated that the restorer line JLR2 was controlled by a single dominant gene, Rf3. By integrating bulk-segregant analysis and next-generation sequencing, a 4 Mb region on chromosome 9 was identified, which was most likely the target region harboring the candidate gene responsible for fertility restoration. This region was further narrowed down to 86.44 Kb via fine mapping in F2 and F3 populations using SSR, InDel, and dCAPS markers. This region contained 10 putative genes (Glyma.09G171100–Glyma.09G172000). Finally, Glyma.09G171200, which encodes a mitochondria-targeted pentatricopeptide repeat protein, was proposed as the potential candidate for Rf3 using sequence alignment and expression analysis in restorer and CMS lines. Based on single-nucleotide polymorphisms in Glyma.09G171200, a CAPS marker co-segregated with Rf3 named CAPS1712 was developed. Our results will be fundamental in the assisted selection and creation of potent lines for the production and rapid selection of novel restorer lines.

Characterization of TaSPP-5A gene associated with sucrose content in wheat (Triticum aestivum L.)

BackgroundSucrose, the major product of photosynthesis and the primary sugar transported as a soluble carbohydrate via the phloem, is a critical determinant for harvest yield in wheat crops. Sucrose-phosphatase (SPP) catalyzes the final step in the sucrose biosynthesis pathway, implying its essential role in the plant.ResultIn this study, wheat SPP homologs genes were isolated from chromosomes 5A, 5B, and 5D, designated as TaSPP-5A, TaSPP-5B, and TaSPP-5D, respectively. Sequence alignment showed one 1-bp Insertion-deletion (InDel) and three single nucleotide polymorphisms (SNPs) at TaSPP-5A coding region, forming two haplotypes, TaSPP-5Aa and TaSPP-5Ab, respectively. A derived cleaved amplified polymorphism sequence (dCAPS) marker, TaSPP-5A-dCAPS, was developed to discriminate allelic variation based on the polymorphism at position 1242 (C-T). A total of 158 varieties were used to perform a TaSPP-5A marker-trait association analysis, where two haplotypes were significantly associated with sucrose content in two environments and with thousand-grain weight (TGW) and grain length (GL) in three environments. Quantitative real-time PCR further revealed that TaSPP-5Aa showed relatively higher expression than TaSPP-5Ab in wheat seedling leaves, generally associating with increased sucrose content and TGW. The expression of TaSPP-5A and sucrose content in TaSPP-5Aa haplotypes were also higher than those in TaSPP-5Ab haplotypes under both 20% PEG-6000 and 100 μM ABA treatment. Sequence alignment showed that the two TaSPP-5A haplotypes comprised 11 SNPs from -395 to -1962 bp at TaSPP-5A promoter locus, participating in the formation of several conserved sequences, may account for the high expression of TaSPP-5A in TaSPP-5Aa haplotypes. In addition, the distribution analysis of TaSPP-5A haplotypes revealed that TaSPP-5Aa was preferred in the natural wheat population, being strongly positively selected in breeding programs.ConclusionAccording to the SNPs detected in the TaSPP-5A sequence, two haplotypes, TaSPP-5Aa and TaSPP-5Ab, were identified among wheat accessions, which potential value for sucrose content selection was validated by association analysis. Our results indicate that the favorable allelic variation TaSPP-5Aa should be valuable in enhancing grain yield by improving the sucrose content. Furthermore, a functional marker, TaSPP-5A-dCAPS, can be used for marker-assisted selection to improve grain weight in wheat and provides insights into the biological function of TaSPP-5A gene.

De Novo Transcriptome Assembly and SNP Discovery for the Development of dCAPS Markers in Oat

Cultivated oat (Avena sativa L.) is an important cereal crop that has captured interest worldwide due to its nutritional properties and associated health benefits. Despite this interest, oat has lagged behind other cereal crops in genome studies and the development of DNA markers due to its large and complex genome. RNA-Seq technology has been widely used for transcriptome analysis, functional gene study, and DNA marker development. In this study, we performed the transcriptome sequencing of 10 oat varieties at the seedling stage using the Illumina platform for the development of DNA markers. In total, 31,187,392~41,304,176 trimmed reads (an average of 34,322,925) were generated from 10 oat varieties. All of the trimmed reads of these varieties were assembled and generated, yielding a total of 128,244 assembled unigenes with an average length of 1071.7 bp and N50 of 1752 bp. According to gene ontology (GO) analysis, 30.7% of unigenes were assigned to the “catalytic activity” of the parent term in the molecular function category. Of the 1273 dCAPS markers developed using 491 genotype-specific SNPs, 30 markers exhibiting polymorphism in 28 oat varieties were finally selected. The transcriptome data of oat varieties could be used for functional studies about the seedling stage of oat and information about sequence variations in DNA marker development. These 30 dCAPS markers will be utilized for oat genetic analysis, cultivar identification, and breeders’ rights protection.

