A transcription factor gene CaMYB114 associated with black seed coat in chickpea

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Chickpeas with black-coloured seed coat is known for their beneficial high antioxidant and fibber content, yet the underlying molecular basis remains poorly understood. Results Here, we examined the grain colour trait of a panel of 261 diverse desi chickpea ( Cicer arietinum ) accessions and specially characterized the development of the black seed coat. We showed that the black colouration emerged on embryo tips at 30 days after flowering (DAF) and expanded to whole grain at 35 DAF. Genome-wide association analyses revealed a single major genetic locus CaBlk3-1 on chromosome Ca3 controlling black seed coat. Candidate gene screening within 0.5 Mb upstream and downstream of CaBlk3-1 identified a single MYB-encoding gene CaMYB114 related to anthocyanin biosynthesis. Phylogeny analyses showed that CaMYB114 was clustered with Arabidopsis MYB90, MYB113, MYB114, consistent with their role in anthocyanin production. Subsequent qRT-PCR analyses suggested that CaMYB114 was abundantly transcribed in black genotypes but weakly in the brown genotypes at 35 DAF, closely linked with black colour development. Genetic variation analyses of CaMYB114 identified a 12-bp deletion containing a GAGA motif in the 5UTR region of black chickpea genotype. A gene-specific marker targeting this deletion was developed to validate its link with the black seed coat in a larger chickpea germplasm collection. Conclusions We identified a single major QTL and the underlying candidate gene CaMYB114 controlling the black seed coat trait in chickpea. Our study has greatly improved our understanding of the genetic basis of chickpea black seed and will unlock the potential for breeding new chickpeas with desired grain colour to meet various market requirements. anthocyanins chickpea black seed coat genome wide association analyses MYB transcription factor molecular markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Population growth and climate change have significantly threatened global food security in recent years. In this context, chickpea ( Cicer arietinum ) becomes a highly valued crop, providing affordable nutritious food for a growing global population [ 1 , 2 ]. It is rich in protein, energy, fibre, minerals, vitamins, and health-promoting phytochemicals [ 3 – 5 ]. Beyond its affordability and rich nutrient profile, the growing interest in chickpea is also driven by its adaptability to drought-prone and semi-arid environments [ 6 , 7 ], making it a sustainable and reliable crop in the face of climate change challenges. The global trade and demand for chickpeas are steadily increasing since early 2000s [ 8 ], and the leading exporters include Australia, Canada, and Argentina [ 9 ]. Grain quality is essential to meet international standards and ensure suitability for the global market. The seed colour is a key visual trait that plays a significant role in market value and consumer preference. Chickpea is commonly classified into two main types: Kabuli, with large seeds, and Desi, with small, rough seeds. The chickpea seed colour can vary widely, ranging from cream, beige for Kabuli, and light brown to darker colours for Desi, depending on both environments and genetic factors. Anthocyanin and proanthocyanidin (PA) biosynthesis are known to play a critical role in determining chickpea seed coat colours [ 10 ]. A few studies have identified some key genes involved in this trait. A study using an integrated genomics approach across diverse Cicer genetic resources identified a seed-colour gene CaMATE23 , which influenced PA accumulation in seed coat through the flavonoid pathway. An 8-bp InDel in the CaMATE23 promoter played a key role in the transition from grey seeds in wild Cicer to the dark brown seeds in cultivated chickpeas [ 11 ]. Another recent study found that natural variants of two anthocyanin and PA biosynthesis genes ( CabHLH and CaMATE1 ) explained the seed colour variation between Kabuli and Desi chickpea [ 10 ]. There is an old type of chickpea that resembling Desi chickpeas in size but features a black seed coat [ 12 ]. The seed colour was showed to be linked to some biochemical compounds, with darker-coloured seeds often containing higher levels of antioxidants [ 13 – 15 ]. Recent studies have incorporated black chickpea wholemeal flour into wheat flour for bread and pasta production. The addition of black chickpea flour not only increased the protein and fibre content but also enriched the final product with higher antioxidant activity [ 16 , 17 ]. The rising demand for healthy food has revived interest in this old black chickpea, highlighting its potential for commercial development [ 18 ]. However, no study to date has explored the genetic basis of black seed colour in chickpea. In common bean ( Phaseolus vulgaris L.), the genetic mechanisms underlying seed colour have been well studied [ 19 , 20 ]. Two key genes, flavonoid 3′5′-hydroxylase and MYB113 transcription factor, were identified as candidates responsible for black seed colour [ 21 ]. MYB transcription factors regulate flavonoid biosynthesis by controlling early and late biosynthetic genes (EBGs and LBGs). In dicots, MYB transcription factors independently activate early biosynthetic genes (EBGs) such as chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), and flavonol synthase (FLS), which are essential for the initial steps in flavonoid production [ 22 ]. While LBGs, including genes that encode enzymes DFR, ANS, ANR, LAR involving in production of PAs and anthocyanins, are regulated by a MBW complex, which includes MYB transcription factors, bHLH proteins, and WD40 repeat proteins [ 23 ]. MYBs can act as either activators or repressors [ 24 ]. A few studies have demonstrated that the MYB gene also served as a key determinant of black seed coat coloration in another two legumes, cowpea ( Vigna unguiculata L.) [ 25 ] and soybean ( Glycine max ) [ 26 ]. Despite its importance, the genetic mechanisms behind chickpea black seed colour remain an area of ongoing research, with no study identifying the key genes responsible for this trait. In this study, we genotyped a panel of 261 diverse chickpea accessions using an SNP chip array and evaluated their seed coat colours. We then performed a genome-wide association study (GWAS) on black seed coat, and examined the major candidate gene through gene expression analysis and 5’ UTR variations. The aim of this project is to provide insights into the genetic basis controlling the black seed in chickpea. Understanding these genetic factors could lead to the development of improved chickpea varieties with desirable colours and enhanced overall quality. Methods Plant materials, phenotyping and genotyping The chickpea materials (261 accessions in total) used in this study were sourced from the Australian Grains Genebank (AGG). These accessions represent a broad genetic diversity, providing a valuable resource for studying seed coat colour diversity. Seed colour was evaluated through a visual assessment. The assessment was conducted under standardized lighting conditions to ensure consistency and accuracy in colour classification. Multiple observers participated in the scoring process to minimize bias and enhance reproducibility. The seed colours were documented and classified into predefined categories, including black, brown, yellow and green. This visual scoring method provided a straightforward yet effective approach for identifying variations in seed coat pigmentation across different chickpea accessions. The seed coat pigment was extracted from a desi chickpea variety (Genesis 836) and a black-seed variety (Brown Rosena) using a water-based anthocyanin extraction method. Seed coats were manually removed, dried, and ground into a fine powder. For each sample, 0.1 g of seed coat powder was mixed with 500 µL of distilled water. The mixture was gently stirred at room temperature for 30 minutes, followed by centrifugation at 10,000 rpm for 10 minutes. The resulting supernatant was collected. High quality DNA was extracted from young leaves for each accession. Genotyping was performed using either the Multispecies Pulse SNP Chip Array (DAV1905-003RTX) or genotyping-by-sequencing (GBS) at Agriculture Victoria, Australia. The SNP chip array includes 4,449 chickpea-specific markers, providing comprehensive genome-wide coverage, with 1,735 SNPs as exclusive to wild Cicer species. This high-density genotyping approach enables the detection of genetic variations associated with key traits, including seed coat colour. To ensure accuracy in downstream analyses, genotyping data was filtered to remove samples with high heterozygosity (> 0.20) before imputation, reducing potential errors in association studies. Phylogeny analyses MYB sequences in chickpea and Arabidopsis reference genome were downloaded from PlantTFDB v5.0 database ( https://planttfdb.gao-lab.org/index.php ) [ 27 ]. Multiple sequence alignment was performed using kalign 3.0 program [ 28 ]. Maximum likelihood (ML) tree was built using fasttree [ 29 ] and branch reliability assessment using maximum-likelihood NNIs with 1000 resampling. A separate ML tree was developed based on sequence alignment for the subclade containing CaMYB114 using IQ-TREE with the substitution model JTTDCMut + F + I + G4 chosen according to best Bayesian Information Criterion [ 30 ]. All tree visualization and annotations were applied in Figtree (v1.4.3, http://tree.bio.ed.ac.uk/software/figtree ) using different colours to distinguish clusters. GWAS and PCA analyses GWAS for black seed coat with the diverse chickpea genotypes were conducted using the rMVP R package [ 31 ], with a significance threshold at 0.05. Fixed and random model