dCAPS Marker Research Articles

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.

DCAPS markers developed for nitrate transporter genes TaNRT2L12s associating with 1 000-grain weight in wheat

Nitrate transporters (NRTs) are regulators of nitrate assimilation and transport. The genome sequences of TaNRT2L12-A, -B and -D were cloned from wheat (Triticum aestivum L.), and polymorphisms were analyzed by sequencing. TaNRT2L12-D in a germplasm population was highly conserved. However, 38 single nucleotide polymorphisms (SNPs) in TaNRT2L12-A coding region and 11 SNPs in TaNRT2L12-B coding region were detected. Two derived cleaved amplified polymorphic sequences (dCAPS) markers A-CSNP1 and A-CSNP2 were developed for TaNRT2L12-A based on SNP-351 and SNP-729, and three haplotypes were identified in the germplasm population. B-CSNP1 and B-CSNP2 were developed for TaNRT2L12-B based on SNP-237 and SNP-1227, and three haplotypes were detected in the germplasm population. Association analyses between the markers and agronomic traits in 30 environments and phenotypic comparisons revealed that A-CSNP2-A is a superior allele of shorter plant height (PH), length of penultimate internode (LPI) and peduncle length (PL), B-CSNP2-G is a superior allele of higher grain number per spike (GNS). Hap-6B-1 containing both superior alleles B-CSNP1-C and B-CSNP2-A is a superior haplotype of 1 000-grain weight (TGW). Expression analysis showed that TaNRT2L12-B is mainly expressed in the root base and regulated by nitrate. Therefore, TaNRT2L12 may be involved in nitrate transport and signaling to regulate TGW in wheat. The superior alleles and dCAPS markers of TaNRT2L12-A/B are beneficial to genetic improvement and germplasm enhancement with molecular markers-assisted selection.

Development of a dCAPS marker based on a minor resistance gene related to head smut in BIN8.03 in maize

Development of a dCAPS marker based on a minor resistance gene related to head smut in BIN8.03 in maize

Functional Analysis Reveals the Regulatory Role of PpTST1 Encoding Tonoplast Sugar Transporter in Sugar Accumulation of Peach Fruit.

Sugar content is related to fruit sweetness, and the complex mechanisms underlying fruit sugar accumulation still remain elusive. Here, we report a peach PpTST1 gene encoding tonoplast sugar transporter that is located in the quantitative trait loci (QTL) interval on Chr5 controlling fruit sucrose content. One derived Cleaved Amplified Polymorphic Sequence (dCAPS) marker was developed based on a nonsynonymous G/T variant in the third exon of PpTST1. Genotyping of peach cultivars with the dCAPS marker revealed a significant difference in fruit sugar content among genotypes. PpTST1 is located in the tonoplast, and substitution of glutamine by histidine caused by the G/T variation has no impact on subcellular location. The expression profile of PpTST1 exhibited a consistency with the sugar accumulation pattern, and its transient silencing significantly inhibited sugar accumulation in peach fruits. All of these results demonstrated the role of PpTST1 in regulating sugar accumulation in peach fruit. In addition, cis-elements for binding of MYB and WRKY transcript factors were found in the promoter sequence of PpTST1, suggesting a gene regulatory network of fruit sugar accumulation. Our results are not only helpful for understanding the mechanisms underlying fruit sugar accumulation, but will also be useful for the genetic improvement of fruit sweetness in peach breeding programs.

Genetic dissection of yield-related traits via genome-wide association analysis across multiple environments in wild soybean (Glycine soja Sieb. and Zucc.).