SNP-based identification of QTL for resistance to black point caused by Bipolaris sorokiniana in bread wheat

Black point disease caused by Bipolaris sorokiniana is a problem in wheat production worldwide. We aimed to identify major quantitative trait loci (QTL) for resistance to black point and develop molecular markers for marker-assisted selection (MAS). A recombinant inbred line (RIL) population derived from a cross between Wanyuanbai 1 (susceptible) and SN4143 (resistant) was evaluated for black point response at three locations during two years under artificial inoculation with B. sorokiniana, providing data for six environments. Thirty resistant and 30 susceptible RILs were selected to form resistant and susceptible bulks, respectively, that were genotyped by the wheat 660K SNP array; 685 single-nucleotide polymorphisms (SNPs) were identified, among which 385 (56.2%) and 115 (16.8%) were located on chromosomes 4A and 2B, respectively. Bulked segregant RNA-Seq analysis identified candidate regions on chromosomes 4A (4.60–40.28 Mb) and 5A (1.22–48.47 Mb). Genetic linkage maps were constructed for chromosomes 2B, 4A, and 5A using 59 polymorphic dCAPS and SSR markers. Finally, two QTL, designated QBB.hau-4A and QBB.hau-5A, were detected on chromosomes 4A and 5A, respectively. The resistance allele of QBB.hau-4A was derived from SN4143, and that of QBB.hau-5A came from Wanyuanbai 1. QBB.hau-4A with a large and consistent effect (15.1%) is likely to be a new locus for black point resistance. The markers linked to QBB.hau-4A and QBB.hau-5A have potential application in MAS-based breeding.

Fine-mapping and identification of a candidate gene controlling seed coat color in melon (Cucumis melo L. var. chinensis Pangalo).

MELO3C019554 encoding a homeobox protein (PHD transcription factor) is a candidate gene that involved in the formation of seed coat color in melon. Seed coat color is related to flavonoid content which is closely related to seed dormancy. According to the genetic analysis of a six-generation population derived from two parents (IC2508 with a yellow seed coat and IC2518 with a brown seed coat), we discovered that the yellow seed coat trait in melon is controlled by a single dominant gene, named CmBS-1. Bulked segregant analysis sequencing (BSA-Seq) revealed that the gene is located at 11,860,000-15,890,000bp (4.03Mb) on Chr 6. The F2 population was genotyped using insertion-deletions (InDels), from which cleaved amplified polymorphic sequence (dCAPS) markers were derived to construct a genetic map. The gene was then fine-mapped to a 233.98kb region containing 12 genes. Based on gene sequence analysis with two parents, we found that the MELO3C019554 gene encoding a homeobox protein (PHD transcription factor) had a nonsynonymous single nucleotide polymorphism (SNP) mutation in the coding sequence (CDS), and the SNP mutation resulted in the conversion of an amino acid (A → T) at residue 534. In addition, MELO3C019554 exhibited lower relative expression levels in the yellow seed coat than in the brown seed coat. Furthermore, we found that MELO3C019554 is related to 12 flavonoid metabolites. Thus, we predicted that MELO3C019554 is a candidate gene controlling seed coat color in melon. The study lays a foundation for further cloning projects and functional analysis of this gene, as well as marker-assisted selection breeding.

Fine mapping and identification of the candidate gene BFS for fruit shape in wax gourd (Benincasa hispida).