Circulating Probability Unification (FarmCPU) were implemented. Kinship and principal component were calculated using the rMVP and used as covariates for GWAS. Population structure analyses were performed using the top 5 calculated principal components. PCA analyses were performed using VCF2PCACluster tool [ 32 ]. Candidate gene expression analysis The seed coat samples of two black lines (Negro and Galbron) and two brown colour Desi lines (PBA Maiden and Genesis 836) were collected 25, 30, 35 days after flowering, to characterize and compare the candidate gene expression in different seed development stages. The leaf, pod, stem and flower samples were also collected in these four chickpea lines to examine the candidate gene expression in different tissues. To further compare the expression difference between black lines and brown lines, the seed coat samples were obtained from another three black lines (IG132577, ILC10392 and Brown Rosena) and three brown Desi lines (PBA Drummond, PBA Slasher and Kyabra) 35 days after flowering. High quality RNA was extracted by TRI Reagent® (Sigma-Aldrich, #T9424) following the instruction. Approximately equal amount of RNA (~ 1 µg) was used for reverse transcription by SensiFAST™ cDNA Synthesis Kit (Bioline, #65053) as per manufacturer instruction. A pair of primers were designed to bind uniquely to the candidate gene and they were made straddle the intron to avoid DNA contamination: MYB114RT_F1 (5′-AGGTTTGAAAAGATGCCGCAAGA-3′) and MYB114RT_R1 (5′-CCTGCAATAAGTGACCACCTGT-3′). The reference gene used in this study was CaCAC as described before [ 33 ]. The real-time PCR was conducted using SensiFAST™ SYBR® Lo-ROX Kit (Bioline, #94005) and ViiA™ 7 Real-Time PCR System (Applied Biosystems), performed in three biological replicates and three technical replicates. The relative expression levels of target gene were determined using comparative Ct method (2 −△Ct ) [ 34 ]. Public transcriptome data from Chickpea Transcriptome Database (CTDB) [ 35 ] was downloaded for the extraction of CaMYB114 in multiple tissues. Variant calling Whole genome shotgun sequencing data and chickpea passport records were downloaded from a previously published study [ 36 ]. Raw reads were trimmed using fastp program [ 37 ] with --qualified_quality_phred 20 and --length_required 30 parameters. The resulted clean reads were mapped to reference genome using BWA-MEM program [ 38 ] with default parameters. Variant calling was performed using GATK4 program [ 39 ] with final quality filtration: "QD 60.0, MQ 4.0, MQRankSum < -12.5" for SNPs and "QD 200.0, SOR > 10.0" for indels. The functional effects of obtained genetic variations were annotated using SnpEff program [ 40 ]. Sequencing and marker design High quality DNA was extracted from five black seed lines (Brown Rosana, Negro, IG132577, ILC10392, Galbron) and five brown seed lines (PBA Maiden, Genesis 836, PBA Drummond, PBA Slasher and Kyabra), using a modified CTAB method described before[ 41 ]. The 5' UTR region of the candidate gene was sequenced using a pair of primers: MYBIndelF2 (5′- AGATCTTTTCTACCGTGCTGT-3′) and MYBIndelR2 (5′- CCATGTGGTAAGCTGCTGAA-3′), by the Sanger sequencing method [ 42 ]. Additionally, this primer pair was used to screen the 261-chickpea diversity panel, to determine the association between the 5' UTR deletion and the black seed coat colour phenotypes across various chickpea accessions. Results Phenotyping and development characterization of black grain trait in chickpea In this study, our laboratory collection of diverse chickpea germplasm lines from AGG were subjected to grain colour phenotyping. Due to the lack of black accession in the Kabuli genotypes, we decide to only use the remaining 261 Desi germplasm lines for further detailed grain colour classification and genetic association analyses. Based on visual assessment, the 261 chickpea lines were generally divided into 4 major colour groups: brown (n = 200), yellow (34), green (14), and black (13) (Fig. 1 A). This category assignment was used for downstream genome wide association analyses. To characterize the development of the black grain trait, we used a black chickpea line Negro (Fig. 1 B) and a brown chickpea cultivar Genesis 836 and sampled the developing seeds at 20 days after flowering (DAF), 25 DAF, 30 DAF, 35 DAF and 40 DAF (Fig. 1 C). As shown in Fig. 1 C, Negro seed displayed deep green at 20 DAF and 25 DAF, started developing black colour at the embryo tip at 30 DAF. The whole seed coat of Negro turned grey colour with green hue at 35 DAF, which further changed to complete black at 40 DAF. In contrast, Genesis 836 seed displayed yellow green at 20 DAF, which changed to deep green at 25 DAF, then gradually ripen with reduced greenness on 30 DAF and 35 DAF and finally become complete brown on 40 DAF (Fig. 1 C). According to literature search, only two types of pigments have been reported responsible for black colouration in plants: anthocyanins and melanins, the latter of which is insoluble [ 43 ]. To determine the chemical nature of black chickpea, we performed pigment extraction of the black chickpea seed coat, which showed that the black pigment was readily soluble in water (Fig. 1 D), supporting the black chickpea pigments as anthocyanin. Identification of QTLs and candidate gene controlling black chickpea grain trait To identify genetic locus controlling the black chickpea grain trait, GWAS analyses were performed. The phenotype was classified into black (1) and non-black (0) for the purpose of GWAS analyses. Genotype data for the 261 chickpea lines using Multispecies pulse SNP chip array was obtained from our recent study [ 44 ]. GWAS analyses using FarmCPU model revealed a single major significant QTLs CaBlk3-1 on chromosome ca3 (Fig. 2 A). The population structure of the used chickpea lines was investigated by PCA analyses (Fig. 2 B), which revealed the presence of three major subgroups. The black chickpea lines were present exclusively in one subgroup, implying a single evolutionary origin. The top five most significant associated SNP markers for CaBlk3-1 and their phenotypic impact were displayed in Fig. 2 C. The most significant SNP AVR-Ca-01147.03-004829307 at 4.83 Mb displayed p-value at 1.05e-36 and was considered the most confident locus controlling the black grain trait in chickpea. To identify potential gene controlling black chickpea grain, we examined the genomic region 0.5 Mb upstream and downstream of AVR-Ca-01147.03-004829307 for candidate genes with annotation function in anthocyanin biosynthesis. Out of the 44 predicted genes, there were only two genes ( LOC101489372 and LOC101500181 ) encoding MYB transcription factors with potential involvement in anthocyanin biosynthesis ( Supplementary File S1 ). Of these, LOC101500181 encoded a MYB114-like transcription factor (XP_004491866.1) with reported function in regulating anthocyanin biosynthesis in several crop species [ 45 ]. In contrast, LOC101489372 was annotated as MYB106-like protein with reported function in cuticle biosynthesis and trichome maturation and was thus excluded for further analysis. LOC101500181 , hence renamed as CaMYB114 , is located at 4.51 Mb on chromosome ca3, 0.32Mb upstream of AVR-Ca-01147.03-004829307, was selected as the candidate gene for CaBlk3-1 for further functional validation. Phylogeny analyses of CaMYB114 To investigate the functional classification of CaMYB114, a phylogenetic tree of chickpea MYB gene family was developed using Arabidopsis MYBs as the references. A total of 166 and 168 MYBs sequences ( Supplementary File S2 ) for chickpea and Arabidopsis were downloaded from PlantTFDB database and were used for phylogeny development. Previously reported soybean GmMYBA2 [ 46 ] and mung bean VrMYB90 [ 47 ] controlling black seed coat trait were also included. A preliminary NJ clustering analyses was performed to identify the subclade containing CaMYB114. Protein sequences for MYBs in the identified subclade were extracted to develop a more robust ML tree. In the developed ML phylogeny (Fig. 3), CaMYB114 (XP_004491866.1), GmMYBA2, and VrMYB90 were clustered together, implying a conserved gene function across different legume species. Using Arabidopsis MYBs as references, this subclade containing CaMYB114, GmMYBA2, and VrMYB90, displayed the closest relationship with Arabidopsis MYB90, MYB113, and MYB114 [ 45 ], which is consistent with their gene annotation as MYB114-like protein involved in anthocyanin biosynthesis. In addition to CaMYB114, another two chickpea MYBs (XP_00449414.1 and XP_00449415.1) annotated as MYB90-like proteins were also clustered in the same clade with CaMYB114. XP_00449414.1 and XP_00449415.1 correspond to two protein isoforms encoded by the same gene LOC101494325 (3.62 Mb) on chromosome ca2. This MYB gene was excluded as candidate for black chickpea trait due to lack of QTL on chromosome ca2. Validation of candidate gene CaMYB114 for black grain trait To investigate the expression profile of CaMYB114 across different tissues, we grow plants for two black (Negro and Galbron) and two brown (Genesis 836 and Maiden) and performed qRT-PCR in leaf, pod, stem, flower, and seed coat tissues. Results showed that CaMYB114 transcription was only observed in stem, flower, and seed coat but not in leaf and pod (Fig. 4 A). In stem, CaMYB114 ’s expression varied significantly across four chickpea genotypes, with abundant expression in Genesis 836, moderately in Negro, and weakly in Maiden and Galbron. In the flower tissue, all four genotypes displayed abundant expression of CaMYB114 , with Negro and Genesis 836 relatively higher than Maiden and Galbron. In the seed coat, CaMYB114 displayed enormous expression in black genotypes Negro and Galbron but only slightly in the brown genotypes Genesis 836 and Maiden. Taken together, the