A total of 41 SNPs were identified as significantly associated with five yield-related traits in wild soybean populations across multiple environments, and the candidate gene GsCID1 was found to be associated with seed weight. These results may facilitate improvements in cultivated soybean. Crop-related wild species contain new sources of genetic diversity for crop improvement. Wild soybean (Glycine soja Sieb. and Zucc.) is the progenitor of cultivated soybean [Glycine max (L.) Merr.] and can be used as an essential genetic resource for yield improvements. In this research, using genome-wide association study (GWAS) in 96 out of 113 wild soybean accessions with 114,090 single nucleotide polymorphisms (SNPs) (with minor allele frequencies ≤ 0.05), SNPs associated with five yield-related traits were identified across multiple environments. In total, 41 SNPs were significantly associated with the traits in two or more environments (significance threshold P ≤ 8.76 × 10-6), with 29, 7, 3, and 2 SNPs detected for 100-seed weight (SW), maturity time (MT), seed yield per plant (SY) and flowering time (FT), respectively. BLAST search against the Glycine soja W05 reference genome was performed, 20 candidate genes were identified based on these 41 significant SNPs. One candidate gene, GsCID1 (Glysoja.04g010563), harbored two significant SNPs-AX-93713187, with a non-synonymous mutation, and AX-93713188, with a synonymous mutation. GsCID1 was highly expressed during seed development based on public information resources. The polymorphisms in this gene were associated with SW. We developed a derived cleaved amplified polymorphic sequence (dCAPS) marker for GsCID1 that was highly associated with SW and was validated as a functional marker. In summary, the revealed SNPs/genes are useful for understanding the genetic architecture of yield-related traits in wild soybean, which could be used as a potential exotic resource to improve cultivated soybean yields.

Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes

ABSTRACT Substantial progress had been made in reducing nornicotine accumulation in burley tobacco, as nornicotine is a precursor of the carcinogen N-nitrosonornicotine (NNN). Three members of the CYP82E2 family encoding nicotine N-demethylase (NND) have been reported to be responsible for the majority of nicotine demethylation that forms nornicotine in burley tobacco. We had obtained a nonsense mutant of each NND member in flue-cured tobacco from an ethyl methanesulfonate (EMS)-mutagenized population. In this study, we developed dCAPS markers for each nonsense mutation. Using marker-assisted selection, NND mutants were crossed with each other to generate a triple mutant GP449. In line with previous reports, the triple knockout caused significantly decreased levels of nornicotine and NNN in flue-cured tobacco. With the decreased nornicotine, the nicotine level was expected to accumulate. However, the nicotine level in GP449 was significantly decreased to 72.80% of wild type. Realtime RT-PCR analysis showed that the nicotine reduction was correlated with inhibited expression of nicotine biosynthetic pathway genes. The triple mutant and dCAPS markers can be utilized to develop new flue-cured tobacco varieties with lower levels of nornicotine and NNN.

Development of diagnostic molecular markers for marker-assisted breeding against bacterial wilt in tomato.

Bacterial wilt, caused by the Ralstonia pseudosolanacearum species complex, is an important vascular disease that limits tomato production in tropical and subtropical regions. Two major quantitative trait loci (QTL) of bacterial wilt resistance on chromosome 6 (Bwr-6) and 12 (Bwr-12) were previously identified in Solanum lycopersicum ‘Hawaii 7996’; however, marker-assisted breeding for bacterial wilt resistance is not well established. To dissect the QTL, six cleaved amplified polymorphic sites (CAPS) and derived CAPS (dCAPS) markers within the Bwr-6 region and one dCAPS marker near Bwr-12 were developed, and resistance levels in 117 tomato cultivars were evaluated. Two markers, RsR6-5 on chromosome 6 and RsR12-1 on chromosome 12, were selected based on the genotypic and phenotypic analysis. The combination of RsR6-5 and RsR12-1 effectively distinguishes resistant and susceptible cultivars. Furthermore, the efficiency of the two markers was validated in the F3 generation derived from the F2 population between E6203 (susceptible) and Hawaii 7998 (resistant). Resistant alleles at both loci led to the resistance to bacterial wilt. These markers will facilitate marker-assisted breeding of tomato resistant to bacterial wilt.