Non-synonymous mutations in the BFS gene, which encodes the IQD protein, are responsible for the shape of wax gourd fruits. Fruit shape is an important agronomic trait in wax gourds. Therefore, in this study, we employed bulked segregant analysis (BSA) to identify a candidate gene for fruit shape in wax gourds within F2 populations derived by crossing GX-71 (long cylindrical fruit, fruit shape index = 4.56) and MY-1 (round fruit, fruit shape index = 1.06) genotypes. According to BSA, the candidate gene is located in the 17.18Mb region on chromosome 2. Meanwhile, kompetitive allele-specific PCR (KASP) markers were used to reduce it to a 19.6Kb region. Only one gene was present within the corresponding region of the reference genome, namely Bch02G016830 (designated BFS). Subsequently, BFS was sequenced in six wax gourd varieties with different fruit shapes. Sequence analysis revealed two non-synonymous mutations in the round wax gourd and one non-synonymous mutation in the cylindrical wax gourd. Quantitative real‑time PCR (qRT-PCR) analysis further showed that the expression of BFS in round fruits was significantly higher than in long cylindrical fruits at the ovary formation stage. Therefore, BFS is a candidate gene for determination wax gourd shape. The predicted protein encoded by the BFS gene belongs to the IQ67-domain protein family, which have the structural characteristics of scaffold proteins and coordinate Ca2+ CaM signaling from the membrane to the nucleus. Ultimately, two derived cleaved amplified polymorphic sequence (dCAPS) markers were developed to facilitate marker-assisted selection for wax gourds breeding.

A novel LOX3-null allele (lox3-b) originated in the aromatic Basmati rice cultivars imparts storage stability to rice bran

Rapid deterioration of rice bran due to the LOX3 enzyme catalysed oxidation of PUFA is the major bottleneck for its utilization in various downstream applications. In the present study, we have identified a set of nine novel LOX3-null rice accessions carrying a deletion of C residue in the exon2 causing a frameshift mutation resulting in a truncated non-functional LOX3 protein. Our study, further manifested the predominance of C deletion based LOX3-null allele, named lox3-b, in the aromatic rice germplasm particularly in the Indian Basmati rice group. The LOX3-null genotypes exhibited significantly reduced rancidity, after six months of storage. They also showed significantly lower percentage reduction of linoleic acid (LA), higher γ-oryzanol content and lower hexanal content. A functional dCAPS marker designed based on the deletion polymorphism clearly differentiated LOX3 and lox3-b alleles, and has the potential application in marker assisted rice breeding programmes to develop cultivars with better bran storability.

Development and utilization of the functional co-dominant KASP marker for thermo-sensitive genic male sterility in rice Oryza sativa L.

Thermo-sensitive genic male sterility (TGMS) is an important genetic means of two-line hybrid rice breeding. Pollen fertility in TGMS lines is regulated by a single point mutation in TGMS genes. Based on single nucleotide polymorphisms (SNPs) and insertion-deletion mutations, Kompetitive Allele-Specific PCR (KASP) markers were developed and utilized in rice molecular breeding via high-throughput detection, which saves time and money. In this study, we converted the SNPs on TGMS genes (including p/tms12-1and tms9-1) to functional co-dominant KASP markers and bred the resultant two-line hybrid rice. We differentiated the TGMS lines carrying p/tms12-1 or tms9-1 from other TGMS lines using the KASP assay. Pei’ai64S and Hua201S (containing p/tms12-1) had a homozygous GG genotype generating a blue signal. HengnongS-1 (containing tms9-1) had a homozygous CC genotype generating a red signal. The KASP assay for tms9-1 was identified as a recessive Mendelian trait. Seed purity was tested using the KASP marker for the two-line hybrid varieties of Liangyoupeijiu and Hualiangyou1206, which was consistent with findings by the dCAPS marker. Moreover, new TGMS lines were generated by pyramiding tms12-1 and tms9-1 gene under the same genetic background. Therefore, the KASP marker to detect the TGMS genes developed in this study can be widely used in two-line hybrid rice breeding. It provides a visually convenient toolkit for breeders to select individual target plants using high-throughput screening in two-line rice breeding.