transcription of CaMYB114 was only positively correlated with the seed colour trait in the seed coat tissue, but not in the other tissues, supporting its potential involvement in the black seed coat trait. To further confirm CaMYB114 ’s involvement in seed coat colour development, we performed qRT-PCR of CaMYB114 at 25 DAF, 30 DAF, 35 DAF in the seed coat of the above 4 chickpea genotypes. Results showed that CaMYB114 was barely expressed at 25 DAF in all chickpea genotypes (Fig. 4 B). At 30 DAF, we observed moderate expression of CaMYB114 in the two black genotypes but only slight transcription in the brown genotypes, consistent with the emergence of black colouration at this stage. Furthermore, at 35 DAF when the black colouration expands to whole seed, we found that the expression of CaMYB114 increased dramatically in the black genotypes but remained low in the brown genotypes. These results matched well with the development changes of the black colouration. To further confirm CaMYB114 ’s role in black chickpea grain, we included another 3 brown and 3 black genotypes for qRT-PCR analyses at 35 DAF, which revealed highly consistent transcriptional pattern as that observed in the other 4 genotypes, i.e CaMYB114 was highly transcribed only in black seed coat but weakly in brown seed coat (Fig. 4 B). In addition, we also extracted the transcriptome data from public database (Fig. 4 C), which showed that the transcription of CaMYB114 was restricted to reproductive tissues but not transcribed in root, vegetive, and seed of the brown chickpea, consistent with our qRT-PCR results. In summary, gene transcriptional analyses of CaMYB114 provided well support its role in controlling black chickpea grain trait and potentially flower colour as well. Genetic variation and molecular marker design analyses of CaMYB114 The gene structure of CaMYB114 was examined. As showed in Fig. 5 A, CaMYB114 contained 3 exons and 2 introns, with a 316 bp 5UTR and 192 bp 3UTR regions. To investigate the genetic variations within CaMYB114 gene regions, potentially those directly linked to the black and non-black chickpea grain traits, we downloaded previously published whole genome resequencing data for 163 diverse chickpea germplasm covering different species, cultivars, landraces, and wild lines and performed variant calling against the reference genome. We identified a total of 301 SNPs and indels within the 5UTR, genic region, and 3UTR ( Supplementary file S3 ). Due to the active expression of CaMYB114 in flower tissue of both black and non-black chickpea lines, we reason that the genetic variations controlling the black colouration most likely alter the transcriptional profile of CaMYB114 , instead of the encoded amino acid sequence. Therefore, we focused on indels present in 5UTR, intron, and 3UTR which are known to modulate gene expression. We designed position-specific molecular markers targeting the 7 indels and run those markers on 13 chickpea genotypes (6 black and 7 non-black). Genotyping results showed that only the marker MYBindel-F2R2 targeting a 12 bp indel on 5UTR region matched perfectly with the grain colour (Fig. 5 B). The black chickpea genotype displayed a deletion at this locus. We further run MYBindel-F2R2 on a larger collection of 1160 chickpea DNA samples and identified additional 9 chickpea lines with the black allele. Grain colour examination showed that all those lines with the black allele displayed a black grain trait, supporting MYBindel-F2R2 as the potential causal variant. To validate the 12 bp deletion in black coloured chickpea, we performed sequencing analysis using the MYBindel-F2R2 primers. Results showed that there were indeed a 12 bp deletion in the black genotypes ILC10392 and IG132577 compared to 2 brown desi cultivars Genesis836 and Slasher (Fig. 5 C). Discussion Grain colour is an important quality trait, particularly in legume species, impacting market value, consumer preference, and nutritional properties [ 48 , 49 ]. Among various legume species, chickpea is playing an increasingly important role in the global food system due to its high nutritional value, climate resilience, and affordability [ 50 ]. In chickpea breeding and research, chickpea genotypes with various coloured grains have gained renewed interests for their various nutritional benefits and superior environmental adaptability in the face of climate change, such as high antioxidant content and potential in drought and heat stress resistance [ 10 , 51 ]. However, our efforts in harnessing these valuable genetic resources of coloured grains in chickpea breeding have been greatly hindered by the poor understanding of their genetic basis and the lack of gene-specific molecular markers. In this study, we employed accurate genotyping and phenotyping of a genetically diverse panel of 261 desi chickpeas and mapped the genetic locus controlling black/dark chickpea grain to a major QTL CaBlk3-1 on chromosome ca3. Based on comprehensive gene functional prediction, protein homology, transcriptional, and genetic variation analyses, we successfully identified a MYB transcription factor CaMYB114 as the underlying candidate gene responsible for the black grain trait in chickpea. Most importantly, we discovered and validated a 12 bp deletion in the promoter region of CaMYB114 as the direct genetic cause of black coloured chickpea. This represents a significant advancement in our understanding of coloured seeds in chickpea. The development of gene-specific molecular markers will unlock the potential for breeding new chickpea varieties with desired grain colour to meet various market requirements. Chickpea grains can naturally develop various colours including cream, beige, light brown, darker browns, yellow, green, and black [ 52 ]. Of these, Kabuli chickpea seeds typically have the light cream or beige colour, whereas Desi chickpeas exhibit a wider range of colours from light brown to deep black. This is consistent with our observation that the black colour was present exclusively in Desi chickpeas. Therefore, we excluded Kabuli chickpeas for GWAS analyses to avoid potential population stratification issue, which may lead to the identification of false positive association [ 53 ]. We identified a single major QTL CaBlk3-1 on ca3 significantly linked to the black grain trait in chickpea. A previous GWAS study [ 52 ] on chickpea seed coat colour employed genotyping by sequencing on a diverse 172 chickpea lines, including 93 cultivars (desi and kabuli) and 79 wild chickpeas, and reported 15 QTLs spanning all chromosomes in chickpea. Despite the presence of black coloured chickpea lines in their collection, none of these 15 QTLs overlap with CaBlk3-1 identified in our study. Our GWAS analyses may be advantageous in two aspects: the exclusive use of desi genotypes and the classification of seed colour into black (1) and non-black (0) for the specific examination of the black trait, which contrasts with the classification of 24 seed colour types in their analyses. In plants, two major types of black pigments have been reported: anthocyanins and melanins, the latter of which are knowns as insoluble compounds [ 43 ] and have been linked to the black hull in barley, wheat, and rice seeds [ 54 , 55 ]. To narrow down the potential candidate gene for CaBlk3-1 , we examined the chemical nature of the black pigments in black chickpea and confirmed that they are readily soluble in extraction buffer, suggesting that the black chickpea trait may be caused by anthocyanins production, instead of melanins. Despite that the anthocyanin composition in black coloured chickpea remain to be characterized, insights may be drawn from several related studies in other legume grains. In black cowpea, comparable amounts of red anthocyanin (cyanidin–3–O–glucoside, 37.2%) and blue anthocyanins (delphinidin–3–O–glucoside, 20.0%; malvidin–3–O–glucoside, 16.5%) were identified [ 56 ]. In contrast, black kidney beans and black soybean seem to contain dominant amount of blue anthocyanin (delphinidin–3–O–glucoside, 60.0%) and red anthocyanin (cyanidin–3–O–glucoside, 98.6%), respectively [ 56 ]. Another report in black bean showed that blue anthocyanins (delphinidin 3-glucoside, 65.7%; maldivin 3 glucoside 8.7%) were dominant [ 57 , 58 ]. These studies suggested that the black seed coat of legumes may involve both red and blue anthocyanins, the latter of which may be more prevalent and may be critical for the black colouration. In contrast to black seed coat, the anthocyanin contents in brown desi chickpea have been determined with dominant yellow anthocyanin (petuinidin), followed by significant amount of red and blue anthocyanins as well [ 10 ]. Future anthocyanin profiling analyses are needed to confirm if blue anthocyanins may be the dominant type in black-coloured chickpea grain. The biosynthesis of anthocyanin in plants is known to be regulated by a complex of MYB, bHLH, and WD40 transcription factors, which are highly conserved across species [ 59 ]. The identification of the MYB transcription factor CaMYB114 is consistent with an anthocyanin basis for the black chickpea trait. Interestingly, the black seed coat trait in soybean [ 46 ] and mung bean [ 47 ] has also been shown to be controlled by MYB transcription factors. Furthermore, all three MYBs displayed the closest homology with Arabidopsis MYB113, MYB114, and MYB90 that have been shown involved in anthocyanin biosynthesis [ 45 ]. These observations suggest that the black seed coat trait may share a common molecular basis. Recently, the brown seed colour trait in chickpea has been characterized to be controlled by a bHLH transcription factor CabHLH and multidrug and toxic compound extrusion transporter CaMATE1 [ 10 ], which were shown to regulate the anthocyanin and proanthocyanin accumulation in both