Development of a PVY Resistant Flue-Cured Tobacco Line via EMS Mutagenesis of eIF4E

Recessive resistance against potyviruses, such as Potato virus Y (PVY), relies on mutations in the eukaryotic translation initiation factor 4E (eIF4E) or one of its isoforms. The eIF4E1-S mutants of burley tobacco (Nicotiana tabacum L.) exhibit recessive resistance against PVY strains. Here, we developed a TILLING population of flue-cured tobacco (N. tabacum cv. Yunyan87) using ethyl methanesulfonate (EMS) to identify eIF4E1-S mutants. M3 plants homozygous for a nonsense mutation in exon 1 of the eIF4E1-S gene demonstrated resistance against PVYMN. These M3 plants were backcrossed to ‘Yunyan87’, and BC4F3 plants were screened using derived cleaved amplified polymorphic sequence (dCAPS) markers. BC4F3 plants showing agronomic traits comparable to the recurrent parent ‘Yunyan87’ and resistance against PVYO, PVYN, and PVYNTN strains were identified. These genotypes would provide useful germplasm for future tobacco improvement and would aid in basic research on PVY resistance in flue-cured tobacco.

A Single Nucleotide Deletion in an ABC Transporter Gene Leads to a Dwarf Phenotype in Watermelon.

Dwarf habit is one of the most important traits in crop plant architecture, as it can increase plant density and improved land utilization, especially for protected cultivation, as well as increasing lodging resistance and economic yield. At least four dwarf genes have been identified in watermelon, but none of them has been cloned. In the current study, the Cldw-1 gene was primary-mapped onto watermelon chromosome 9 by next-generation sequencing-aided bulked-segregant analysis (BSA-seq) of F2 plants derived from a cross between a normal-height line, WT4, and a dwarf line, WM102, in watermelon. The candidate region identified by BSA-seq was subsequently validated and confirmed by linkage analysis using 30 simple sequence repeat (SSR) markers in an F2 population of 124 plants. The Cldw-1 gene was further fine-mapped by chromosome walking in a large F2 population of 1,053 plants and was delimited into a candidate region of 107.00 kb. Six genes were predicted to be in the candidate region, and only one gene, Cla010337, was identified to have two single nucleotide polymorphisms (SNPs) and a single nucleotide deletion in the exons in the dwarf line, WM102. A derived cleaved amplified polymorphic sequence (dCAPS) marker was developed from the single nucleotide deletion, co-segregated with the dwarf trait in both the F2 population and a germplasm collection of 165 accessions. Cla010337 encoded an ATP-binding cassette transporter (ABC transporter) protein, and the expression levels of Cla010337 were significantly reduced in all the tissues tested in the dwarf line, WM102. The results of this study will be useful in achieving a better understanding of the molecular mechanism of the dwarf plant trait in watermelon and for the development of marker-assisted selection (MAS) for new dwarf cultivars.

Genome-Wide Association Study to Identify Favorable SNP Allelic Variations and Candidate Genes That Control the Timing of Spring Bud Flush of Tea (Camellia sinensis) Using SLAF-seq.

The timing of spring bud flush (TBF) is of economic importance for tea plant (Camellia sinensis) breeding. We employed a genome-wide association study (GWAS) to identify favorable single nucleotide polymorphism (SNP) allelic variations as well as candidate genes that control TBF of C. sinensis using specific-locus-amplified fragment sequencing (SLAF-seq) in a diversity panel comprising 151 tea plant germplasm resources. GWAS analysis revealed 26 SNPs associated with TBF in three years, and we eventually identified a final significant SNP for TBF. To identify candidate genes possibly related to TBF, we screened seven candidate genes within 100 kb regions surrounding the trait-related SNP loci. Furthermore, the favorable allelic variation, the "TT" genotype in the SNP loci, was discovered, and a derived cleaved amplified polymorphism (dCAPS) marker was designed that cosegregated with TBF, which could be used for marker-assisted selection (MAS) breeding in C. sinensis. The results obtained from this study can provide a theoretical and applied basis for the MAS of early breeding in tea plants in the future.