Elite sd1 alleles in japonica rice and their breeding applications in northeast China

The Green Revolution gene sd1 has been used extensively in modern rice breeding, especially in indica cultivars. However, elite sd1 alleles and related germplasm resources used for japonica rice breeding have not been identified, and extensive efforts are needed for japonica rice breeding to obtain new dwarfing sources. Data from MBKbase-Rice revealed seven sd1 haplotypes in indica and four in japonica rice. Two new sd1 alleles were identified in indica rice. In 295 japonica accessions from northeast Asia, except for the weak functional allele SD1-EQ, sd1-r was the major allele, reducing plant height in comparison with SD1-EQ. Japonica germplasm resources carrying reported sd1 alleles were identified by genotype searching and further verified by literature search, genealogical analysis, and dCaps markers. Pedigrees and geographic distribution showed that sd1-r is an excellent allele widely used in northern China and Tohoku in Japan, and sd1-j is commonly used in east China and Kyushu in Japan. Dongnong- and Xiushui-series cultivars carrying sd1-r and sd1-j, respectively, are essential branches of the backbone parents of Chinese japonica rice, Akihikari and Ce21, with the largest number of descendants and derived generations. In semi-dwarf japonica rice breeding, sd1-d was introgressed into Daohuaxiang 2 (DHX2). Dwarf and semi-dwarf lines carrying sd1-d were selected and designated as 1279 and 1280, respectively, after withstanding typhoon-induced strong winds and heavy rains in 2020, and are anticipated to become useful intermediate materials for future genetic research and breeding. This work will facilitate the introduction, parental selection, and marker-assisted breeding, and provide a material basis for the next step in identifying favorable genes that selected together with the sd1 alleles in japonica backbone parents.

Molecular Mapping of a Novel Quantitative Trait Locus Conferring Adult Plant Resistance to Stripe Rust in Chinese Wheat Landrace Guangtoumai.

Stripe rust (yellow rust), caused by Puccinia striiformis f. sp. tritici, is one of the most destructive diseases of wheat worldwide. Chinese wheat landrace Guangtoumai (GTM) exhibited a high level of resistance against predominant P. striiformis f. sp. tritici races in China at the adult plant stage. The objective of this research was to identify and map the major locus/loci for stripe rust resistance in GTM. A set of 212 recombinant inbred lines (RILs) was developed from a cross between GTM and Avocet S. The parents and RILs were evaluated in three field tests (2018, 2019, and 2020 at Chongzhou, Sichuan) with the currently predominant P. striiformis f. sp. tritici races for final disease severity and genotyped with the Wheat 55K single nucleotide polymorphism (SNP) array to construct a genetic map with 1,031 SNP markers. A major locus, named QYr.GTM-5DL, was detected on chromosome 5DL in GTM. The locus was mapped in a 2.75-cM interval flanked by SNP markers AX-109855976 and AX-109453419, explaining up to 44.4% of the total phenotypic variation. Since no known Yr genes have been reported on chromosome 5DL, QYr.GTM-5DL is very likely a novel adult plant resistance locus. Haplotype analysis revealed that the resistance allele displayed enhanced levels of stripe rust resistance and is likely present in 5.3% of the 247 surveyed Chinese wheat landraces. The derived cleaved amplified polymorphic sequence (dCAPS) marker dCAPS-5722, converted from a SNP marker tightly linked to QYr.GTM-5DL with 0.3 cM, was validated on a subset of RILs and 48 commercial wheat cultivars developed in Sichuan. The results indicated that QYr.GTM-5DL with its linked dCAPS marker could be used in marker-assisted selection to improve stripe rust resistance in breeding programs, and this quantitative trait locus will provide new and possibly durable resistance to stripe rust.

A loss-of-function of the dirigent gene TaDIR-B1 improves resistance to Fusarium crown rot in wheat.

A loss-of-function of the dirigent gene TaDIR-B1 improves resistance to Fusarium crown rot in wheat.

A PCR plus restriction enzyme-based technique for detecting target-enzyme mutations at position Pro-106 in glyphosate-resistant Lolium perenne.

The first step in managing herbicide-resistant weeds is to confirm their resistance status. It is, therefore, crucial to have a rapid, reliable and cost-effective technique to assess samples for herbicide resistance. We designed and evaluated three derived cleaved amplified polymorphic sequence (dCAPS) markers for detecting glyphosate resistance in Lolium perenne. conferred by non-synonymous mutations at codon-106 in the enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. The dCAPS markers involve amplification of the target region, digestion of the amplified products with restriction enzymes and gel-based visualisation of the digested products. The results showed that all three dCAPS markers could successfully detect mutations at codon-106 in the target enzyme. The dCAPS markers can also inform us of the zygosity state of the resistance allele and was confirmed by sequencing the target region of the EPSPS gene. The markers described here are effective quick tests for the monitoring and evaluation of the target-enzyme mechanism of glyphosate resistance in Lolium perenne.