seed coat and flower. All candidate genes associated with chickpea grain colour and flower colour to date are linked to anthocyanin production. In addition to black seed coat, we observed that CaMYB114 was also highly expressed in flower of both black and brown grain chickpea. It is intriguing to investigate if CaMYB114 and the previously reported CabHLH may interact to control flower colour. In addition to the transcription factors, downstream structural genes required for anthocyanin biosynthesis may also be explored to fully understand the molecular mechanisms of black seed coat in chickpea. Specifically, the transcription of two candidate genes F3’H and F3’5’H responsible for the biosynthesis of red and blue anthocyanins [ 60 ], respectively, need to be investigated. This can be achieved through comparative transcriptome and metabolome analyses between black and non-black chickpeas. Genetic variations such as SNPs and indels in key anthocyanin biosynthesis genes have been reported to be associated with seed colours in many species, such as soybean [ 10 ], cowpea [ 61 ], lupin [ 62 ], common bean [ 21 , 63 ], pumkin [ 64 ], bitter gourd [ 65 ], lettuce [ 66 ], watermelon [ 67 ], and Chinese cabbage [ 68 ]. Many of these traits and variations have been suggested to be under selection during crop domestication. In this study, we found a similar case for the black coloured chickpea which is closely linked with a 12 bp deletion in the promoter region of CaMYB114 . It would be interesting for future studies to investigate the geographical origin and selection pressure of this allele during chickpea evolution and domestication. Conclusions Genome-wide association analyses using a diverse set of germplasm revealed a single dominant QTL CaBlk3-1 on chromosome Ca3 controlling the black seed coat in chickpea. We identified a MYB transcription factor CaMYB114 as the underlying candidate gene for CaBlk3-1 . Transcriptional analyses in various tissues of brown and black chickpea genotypes confirmed that the expression of CaMYB114 was closely linked with the development of the black seed coat. In addition, CaMYB114 was also found actively expressed in coloured chickpea flower both black and non-black chickpea lines. We identified a 12 bp deletion in the promoter region of CaMYB114 as the potential cause of black-coloured chickpea, which may have altered the transcription pattern of CaMYB114 in black seed coat chickpea. This study will unlock the potential for breeding new chickpea varieties with desired grain colour to meet various market requirements. Abbreviations Proanthocyanidin (PA) Early biosynthetic genes (EBGs) Chalcone synthase (CHS) Chalcone isomerase (CHI) Flavanone 3-hydroxylase (F3H) Flavonoid 3′-hydroxylase (F3′H) Flavonol 96 synthase (FLS) genome-wide association study (GWAS) Australian Grains Genebank (AGG) Genotyping-by-sequencing (GBS) Maximum likelihood (ML) Fixed and random model Circulating Probability Unification (FarmCPU) Days after flowering (DAF) Declarations Data availability The datasets generated and/or analysed during the current study are not publicly available due to intellectual property protection by funding body but are available from the corresponding author on reasonable request. Author contribution CL, YJ, DS supervised the study. HL, YJ wrote the manuscript. LAM, JB, HL responsible for phenotyping. ZL, YJ, LL, CT for GWAS and variant calling. SK for SNP-chip array genotyping. HL, GR, WW for gene expression, sequencing, and genotyping analyses. All authors have read the manuscript. Acknowledgement and Funding The authors would like to thank the chickpea research community for making the genomic data available to the public. The work was funded through Grains Research and Development Corporation (GRDC) project DAW2205-004RTX and UMU2303-003RTX. Seeds were made available through SMTA with the Australian Grains Gene bank. Conflict of interests The authors declared no conflict of interests. Ethics, Consent to Participate, and Consent to Publish declarations Not applicable Clinical trial number Not applicable References Wallace TC, Murray R, Zelman KM: The Nutritional Value and Health Benefits of Chickpeas and Hummus . Nutrients 2016, 8 (12). Yegrem L: Nutritional Composition, Antinutritional Factors, and Utilization Trends of Ethiopian Chickpea (Cicer arietinum L.) . Int J Food Sci 2021, 2021 . 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Li BB, Lu XQ, Gebremeskel H, Zhao SJ, He N, Yuan PL, Gong CS, Mohammed U, Liu WG: Genetic Mapping and Discovery of the Candidate Gene for Black Seed Coat Color in Watermelon (Citrullus lanatus) . Frontiers in Plant Science 2020, 10 . Ren YJ, He Q, Ma XM, Zhang LG: Characteristics of Color Development in Seeds of Brown- and Yellow-Seeded Heading Chinese Cabbage and Molecular Analysis of the Candidate Gene Controlling Seed Coat Color . Front Plant Sci 2017, 8 . Additional Declarations No competing interests reported. Supplementary Files Supplementaryfiles.xlsx Supplementary materials Supplementary file S1. Annotated candidate genes within 0.5 Mb upstream and downstream of CaBlk3-1 QTL. Supplementary file S2. Full list of protein sequences of MYBs in chickpea and Arabidopsis. Supplementary file S3. Genetic variations identified in the genic region of CaMYB114 in diverse chickpea lines. Cite Share Download PDF Status: Published Journal Publication published 11 Nov, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 27 Aug, 2025 Reviews received at journal 21 Aug, 2025 Reviewers agreed at journal 17 Aug, 2025 Reviews received at journal 15 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers invited by journal 10 Aug, 2025 Editor assigned by journal 10 Aug, 2025 Editor invited by journal 08 Aug, 2025 Submission checks completed at journal 08 Aug, 2025 First submitted to journal 08 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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04:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7248383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7248383/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07544-0","type":"published","date":"2025-11-11T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89071645,"identity":"89c4e7c0-87b8-47f7-951f-a669b7a45469","added_by":"auto","created_at":"2025-08-14 11:17:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":427950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotyping of the black seed coat trait in chickpea. A\u003c/strong\u003e) The four different seed coat colours scored across the examined chickpea germplasms. \u003cstrong\u003eB\u003c/strong\u003e) The plant of Negro, a black seed chickpea line. \u003cstrong\u003eC\u003c/strong\u003e) The chickpea seed development of a black seed line (Negro) and a brown seed Desi line (Genesis 836). \u003cstrong\u003eD\u003c/strong\u003e) Pigment extraction from brown and black chickpea seeds.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7248383/v1/fd6b8821c8859aa82464b9ed.png"},{"id":89071647,"identity":"a40ad659-4764-414b-83c0-1d9818037975","added_by":"auto","created_at":"2025-08-14 11:17:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":361164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGWAS analysis of black seed trait in chickpea germplasm collection. A\u003c/strong\u003e) Manhattan plot showing the significant QTLs associated with black seed coat in chickpea. The coloured bars displayed the SNP density/number within 1Mb window size. \u003cstrong\u003eB\u003c/strong\u003e) PCA plot of the chickpea lines used for GWAS analysis. Black seed coat chickpea lines were coloured in black. \u003cstrong\u003eC\u003c/strong\u003e) The most significant SNP markers associated with black seed.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7248383/v1/1f04174e0cede4464a14910e.png"},{"id":89071655,"identity":"bac92101-ea70-4bd0-9b1d-aaca36e9b539","added_by":"auto","created_at":"2025-08-14 11:17:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":124530,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisplays the phylogeny clustering of CaMYB114. \u003c/strong\u003eThe ML phylogeny of chickpea MYBs (orange) was developed using Arabidopsis MYBs (blue) as references. CaMYB114 (XP_004491866.1), GmMYBA2, VrMYB90 were highlighted in red colour. The clade encompassing Arabidopsis MYB90, MYB113, MYB114 were coloured in red.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7248383/v1/21c0f543bf5fdb4ca6ad31c4.png"},{"id":89071663,"identity":"7ac1706d-4504-4d1d-b365-545c95cebcaf","added_by":"auto","created_at":"2025-08-14 11:17:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":359492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaMYB114 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression in chickpea seed coat and other tissues. A\u003c/strong\u003e) Comparing the \u003cem\u003eCaMYB114\u003c/em\u003egene expression in different tissues of black and brown chickpea. \u003cstrong\u003eB\u003c/strong\u003e) Comparing the \u003cem\u003eCaMYB114\u003c/em\u003e in seed coat at different development stages of black seed and brown seed lines. \u003cstrong\u003eC\u003c/strong\u003e) Public transcriptome profile of \u003cem\u003eCaMYB114\u003c/em\u003ein various tissues extracted from CTDB. DAF: days after flowering; RQ: relative quantification; SC: seed coat.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7248383/v1/3647d7fe98d14a52e857e879.png"},{"id":89071648,"identity":"6b40bb20-c5c6-4ebc-bc78-195aabd8ec31","added_by":"auto","created_at":"2025-08-14 11:17:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":325439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisplays the gene structure, indel variations, and genotyping of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaMYB114\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. A\u003c/strong\u003e) Displays the gene structure of \u003cem\u003eCaMYB114\u003c/em\u003e and the location of the identified 12 bp deletion. \u003cstrong\u003eB\u003c/strong\u003e) Displays the genotyping results of MYBindel-F2R2 in brown (white ID) and black (blue ID) chickpea lines. \u003cstrong\u003eC\u003c/strong\u003e) Sanger sequencing of the promoter region of CaMYB114 in brown (Genesis 836, Slasher) and black (ILC10392, IG132577) chickpea lines.