A putative bHLH transcription factor is a candidate gene for male sterile 32, a locus affecting pollen and tapetum development in tomato

The tomato (Solanum lycopersicum) male sterile 32 (ms32) mutant has been used in hybrid seed breeding programs largely because it produces no pollen and has exserted stigmas. In this study, histological examination of anthers revealed dysfunctional pollen and tapetum development in the ms32 mutant. The ms32 locus was fine mapped to a 28.5 kb interval that encoded four putative genes. Solyc01g081100, a homolog of Arabidopsis bHLH10/89/90 and rice EAT1, was proposed to be the candidate gene of MS32 because it contained a single nucleotide polymorphism (SNP) that led to the formation of a premature stop codon. A codominant derived cleaved amplified polymorphic sequence (dCAPS) marker, MS32D, was developed based on the SNP. Real-time quantitative reverse-transcription PCR showed that most of the genes, which were proposed to be involved in pollen and tapetum development in tomato, were downregulated in the ms32 mutant. These findings may aid in marker-assisted selection of ms32 in hybrid breeding programs and facilitate studies on the regulatory mechanisms of pollen and tapetum development in tomato.

Genome-wide association study of pre-harvest sprouting tolerance using a 90K SNP array in common wheat (Triticum aestivum L.).

Three major loci for pre-harvest sprouting tolerance (PHST) were mapped on chromosomes 1AL, 3BS, and 6BL, and two CAPS and one dCAPS markers were validated. Sixteen lines with favorable alleles and increased PHST were identified. Pre-harvest sprouting (PHS) significantly affects wheat grain yield and quality. In the present study, the PHS tolerance (PHST) of 192 wheat varieties (lines) was evaluated by assessment of field sprouting, seed germination index, and period of dormancy in different environments. A high-density Illumina iSelect 90K SNP array was used to genotype the panel. A genome-wide association study (GWAS) based on single- and multi-locus mixed linear models was used to detect loci for PHST. The single-locus model identified 23 loci for PHST (P < 0.0001) and explained 6.0-18.9% of the phenotypic variance. Twenty loci were consistent with known quantitative trait loci (QTLs). Three single-nucleotide polymorphism markers closely linked with three major loci (Qphs.ahau-1A, Qphs.ahau-3B, and Qphs.ahau-6B) on chromosomes 1AL, 3BS, and 6BL, respectively, were converted to two cleaved amplified polymorphic sequences (CAPS) and one derived-CAPS markers, and validated in 374 wheat varieties (lines). The CAPS marker EX06323 for Qphs.ahau-6B co-segregated with a novel major QTL underlying PHST in a recombinant inbred line population raised from the cross Jing 411 × Wanxianbaimaizi. Linear regression showed a clear dependence of PHST on the number of favorable alleles. Sixteen varieties showing an elevated degree of PHST were identified and harbored more than 16 favorable alleles. The multi-locus model detected 39 marker-trait associations for PHST (P < 0.0001), of which five may be novel. Six loci common to the two models were identified. The combination of the two GWAS methods contributes to efficient dissection of the complex genetic mechanism of PHST.

Identification of Loci and Candidate Genes Responsible for Pod Dehiscence in Soybean via Genome-Wide Association Analysis Across Multiple Environments.

Pod dehiscence (shattering) is the main cause of serious yield loss during the soybean mechanical harvesting process. A better understanding of the genetic architecture and molecular mechanisms of pod dehiscence is of great significance for soybean breeding. In this study, genome-wide association analysis (GWAS) with NJAU 355K SoySNP array was performed to detect single nucleotide polymorphisms (SNPs) associated with pod dehiscence in an association panel containing 211 accessions across five environments. A total of 163 SNPs were identified as significantly associated with pod dehiscence. Among these markers, 136 SNPs identified on chromosome 16 were located in the known QTL qPDH1. One, one, three, eleven, three, one, three, three and one SNPs were distributed on chromosome 1, 4, 6, 8, 9, 11, 17, 18, and 20, respectively. Favorable SNPs and six haplotypes were identified based on ten functional SNPs; among those Hap2 and Hap3 were considered as optimal haplotypes. In addition, based on GWAS results, the candidate gene Glyma09g06290 was identified. Quantitative real-time PCR (qRT-PCR) results and polymorphism analysis suggested that Glyma09g06290 might be involved in pod dehiscence. Furthermore, a derived cleaved amplified polymorphic sequences (dCAPS) marker for Glyma09g06290 was developed. Overall, the loci and genes identified in this study will be helpful in breeding soybean accessions resistant to pod dehiscence.