Non-Destructive Genotyping of Honeybee Queens to Support Selection and Breeding

Simple SummaryGenotyping of an individual usually requires tissue samples obtained by methods that may cause an injury. In the case of honeybee queens, most of such injuries cause supersedure of the queen. We have investigated the possibility of using two non-destructive sources to obtain genetic information from an individual queen: feces and exuviae. Both sources turned out to be acceptable in terms of quality of DNA and usefulness in genotyping. In practical use, any of the two could be used to shorten generation interval in queen breeding and decrease costs of the selection process. In traditional bee breeding, the honeybee queen is chosen for breeding based on the performance of the colony produced by its mother. However, we cannot be entirely certain that a specific queen will produce offspring with desirable traits until we observe the young queen’s new colony. Collecting the queen’s genetic material enables quick and reliable determination of the relevant information. We sampled exuviae, feces, and wingtips for DNA extraction to avoid fatally injuring the queen when using tissue samples. Quantity and purity of extracted DNA were measured. Two mitochondrial markers were used to determine the lineage affiliation and exclude possible contamination of DNA extracts with non-honeybee DNA. dCAPS (derived Cleaved Amplified Polymorphic Sequences) markers allowed detection of single nucleotide polymorphisms (SNPs) in nuclear DNA regions presumably associated with Varroa sensitive hygiene and set the example of successful development of genotyping protocol from non-destructive DNA sources. One of the logical future steps in honeybee breeding is introducing genomic selection and non-destructive sampling methods of genetic material may be the prerequisite for successful genotyping. Our results demonstrate that the extraction of DNA from feces and exuviae can be introduced into practice. The advantage of these two sources over wingtips is reducing the time window for processing the samples, thus enabling genotyping directly after the queen’s emergence.

Host reaction of tomato varieties and applicability of Sm-linked DNA markers to Stemphylium lycopersici

Gray leaf spot (GLS) disease, caused by the Stemphylium (Sm) fungus, affects a wide range of horticultural crops including tomato. In this study, the South Korean Sm isolate (PNU-SM01) was identified, and the applicability of Sm-resistance gene (Sm)-linked DNA markers in marker-assisted selection was validated. ITS/gpd sequencing and morphological observation identified the isolate as S. lycopersici. A GLS disease assay was conducted on 80 tomato varieties, including commercial F1 hybrids and elite inbred lines. The reference line ‘Motelle’ was highly resistant (R, DSI = 4.0), whereas the ‘Moneymaker’ line was susceptible (S) and showed severe disease symptoms (DSI = 79.3). Among 30 commercial F1 hybrids, five were scored as S by DSI; whereas the rest, including all 17 hybrids claimed as Sm-resistant by the supplier, were R or moderately resistant (MR). Among 48 inbred lines, four were scored as S and 44, including five Lycopersicon accessions reported as R, were R or MR. Three known Sm-linked DNA markers (CT55, D5, and Sm-InDel) were genotyped in all 80 cultivars; the highest match (92.5%) to the GLS disease assay was observed for Sm-InDel. We converted the recessive marker CT55 into a codominant marker (CT55-Co) and developed dCAPS (Sm-SNP) and KASP (Sm-KASP) markers from a previously identified Sm-linked SNP. Sm-SNP and Sm-KASP showed a similar level of phenotype to marker genotype association as Sm-InDel, whereas CT55-Co displayed a high level of mismatch. To screen for the source of Sm resistance, 138 tomato accessions were genotyped with Sm-InDel and Sm-SNP: 15 were homozygous for the resistance allele of both markers. In this study, resistance to the Korean isolate of S. lycopersici in commercial F1 hybrids and breeding sources was characterized to reveal which DNA markers can be effectively applied for marker-assisted selection of Sm-resistance.