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7248383/v1/c1b2710455256c2d9d3b718c.png"},{"id":96105373,"identity":"b59b25e5-9fa7-492a-8b48-ca8c3189a92b","added_by":"auto","created_at":"2025-11-17 16:11:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5475013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7248383/v1/f0b378fc-cf28-4db3-bfcc-0baf9e599ec2.pdf"},{"id":89072574,"identity":"998c582f-1f2e-4e89-8b93-23a41a30460f","added_by":"auto","created_at":"2025-08-14 11:25:33","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":269572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary file S1\u003c/strong\u003e. Annotated candidate genes within 0.5 Mb upstream and downstream of \u003cem\u003eCaBlk3-1\u003c/em\u003eQTL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary file S2\u003c/strong\u003e. Full list of protein sequences of MYBs in chickpea and Arabidopsis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary file S3\u003c/strong\u003e. Genetic variations identified in the genic region of CaMYB114 in diverse chickpea lines.\u003c/p\u003e","description":"","filename":"Supplementaryfiles.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7248383/v1/27976aea91a00990a9c2edb0.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A transcription factor gene CaMYB114 associated with black seed coat in chickpea","fulltext":[{"header":"Background","content":"\u003cp\u003ePopulation growth and climate change have significantly threatened global food security in recent years. In this context, chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e) becomes a highly valued crop, providing affordable nutritious food for a growing global population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is rich in protein, energy, fibre, minerals, vitamins, and health-promoting phytochemicals [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Beyond its affordability and rich nutrient profile, the growing interest in chickpea is also driven by its adaptability to drought-prone and semi-arid environments [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], making it a sustainable and reliable crop in the face of climate change challenges.\u003c/p\u003e\u003cp\u003eThe global trade and demand for chickpeas are steadily increasing since early 2000s [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and the leading exporters include Australia, Canada, and Argentina [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Grain quality is essential to meet international standards and ensure suitability for the global market. The seed colour is a key visual trait that plays a significant role in market value and consumer preference. Chickpea is commonly classified into two main types: Kabuli, with large seeds, and Desi, with small, rough seeds. The chickpea seed colour can vary widely, ranging from cream, beige for Kabuli, and light brown to darker colours for Desi, depending on both environments and genetic factors. Anthocyanin and proanthocyanidin (PA) biosynthesis are known to play a critical role in determining chickpea seed coat colours [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A few studies have identified some key genes involved in this trait. A study using an integrated genomics approach across diverse Cicer genetic resources identified a seed-colour gene \u003cem\u003eCaMATE23\u003c/em\u003e, which influenced PA accumulation in seed coat through the flavonoid pathway. An 8-bp InDel in the \u003cem\u003eCaMATE23\u003c/em\u003e promoter played a key role in the transition from grey seeds in wild Cicer to the dark brown seeds in cultivated chickpeas [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Another recent study found that natural variants of two anthocyanin and PA biosynthesis genes (\u003cem\u003eCabHLH\u003c/em\u003e and \u003cem\u003eCaMATE1\u003c/em\u003e) explained the seed colour variation between Kabuli and Desi chickpea [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThere is an old type of chickpea that resembling Desi chickpeas in size but features a black seed coat [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The seed colour was showed to be linked to some biochemical compounds, with darker-coloured seeds often containing higher levels of antioxidants [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recent studies have incorporated black chickpea wholemeal flour into wheat flour for bread and pasta production. The addition of black chickpea flour not only increased the protein and fibre content but also enriched the final product with higher antioxidant activity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The rising demand for healthy food has revived interest in this old black chickpea, highlighting its potential for commercial development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, no study to date has explored the genetic basis of black seed colour in chickpea. In common bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.), the genetic mechanisms underlying seed colour have been well studied [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Two key genes, flavonoid 3\u0026prime;5\u0026prime;-hydroxylase and MYB113 transcription factor, were identified as candidates responsible for black seed colour [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. MYB transcription factors regulate flavonoid biosynthesis by controlling early and late biosynthetic genes (EBGs and LBGs). In dicots, MYB transcription factors independently activate early biosynthetic genes (EBGs) such as chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3\u0026prime;-hydroxylase (F3\u0026prime;H), and flavonol synthase (FLS), which are essential for the initial steps in flavonoid production [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While LBGs, including genes that encode enzymes DFR, ANS, ANR, LAR involving in production of PAs and anthocyanins, are regulated by a MBW complex, which includes MYB transcription factors, bHLH proteins, and WD40 repeat proteins [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. MYBs can act as either activators or repressors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A few studies have demonstrated that the MYB gene also served as a key determinant of black seed coat coloration in another two legumes, cowpea (\u003cem\u003eVigna unguiculata\u003c/em\u003e L.) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and soybean (\u003cem\u003eGlycine max\u003c/em\u003e) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Despite its importance, the genetic mechanisms behind chickpea black seed colour remain an area of ongoing research, with no study identifying the key genes responsible for this trait.\u003c/p\u003e\u003cp\u003eIn this study, we genotyped a panel of 261 diverse chickpea accessions using an SNP chip array and evaluated their seed coat colours. We then performed a genome-wide association study (GWAS) on black seed coat, and examined the major candidate gene through gene expression analysis and 5\u0026rsquo; UTR variations. The aim of this project is to provide insights into the genetic basis controlling the black seed in chickpea. Understanding these genetic factors could lead to the development of improved chickpea varieties with desirable colours and enhanced overall quality.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials, phenotyping and genotyping\u003c/h2\u003e\u003cp\u003eThe chickpea materials (261 accessions in total) used in this study were sourced from the Australian Grains Genebank (AGG). These accessions represent a broad genetic diversity, providing a valuable resource for studying seed coat colour diversity.\u003c/p\u003e\u003cp\u003eSeed colour was evaluated through a visual assessment. The assessment was conducted under standardized lighting conditions to ensure consistency and accuracy in colour classification. Multiple observers participated in the scoring process to minimize bias and enhance reproducibility. The seed colours were documented and classified into predefined categories, including black, brown, yellow and green. This visual scoring method provided a straightforward yet effective approach for identifying variations in seed coat pigmentation across different chickpea accessions. The seed coat pigment was extracted from a desi chickpea variety (Genesis 836) and a black-seed variety (Brown Rosena) using a water-based anthocyanin extraction method. Seed coats were manually removed, dried, and ground into a fine powder. For each sample, 0.1 g of seed coat powder was mixed with 500 \u0026micro;L of distilled water. The mixture was gently stirred at room temperature for 30 minutes, followed by centrifugation at 10,000 rpm for 10 minutes. The resulting supernatant was collected.\u003c/p\u003e\u003cp\u003eHigh quality DNA was extracted from young leaves for each accession. Genotyping was performed using either the Multispecies Pulse SNP Chip Array (DAV1905-003RTX) or genotyping-by-sequencing (GBS) at Agriculture Victoria, Australia. The SNP chip array includes 4,449 chickpea-specific markers, providing comprehensive genome-wide coverage, with 1,735 SNPs as exclusive to wild Cicer species. This high-density genotyping approach enables the detection of genetic variations associated with key traits, including seed coat colour. To ensure accuracy in downstream analyses, genotyping data was filtered to remove samples with high heterozygosity (\u0026gt;\u0026thinsp;0.20) before imputation, reducing potential errors in association studies.