Development and Validation of a Gene-Targeted dCAPS Marker for Marker-Assisted Selection of Low-Alkaloid Content in Seeds of Narrow-Leafed Lupin (Lupinus angustifolius L.).

Low-alkaloid content is an important breeding target to improve the quality of lupin seeds. An APETALA2/ethylene response transcription factor, RAP2-7, is likely a candidate gene for the major alkaloid locus iucundus, and plays a crucial role in regulation of seed alkaloid content in narrow-leafed lupin (NLL; Lupinus angustifolius L.). Here, we exploited a single-nucleotide polymorphism within RAP2-7 credibly associated with seed alkaloid content, to develop the co-dominant derived cleaved amplified polymorphic sequence (dCAPS) marker iuc_RAP2-7. Marker validation in 202 NLL accessions demonstrated that seed alkaloid content ≥0.9% of the seed dry weight was associated with the high-alkaloid marker band (Iucundus genotypes), whereas alkaloid content up to 0.5% of the seed dry weight was associated with the low-alkaloid marker band (iucundus genotypes). Within a given detection limit, iuc_RAP2-7 unambiguously identified all but three low-alkaloid accessions. The latter accessions apparently have a different regulatory mechanism for seed alkaloid content because the RAP2-7 gene/putative promoter sequence and expression of alkaloid-associated genes in the leaves of the three ambiguous accessions were similar to those of bitter Iucundus lines. We consider the iuc_RAP2-7 marker is a powerful tool that will facilitate NLL marker-assisted selection by rapid rejection of bitter Iucundus genotypes and thus accelerate development of new low-alkaloid cultivars.

Rice Genome Resequencing Reveals a Major Quantitative Trait Locus for Resistance to Bakanae Disease Caused by Fusarium fujikuroi.

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, has become a serious threat in rice-cultivating regions worldwide. In the present study, quantitative trait locus (QTL) mapping was performed using F2 and F3 plants derived after crossing a BD-resistant and a BD-susceptible Korean japonica rice variety, ‘Samgwang’ and ‘Junam’, respectively. Resequencing of ‘Junam’ and ‘Samgwang’ genomes revealed 151,916 DNA polymorphisms between the two varieties. After genotyping 188 F2 plants, we constructed a genetic map comprising 184 markers, including 175 kompetitive allele-specific PCR markers, eight cleaved amplified polymorphic sequence (CAPS) markers, and a derived CAPS (dCAPS) marker. The degree of BD susceptibility of each F2 plant was evaluated on the basis of the mortality rate measured with corresponding F3 progeny seedlings by in vitro screening. Consequently, qFfR9, a major QTL, was discovered at 30.1 centimorgan (cM) on chromosome 9 with a logarithm of the odds score of 60.3. For the QTL interval, 95% probability lay within a 7.24–7.56 Mbp interval. In this interval, we found that eight genes exhibited non-synonymous single nucleotide polymorphisms (SNPs) by comparing the ‘Junam’ and ‘Samgwang’ genome sequence data, and are possibly candidate genes for qFfR9; therefore, qFfR9 could be utilized as a valuable resource for breeding BD-resistant rice varieties.

Genetic analysis and gene mapping of the orange flower trait in Chinese cabbage (Brassica rapa L.)