Favorable allele mining and breeding utilization of ALK in rice

ALK is the key gene of controlling rice gelatinization temperature (GT), and at least three ALK alleles are thought to be responsible for the diversity of GT among rice cultivars. But whether the sequence polymorphism of the promoter region also associated with GT is not clearly studied. In this study, we analyzed sequence variation in the promoter region of ALK and mainly two types of promoter were identified. ALK type I promoter prevails among thousands of indica and japonica rice varieties, while ALK type II promoter predominates over varieties from Aus/boro and Basmati ecotypes. Phenotypic analysis and quality characterization of CSSLs with different alleles of ALK and promoter activity assays revealed that type II promoter downregulates the expression level of ALK and subsequently, negatively affects ASV values. ALKQ1882, owning type II promoter region and TT SNP at 4329/4330 bp of ALK gene, was identified as a rare and elite ALK allele for rice quality improvement. Transcriptional detection in hybrid triploid endosperm revealed that ALK is simultaneously expressed by gene copes from both parents, the ASV of hybrid rice varieties can be estimated through the genotypes of two parents. More importantly, a new dCAPS marker was developed to discriminate the functional SNP of TT at 4329/4330 bp, which is an efficient tool for improving the grain quality with high accuracy on low testing cost.

Development of SNP-based high-density genetic map and gene mapping of pod colour trait in Cowpea (Vigna unguiculata L. Walp.)

ABSTRACT The colour of fresh pods is one of the most important traits that affects the commercial value of cowpea (Vigna unguiculata) beans. To investigate the genetic architecture, we firstly scored the fresh pod colour of cowpea for six generations in field trials. Afterwards, an F2 population of 150 recombinant individuals was derived from crosses between two high-generation inbred cowpea lines, ‘Baojia’ (green pod colour, P1) and ‘Qilong’ (purple pod colour, P2). Ahigh-density genetic map based on 9,493 single nucleotide polymorphisms was constructed through SLAF sequencing and used for gene/s mapping of pod colour trait. We observed 59 single nucleotide polymorphism markers in one candidate gene region that may be associated with fresh pod colour trait located on LG6. The locus of pc was confirmed on chromosome 5 of the cowpea genome, which was corresponded to the genes encoding anthocyanin 5-aromatic acyltransferase and MYB-90 and MYB-114 domain proteins, that play important roles in the anthocyanin biosynthesis. Lastly, dCAPS markers were developed that explained the high rate of identification for the phenotypes of the F2 population. This study provides new knowledge of the genomic regions and will accelerate the genetic improvements, regarding the fresh pod colour (pc) trait in cowpea and marker-assisted selection.

Discovery of a Novel Induced Polymorphism in SD1 Gene Governing Semi-Dwarfism in Rice and Development of a Functional Marker for Marker-Assisted Selection

The semi-dwarfing allele, sd1-d, has been widely utilized in developing high-yielding rice cultivars across the world. Originally identified from the rice cultivar Dee-Geo-Woo-Gen (DGWG), sd1-d, derived from a spontaneous mutation, has a 383-bp deletion in the SD1 gene. To date, as many as seven alleles of the SD1 gene have been identified and used in rice improvement, either with a functional single-nucleotide polymorphism (SNP), with insertion–deletions (InDels), or both. Here, we report discovery of a novel SNP in the SD1 gene from the rice genotype, Pusa 1652. Genetic analysis revealed that the inheritance of the semi-dwarfism in Pusa 1652 is monogenic and recessive, but it did not carry the sd1-d allele. However, response to exogenous gibberellic acid (GA3) application and the subsequent bulked segregant and linkage analyses confirmed that the SD1 gene is involved in the plant height reduction in Pusa 1652. Sequencing of the SD1 gene from Pusa 1652 revealed a novel transition in exon 3 (T/A) causing a nonsense mutation at the 300th codon. The stop codon leads to premature termination, resulting in a truncated protein of OsGA20ox2 obstructing the GA3 biosynthesis pathway. This novel recessive allele, named sd1-bm, is derived from Bindli Mutant 34 (BM34), a γ-ray induced mutant of a short-grain aromatic landrace, Bindli. BM34 is the parent of an aromatic semi-dwarf cultivar, Pusa 1176, from which Pusa 1652 is derived. The semi-dwarfing allele, sd1-bm, was further validated by developing a derived cleaved amplified polymorphic sequence (dCAPS) marker, AKS-sd1. This allele provides an alternative to the most widely used sd1-d in rice improvement programs and the functional dCAPS marker will facilitate marker-assisted introgression of the semi-dwarf trait into tall genotypes.