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhylogeny analyses\u003c/h3\u003e\n\u003cp\u003eMYB sequences in chickpea and Arabidopsis reference genome were downloaded from PlantTFDB v5.0 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://planttfdb.gao-lab.org/index.php\u003c/span\u003e\u003cspan address=\"https://planttfdb.gao-lab.org/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Multiple sequence alignment was performed using kalign 3.0 program [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Maximum likelihood (ML) tree was built using fasttree [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and branch reliability assessment using maximum-likelihood NNIs with 1000 resampling. A separate ML tree was developed based on sequence alignment for the subclade containing CaMYB114 using IQ-TREE with the substitution model JTTDCMut\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4 chosen according to best Bayesian Information Criterion [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. All tree visualization and annotations were applied in Figtree (v1.4.3, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using different colours to distinguish clusters.\u003c/p\u003e\n\u003ch3\u003eGWAS and PCA analyses\u003c/h3\u003e\n\u003cp\u003eGWAS for black seed coat with the diverse chickpea genotypes were conducted using the rMVP R package [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], with a significance threshold at 0.05. Fixed and random model Circulating Probability Unification (FarmCPU) were implemented. Kinship and principal component were calculated using the rMVP and used as covariates for GWAS. Population structure analyses were performed using the top 5 calculated principal components. PCA analyses were performed using VCF2PCACluster tool [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCandidate gene expression analysis\u003c/h3\u003e\n\u003cp\u003eThe seed coat samples of two black lines (Negro and Galbron) and two brown colour Desi lines (PBA Maiden and Genesis 836) were collected 25, 30, 35 days after flowering, to characterize and compare the candidate gene expression in different seed development stages. The leaf, pod, stem and flower samples were also collected in these four chickpea lines to examine the candidate gene expression in different tissues. To further compare the expression difference between black lines and brown lines, the seed coat samples were obtained from another three black lines (IG132577, ILC10392 and Brown Rosena) and three brown Desi lines (PBA Drummond, PBA Slasher and Kyabra) 35 days after flowering.\u003c/p\u003e\u003cp\u003eHigh quality RNA was extracted by TRI Reagent\u0026reg; (Sigma-Aldrich, #T9424) following the instruction. Approximately equal amount of RNA (~\u0026thinsp;1 \u0026micro;g) was used for reverse transcription by SensiFAST\u0026trade; cDNA Synthesis Kit (Bioline, #65053) as per manufacturer instruction. A pair of primers were designed to bind uniquely to the candidate gene and they were made straddle the intron to avoid DNA contamination: MYB114RT_F1 (5\u0026prime;-AGGTTTGAAAAGATGCCGCAAGA-3\u0026prime;) and MYB114RT_R1 (5\u0026prime;-CCTGCAATAAGTGACCACCTGT-3\u0026prime;). The reference gene used in this study was CaCAC as described before [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The real-time PCR was conducted using SensiFAST\u0026trade; SYBR\u0026reg; Lo-ROX Kit (Bioline, #94005) and ViiA\u0026trade; 7 Real-Time PCR System (Applied Biosystems), performed in three biological replicates and three technical replicates. The relative expression levels of target gene were determined using comparative Ct method (2\u003csup\u003e\u0026minus;△Ct\u003c/sup\u003e) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePublic transcriptome data from Chickpea Transcriptome Database (CTDB) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] was downloaded for the extraction of CaMYB114 in multiple tissues.\u003c/p\u003e\n\u003ch3\u003eVariant calling\u003c/h3\u003e\n\u003cp\u003eWhole genome shotgun sequencing data and chickpea passport records were downloaded from a previously published study [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Raw reads were trimmed using fastp program [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] with --qualified_quality_phred 20 and --length_required 30 parameters. The resulted clean reads were mapped to reference genome using BWA-MEM program [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] with default parameters. Variant calling was performed using GATK4 program [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] with final quality filtration: \"QD\u0026thinsp;\u0026lt;\u0026thinsp;2.0, FS\u0026thinsp;\u0026gt;\u0026thinsp;60.0, MQ\u0026thinsp;\u0026lt;\u0026thinsp;40.0, SOR\u0026thinsp;\u0026gt;\u0026thinsp;4.0, MQRankSum \u0026lt; -12.5\" for SNPs and \"QD\u0026thinsp;\u0026lt;\u0026thinsp;2.0, FS\u0026thinsp;\u0026gt;\u0026thinsp;200.0, SOR\u0026thinsp;\u0026gt;\u0026thinsp;10.0\" for indels. The functional effects of obtained genetic variations were annotated using SnpEff program [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSequencing and marker design\u003c/h2\u003e\u003cp\u003eHigh quality DNA was extracted from five black seed lines (Brown Rosana, Negro, IG132577, ILC10392, Galbron) and five brown seed lines (PBA Maiden, Genesis 836, PBA Drummond, PBA Slasher and Kyabra), using a modified CTAB method described before[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The 5' UTR region of the candidate gene was sequenced using a pair of primers: MYBIndelF2 (5\u0026prime;- AGATCTTTTCTACCGTGCTGT-3\u0026prime;) and MYBIndelR2 (5\u0026prime;- CCATGTGGTAAGCTGCTGAA-3\u0026prime;), by the Sanger sequencing method [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, this primer pair was used to screen the 261-chickpea diversity panel, to determine the association between the 5' UTR deletion and the black seed coat colour phenotypes across various chickpea accessions.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003ePhenotyping and development characterization of black grain trait in chickpea\u003c/h2\u003e\u003cp\u003eIn this study, our laboratory collection of diverse chickpea germplasm lines from AGG were subjected to grain colour phenotyping. Due to the lack of black accession in the Kabuli genotypes, we decide to only use the remaining 261 Desi germplasm lines for further detailed grain colour classification and genetic association analyses. Based on visual assessment, the 261 chickpea lines were generally divided into 4 major colour groups: brown (n\u0026thinsp;=\u0026thinsp;200), yellow (34), green (14), and black (13) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This category assignment was used for downstream genome wide association analyses.\u003c/p\u003e\u003cp\u003eTo characterize the development of the black grain trait, we used a black chickpea line Negro (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and a brown chickpea cultivar Genesis 836 and sampled the developing seeds at 20 days after flowering (DAF), 25 DAF, 30 DAF, 35 DAF and 40 DAF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Negro seed displayed deep green at 20 DAF and 25 DAF, started developing black colour at the embryo tip at 30 DAF. The whole seed coat of Negro turned grey colour with green hue at 35 DAF, which further changed to complete black at 40 DAF. In contrast, Genesis 836 seed displayed yellow green at 20 DAF, which changed to deep green at 25 DAF, then gradually ripen with reduced greenness on 30 DAF and 35 DAF and finally become complete brown on 40 DAF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eAccording to literature search, only two types of pigments have been reported responsible for black colouration in plants: anthocyanins and melanins, the latter of which is insoluble [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. To determine the chemical nature of black chickpea, we performed pigment extraction of the black chickpea seed coat, which showed that the black pigment was readily soluble in water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), supporting the black chickpea pigments as anthocyanin.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of QTLs and candidate gene controlling black chickpea grain trait\u003c/h2\u003e\u003cp\u003eTo identify genetic locus controlling the black chickpea grain trait, GWAS analyses were performed. The phenotype was classified into black (1) and non-black (0) for the purpose of GWAS analyses. Genotype data for the 261 chickpea lines using Multispecies pulse SNP chip array was obtained from our recent study [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. GWAS analyses using FarmCPU model revealed a single major significant QTLs \u003cem\u003eCaBlk3-1\u003c/em\u003e on chromosome ca3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The population structure of the used chickpea lines was investigated by PCA analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), which revealed the presence of three major subgroups. The black chickpea lines were present exclusively in one subgroup, implying a single evolutionary origin. The top five most significant associated SNP markers for \u003cem\u003eCaBlk3-1\u003c/em\u003e and their phenotypic impact were displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. The most significant SNP AVR-Ca-01147.03-004829307 at 4.83 Mb displayed p-value at 1.05e-36 and was considered the most confident locus controlling the black grain trait in chickpea.