Flower color is considered an important appealing signal to pollinators and also a marker trait in Brassica crop breeding. However, the genetic basis of orange flower trait remains poorly understood in Brassica rapa. In this study, we conducted a genetic analysis of orange flower trait and fine mapped the underlying gene in B. rapa. Two populations, BC1F1 and BC1F2 with 478 and 443 individuals, respectively, were constructed from a cross between 94C9 (orange flower) and 92S105 (yellow flower). Genetic analysis showed that a single recessive gene, BrOF, controlled the orange flower trait. Using Indel and dCAPS markers developed from whole-genome resequencing data of 94C9 and 92S105, BrOF was mapped to a 41.5-kb region on chromosome A09 delimited by InDel409 and dCAPS425 containing six putative genes. Among them, only Bra037124 and Bra037125, which encode an AP2 domain–containing transcription factor and an SEC-C motif–containing protein/OTU-like cysteine protease family protein, respectively, were successfully cloned. The sequence analysis revealed two SNPs resulting in amino acid residue changes in the coding region of Bra037124, as well as seven SNPs and one insertion leading to amino acid residue mutations in the coding region of Bra037125, between 94C9 and 92S105. The reliability of a co-segregating marker InDel314 in marker-assisted selection (MAS) was confirmed by testing different yellow/orange flower Chinese cabbage lines. These results provide a good foundation to identify BrOF and facilitate our understanding of the genetic basis underlying the development of orange flowers in Chinese cabbage.

B-class MADS-box TM6 is a candidate gene for tomato male sterile-1526.

Tomato male sterile-1526 locus was fine-mapped to an interval of 44.6kb, and a B-class MADS-box gene TM6 was identified as the candidate gene. Male sterile lines have been widely used for hybrid seed production in many crop plants. The tomato male sterile-1526 (ms-1526) mutant displays abnormal stamens and exerted stigmas and is suitable for practical use. In this study, the ms-1526 locus was fine-mapped to a 44.6kb interval that contained four putative genes. Thereinto, Solyc02g084630 encodes tomato B-class MADS-box gene TM6 (syn. TDR6), which plays an important role in stamen development. Sequencing revealed that there was a 12.7kb deletion in the ms-1526 region, where the promoter and first four exons of the TM6 gene were absent. ms-1547, an allele of ms-1526, also contained the same deletion in the TM6 gene. And the other allele ms-15 mutant contained a single-nucleotide polymorphism (SNP, C to A) in the coding region of the TM6 gene, which led to a missense mutation (G to W). The codominant insertion/deletion (InDel) marker MS26D and codominant derived cleaved amplified polymorphic sequence (dCAPS) marker MS15C were developed based on the deletion and SNP, respectively. A real-time quantitative reverse-transcription PCR showed that expression of the TM6 gene was barely detectable in the flowers of the ms-1526 and ms-1547 mutants. In addition, other floral organ identity genes, pollen development marker genes, and pistil marker genes were differentially expressed between wild type and mutant flowers. These findings may facilitate functional analysis of the TM6 gene and help in the marker-assisted selection of ms-15 and its alleles in tomatobreeding.

Mapping-by-sequencing using NGS-based 3'-MACE-Seq reveals a new mutant allele of the essential nodulation gene Sym33 (IPD3) in pea (Pisum sativum L.).

Large collections of pea symbiotic mutants were accumulated in the 1990s, but the causal genes for a large portion of the mutations are still not identified due to the complexity of the task. We applied a Mapping-by-Sequencing approach including Bulk Segregant Analysis and Massive Analysis of cDNA Ends (MACE-Seq) sequencing technology for genetic mapping the Sym11 gene of pea which controls the formation of symbioses with both nodule bacteria and arbuscular-mycorrhizal fungi. For mapping we developed an F2-population from the cross between pea line N24 carrying the mutant allele of sym11 and the wild type NGB1238 (=JI0073) line. Sequencing libraries were prepared from bulks of 20 plants with mutant and 12 with wild-type phenotype. MACE-Seq differential gene expression analysis between mutant-phenotype and wild-type-phenotype bulks revealed 2,235 genes, of which 514 (23%) were up-regulated and 1,721 (77%) were down-regulated in plant roots inoculated with rhizobia as a consequence of sym11 mutation. MACE-Seq also detected single nucleotide variants between bulks in 217 pea genes. Using a novel mathematical model we calculated the recombination frequency (RF) between the Sym11 gene and these 217 polymorphic genes. Six genes with the lowest RF were converted into CAPS or dCAPS markers and genetically mapped on the complete mapping population of 108 F2-plants which confirmed their tight linkage to Sym11 and to each other. The Medicago truncatula Gaertn. (Mt) homologs of these genes are located in a distinct region of Mt chromosome 5, which corresponds to linkage group I of pea. Among 94 candidate genes from this region only one was down-regulated—the pea Sym33 homolog of the Mt IPD3 gene which is essential for nodulation. Sequencing of the Sym33 allele of the N24 (sym11) mutant revealed a single nucleotide deletion (c.C319del) in its third exon resulting in a codon shift in the open reading frame and premature translation termination. Thus, we identified a novel mutant allele sym33-4 most probably responsible for the mutant phenotype of the N24 (sym11) line, thereby demonstrating that mapping by MACE-Seq can be successfully used for genetic mapping of mutations and identification of candidate genes in pea.