\u003c/p\u003e\u003cp\u003eTo identify potential gene controlling black chickpea grain, we examined the genomic region 0.5 Mb upstream and downstream of AVR-Ca-01147.03-004829307 for candidate genes with annotation function in anthocyanin biosynthesis. Out of the 44 predicted genes, there were only two genes (\u003cem\u003eLOC101489372\u003c/em\u003e and \u003cem\u003eLOC101500181\u003c/em\u003e) encoding MYB transcription factors with potential involvement in anthocyanin biosynthesis (\u003cb\u003eSupplementary File S1\u003c/b\u003e). Of these, \u003cem\u003eLOC101500181\u003c/em\u003e encoded a MYB114-like transcription factor (XP_004491866.1) with reported function in regulating anthocyanin biosynthesis in several crop species [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In contrast, \u003cem\u003eLOC101489372\u003c/em\u003e was annotated as MYB106-like protein with reported function in cuticle biosynthesis and trichome maturation and was thus excluded for further analysis. \u003cem\u003eLOC101500181\u003c/em\u003e, hence renamed as \u003cem\u003eCaMYB114\u003c/em\u003e, is located at 4.51 Mb on chromosome ca3, 0.32Mb upstream of AVR-Ca-01147.03-004829307, was selected as the candidate gene for \u003cem\u003eCaBlk3-1\u003c/em\u003e for further functional validation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePhylogeny analyses of CaMYB114\u003c/h2\u003e\u003cp\u003eTo investigate the functional classification of CaMYB114, a phylogenetic tree of chickpea MYB gene family was developed using Arabidopsis MYBs as the references. A total of 166 and 168 MYBs sequences (\u003cb\u003eSupplementary File S2\u003c/b\u003e) for chickpea and Arabidopsis were downloaded from PlantTFDB database and were used for phylogeny development. Previously reported soybean GmMYBA2 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and mung bean VrMYB90 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] controlling black seed coat trait were also included. A preliminary NJ clustering analyses was performed to identify the subclade containing CaMYB114. Protein sequences for MYBs in the identified subclade were extracted to develop a more robust ML tree. In the developed ML phylogeny (Fig.\u0026nbsp;3), CaMYB114 (XP_004491866.1), GmMYBA2, and VrMYB90 were clustered together, implying a conserved gene function across different legume species. Using Arabidopsis MYBs as references, this subclade containing CaMYB114, GmMYBA2, and VrMYB90, displayed the closest relationship with Arabidopsis MYB90, MYB113, and MYB114 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], which is consistent with their gene annotation as MYB114-like protein involved in anthocyanin biosynthesis. In addition to CaMYB114, another two chickpea MYBs (XP_00449414.1 and XP_00449415.1) annotated as MYB90-like proteins were also clustered in the same clade with CaMYB114. XP_00449414.1 and XP_00449415.1 correspond to two protein isoforms encoded by the same gene LOC101494325 (3.62 Mb) on chromosome ca2. This MYB gene was excluded as candidate for black chickpea trait due to lack of QTL on chromosome ca2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eValidation of candidate gene CaMYB114 for black grain trait\u003c/h2\u003e\u003cp\u003eTo investigate the expression profile of \u003cem\u003eCaMYB114\u003c/em\u003e across different tissues, we grow plants for two black (Negro and Galbron) and two brown (Genesis 836 and Maiden) and performed qRT-PCR in leaf, pod, stem, flower, and seed coat tissues. Results showed that \u003cem\u003eCaMYB114\u003c/em\u003e transcription was only observed in stem, flower, and seed coat but not in leaf and pod (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In stem, \u003cem\u003eCaMYB114\u003c/em\u003e\u0026rsquo;s expression varied significantly across four chickpea genotypes, with abundant expression in Genesis 836, moderately in Negro, and weakly in Maiden and Galbron. In the flower tissue, all four genotypes displayed abundant expression of \u003cem\u003eCaMYB114\u003c/em\u003e, with Negro and Genesis 836 relatively higher than Maiden and Galbron. In the seed coat, \u003cem\u003eCaMYB114\u003c/em\u003e displayed enormous expression in black genotypes Negro and Galbron but only slightly in the brown genotypes Genesis 836 and Maiden. Taken together, the transcription of \u003cem\u003eCaMYB114\u003c/em\u003e was only positively correlated with the seed colour trait in the seed coat tissue, but not in the other tissues, supporting its potential involvement in the black seed coat trait.\u003c/p\u003e\u003cp\u003eTo further confirm \u003cem\u003eCaMYB114\u003c/em\u003e\u0026rsquo;s involvement in seed coat colour development, we performed qRT-PCR of \u003cem\u003eCaMYB114\u003c/em\u003e at 25 DAF, 30 DAF, 35 DAF in the seed coat of the above 4 chickpea genotypes. Results showed that \u003cem\u003eCaMYB114\u003c/em\u003e was barely expressed at 25 DAF in all chickpea genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). At 30 DAF, we observed moderate expression of \u003cem\u003eCaMYB114\u003c/em\u003e in the two black genotypes but only slight transcription in the brown genotypes, consistent with the emergence of black colouration at this stage. Furthermore, at 35 DAF when the black colouration expands to whole seed, we found that the expression of \u003cem\u003eCaMYB114\u003c/em\u003e increased dramatically in the black genotypes but remained low in the brown genotypes. These results matched well with the development changes of the black colouration. To further confirm \u003cem\u003eCaMYB114\u003c/em\u003e\u0026rsquo;s role in black chickpea grain, we included another 3 brown and 3 black genotypes for qRT-PCR analyses at 35 DAF, which revealed highly consistent transcriptional pattern as that observed in the other 4 genotypes, i.e \u003cem\u003eCaMYB114\u003c/em\u003e was highly transcribed only in black seed coat but weakly in brown seed coat (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, we also extracted the transcriptome data from public database (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), which showed that the transcription of \u003cem\u003eCaMYB114\u003c/em\u003e was restricted to reproductive tissues but not transcribed in root, vegetive, and seed of the brown chickpea, consistent with our qRT-PCR results.\u003c/p\u003e\u003cp\u003eIn summary, gene transcriptional analyses of \u003cem\u003eCaMYB114\u003c/em\u003e provided well support its role in controlling black chickpea grain trait and potentially flower colour as well.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGenetic variation and molecular marker design analyses of CaMYB114\u003c/h2\u003e\u003cp\u003eThe gene structure of \u003cem\u003eCaMYB114\u003c/em\u003e was examined. As showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, CaMYB114 contained 3 exons and 2 introns, with a 316 bp 5UTR and 192 bp 3UTR regions. To investigate the genetic variations within CaMYB114 gene regions, potentially those directly linked to the black and non-black chickpea grain traits, we downloaded previously published whole genome resequencing data for 163 diverse chickpea germplasm covering different species, cultivars, landraces, and wild lines and performed variant calling against the reference genome. We identified a total of 301 SNPs and indels within the 5UTR, genic region, and 3UTR (\u003cb\u003eSupplementary file S3\u003c/b\u003e). Due to the active expression of \u003cem\u003eCaMYB114\u003c/em\u003e in flower tissue of both black and non-black chickpea lines, we reason that the genetic variations controlling the black colouration most likely alter the transcriptional profile of \u003cem\u003eCaMYB114\u003c/em\u003e, instead of the encoded amino acid sequence. Therefore, we focused on indels present in 5UTR, intron, and 3UTR which are known to modulate gene expression. We designed position-specific molecular markers targeting the 7 indels and run those markers on 13 chickpea genotypes (6 black and 7 non-black). Genotyping results showed that only the marker MYBindel-F2R2 targeting a 12 bp indel on 5UTR region matched perfectly with the grain colour (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The black chickpea genotype displayed a deletion at this locus. We further run MYBindel-F2R2 on a larger collection of 1160 chickpea DNA samples and identified additional 9 chickpea lines with the black allele. Grain colour examination showed that all those lines with the black allele displayed a black grain trait, supporting MYBindel-F2R2 as the potential causal variant. To validate the 12 bp deletion in black coloured chickpea, we performed sequencing analysis using the MYBindel-F2R2 primers. Results showed that there were indeed a 12 bp deletion in the black genotypes ILC10392 and IG132577 compared to 2 brown desi cultivars Genesis836 and Slasher (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGrain colour is an important quality trait, particularly in legume species, impacting market value, consumer preference, and nutritional properties [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Among various legume species, chickpea is playing an increasingly important role in the global food system due to its high nutritional value, climate resilience, and affordability [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In chickpea breeding and research, chickpea genotypes with various coloured grains have gained renewed interests for their various nutritional benefits and superior environmental adaptability in the face of climate change, such as high antioxidant content and potential in drought and heat stress resistance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, our efforts in harnessing these valuable genetic resources of coloured grains in chickpea breeding have been greatly hindered by the poor understanding of their genetic basis and the lack of gene-specific molecular markers. In this study, we employed accurate genotyping and phenotyping of a genetically diverse panel of 261 desi chickpeas and mapped the genetic locus controlling black/dark chickpea grain to a major QTL \u003cem\u003eCaBlk3-1\u003c/em\u003e on chromosome ca3. Based on comprehensive gene functional prediction, protein homology, transcriptional, and genetic variation analyses, we successfully identified a MYB transcription factor \u003cem\u003eCaMYB114\u003c/em\u003e as the underlying candidate gene responsible for the black grain trait in chickpea. Most importantly, we discovered and validated a 12 bp deletion in the promoter region of \u003cem\u003eCaMYB114\u003c/em\u003e as the direct genetic cause of black coloured chickpea. This represents a significant advancement in our understanding of coloured seeds in chickpea. The development of gene-specific molecular markers will unlock the potential for breeding new chickpea varieties with desired grain colour to meet various market requirements.