Genome-wide association study identifies favorable SNP alleles and candidate genes for waterlogging tolerance in chrysanthemums

Chrysanthemums are sensitive to waterlogging stress, and the development of screening methods for tolerant germplasms or genes and the breeding of tolerant new varieties are of great importance in chrysanthemum breeding. To understand the genetic basis of waterlogging tolerance (WT) in chrysanthemums, we performed a genome-wide association study (GWAS) using 92,811 single nucleotide polymorphisms (SNPs) in a panel of 88 chrysanthemum accessions, including 64 spray cut and 24 disbud chrysanthemums. The results showed that the average MFVW (membership function value of waterlogging) of the disbud type (0.65) was significantly higher than that of the spray type (0.55) at P < 0.05, and the MFVW of the Asian accessions (0.65) was significantly higher than that of the European accessions (0.48) at P < 0.01. The GWAS performed using the general linear model (GLM) and mixed linear model (MLM) identified 137 and 14 SNP loci related to WT, respectively, and 11 associations were commonly predicted. By calculating the phenotypic effect values for 11 common SNP loci, six highly favorable SNP alleles that explained 12.85—21.85% of the phenotypic variations were identified. Furthermore, the dosage-pyramiding effects of the favorable alleles and the significant linear correlations between the numbers of highly favorable alleles and phenotypic values were identified (r2 = 0.45; P < 0.01). A major SNP locus (Marker6619-75) was converted into a derived cleaved amplified polymorphic sequence (dCAPS) marker that cosegregated with WT with an average efficiency of 78.9%. Finally, four putative candidate genes in the WT were identified via quantitative real-time PCR (qRT-PCR). The results presented in this study provide insights for further research on WT mechanisms and the application of molecular marker-assisted selection (MAS) in chrysanthemum WT breeding programs.

Identification of favorable SNP alleles and candidate genes responsible for inflorescence-related traits via GWAS in chrysanthemum

81 SNPs were identified for three inflorescence-related traits, in which 15 were highly favorable. Two dCAPS markers were developed for future MAS breeding, and six candidate genes were predicted. Chrysanthemum is a leading ornamental species worldwide and demonstrates a wealth of morphological variation. Knowledge about the genetic basis of its phenotypic variation for key horticultural traits can contribute to its effective management and genetic improvement. In this study, we conducted a genome-wide association study (GWAS) based on two years of phenotype data and a set of 92,617 single nucleotide polymorphisms (SNPs) using a panel of 107 diverse cut chrysanthemums to dissect the genetic control of three inflorescence-related traits. A total of 81 SNPs were significantly associated with the three inflorescence-related traits (capitulum diameter, number of ray florets and flowering time) in at least one environment, with an individual allele explaining 22.72-38.67% of the phenotypic variation. Fifteen highly favorable alleles were identified for the three target traits by computing the phenotypic effect values for the stable associations detected in 2 year-long trials at each locus. Dosage pyramiding effects of the highly favorable SNP alleles and significant linear correlations between highly favorable allele numbers and corresponding phenotypic performance were observed. Two highly favorable SNP alleles correlating to flowering time and capitulum diameter were converted to derived cleaved amplified polymorphic sequence (dCAPS) markers to facilitate future breeding. Finally, six putative candidate genes were identified that contribute to flowering time and capitulum diameter. These results serve as a foundation for analyzing the genetic mechanisms underlying important horticultural traits and provide valuable insights into molecular marker-assisted selection (MAS) in chrysanthemum breeding programs.