\u003c/p\u003e\u003cp\u003eChickpea grains can naturally develop various colours including cream, beige, light brown, darker browns, yellow, green, and black [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Of these, Kabuli chickpea seeds typically have the light cream or beige colour, whereas Desi chickpeas exhibit a wider range of colours from light brown to deep black. This is consistent with our observation that the black colour was present exclusively in Desi chickpeas. Therefore, we excluded Kabuli chickpeas for GWAS analyses to avoid potential population stratification issue, which may lead to the identification of false positive association [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. We identified a single major QTL \u003cem\u003eCaBlk3-1\u003c/em\u003eon ca3 significantly linked to the black grain trait in chickpea. A previous GWAS study [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] on chickpea seed coat colour employed genotyping by sequencing on a diverse 172 chickpea lines, including 93 cultivars (desi and kabuli) and 79 wild chickpeas, and reported 15 QTLs spanning all chromosomes in chickpea. Despite the presence of black coloured chickpea lines in their collection, none of these 15 QTLs overlap with \u003cem\u003eCaBlk3-1\u003c/em\u003e identified in our study. Our GWAS analyses may be advantageous in two aspects: the exclusive use of desi genotypes and the classification of seed colour into black (1) and non-black (0) for the specific examination of the black trait, which contrasts with the classification of 24 seed colour types in their analyses. In plants, two major types of black pigments have been reported: anthocyanins and melanins, the latter of which are knowns as insoluble compounds [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and have been linked to the black hull in barley, wheat, and rice seeds [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. To narrow down the potential candidate gene for \u003cem\u003eCaBlk3-1\u003c/em\u003e, we examined the chemical nature of the black pigments in black chickpea and confirmed that they are readily soluble in extraction buffer, suggesting that the black chickpea trait may be caused by anthocyanins production, instead of melanins. Despite that the anthocyanin composition in black coloured chickpea remain to be characterized, insights may be drawn from several related studies in other legume grains. In black cowpea, comparable amounts of red anthocyanin (cyanidin\u0026ndash;3\u0026ndash;O\u0026ndash;glucoside, 37.2%) and blue anthocyanins (delphinidin\u0026ndash;3\u0026ndash;O\u0026ndash;glucoside, 20.0%; malvidin\u0026ndash;3\u0026ndash;O\u0026ndash;glucoside, 16.5%) were identified [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In contrast, black kidney beans and black soybean seem to contain dominant amount of blue anthocyanin (delphinidin\u0026ndash;3\u0026ndash;O\u0026ndash;glucoside, 60.0%) and red anthocyanin (cyanidin\u0026ndash;3\u0026ndash;O\u0026ndash;glucoside, 98.6%), respectively [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Another report in black bean showed that blue anthocyanins (delphinidin 3-glucoside, 65.7%; maldivin 3 glucoside 8.7%) were dominant [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. These studies suggested that the black seed coat of legumes may involve both red and blue anthocyanins, the latter of which may be more prevalent and may be critical for the black colouration. In contrast to black seed coat, the anthocyanin contents in brown desi chickpea have been determined with dominant yellow anthocyanin (petuinidin), followed by significant amount of red and blue anthocyanins as well [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Future anthocyanin profiling analyses are needed to confirm if blue anthocyanins may be the dominant type in black-coloured chickpea grain.\u003c/p\u003e\u003cp\u003eThe biosynthesis of anthocyanin in plants is known to be regulated by a complex of MYB, bHLH, and WD40 transcription factors, which are highly conserved across species [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The identification of the MYB transcription factor CaMYB114 is consistent with an anthocyanin basis for the black chickpea trait. Interestingly, the black seed coat trait in soybean [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and mung bean [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] has also been shown to be controlled by MYB transcription factors. Furthermore, all three MYBs displayed the closest homology with Arabidopsis MYB113, MYB114, and MYB90 that have been shown involved in anthocyanin biosynthesis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. These observations suggest that the black seed coat trait may share a common molecular basis. Recently, the brown seed colour trait in chickpea has been characterized to be controlled by a bHLH transcription factor CabHLH and multidrug and toxic compound extrusion transporter CaMATE1 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which were shown to regulate the anthocyanin and proanthocyanin accumulation in both seed coat and flower. All candidate genes associated with chickpea grain colour and flower colour to date are linked to anthocyanin production. In addition to black seed coat, we observed that CaMYB114 was also highly expressed in flower of both black and brown grain chickpea. It is intriguing to investigate if CaMYB114 and the previously reported CabHLH may interact to control flower colour. In addition to the transcription factors, downstream structural genes required for anthocyanin biosynthesis may also be explored to fully understand the molecular mechanisms of black seed coat in chickpea. Specifically, the transcription of two candidate genes F3\u0026rsquo;H and F3\u0026rsquo;5\u0026rsquo;H responsible for the biosynthesis of red and blue anthocyanins [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], respectively, need to be investigated. This can be achieved through comparative transcriptome and metabolome analyses between black and non-black chickpeas. Genetic variations such as SNPs and indels in key anthocyanin biosynthesis genes have been reported to be associated with seed colours in many species, such as soybean [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], cowpea [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], lupin [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], common bean [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], pumkin [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], bitter gourd [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], lettuce [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], watermelon [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], and Chinese cabbage [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Many of these traits and variations have been suggested to be under selection during crop domestication. In this study, we found a similar case for the black coloured chickpea which is closely linked with a 12 bp deletion in the promoter region of \u003cem\u003eCaMYB114\u003c/em\u003e. It would be interesting for future studies to investigate the geographical origin and selection pressure of this allele during chickpea evolution and domestication.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eGenome-wide association analyses using a diverse set of germplasm revealed a single dominant QTL \u003cem\u003eCaBlk3-1\u003c/em\u003e on chromosome Ca3 controlling the black seed coat in chickpea. We identified a MYB transcription factor \u003cem\u003eCaMYB114\u003c/em\u003e as the underlying candidate gene for \u003cem\u003eCaBlk3-1\u003c/em\u003e. Transcriptional analyses in various tissues of brown and black chickpea genotypes confirmed that the expression of \u003cem\u003eCaMYB114\u003c/em\u003e was closely linked with the development of the black seed coat. In addition, \u003cem\u003eCaMYB114\u003c/em\u003e was also found actively expressed in coloured chickpea flower both black and non-black chickpea lines. We identified a 12 bp deletion in the promoter region of \u003cem\u003eCaMYB114\u003c/em\u003e as the potential cause of black-coloured chickpea, which may have altered the transcription pattern of \u003cem\u003eCaMYB114\u003c/em\u003e in black seed coat chickpea. This study will unlock the potential for breeding new chickpea varieties with desired grain colour to meet various market requirements.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eProanthocyanidin (PA)\u003c/p\u003e\n\u003cp\u003eEarly biosynthetic genes (EBGs)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChalcone synthase (CHS)\u003c/p\u003e\n\u003cp\u003eChalcone isomerase (CHI)\u003c/p\u003e\n\u003cp\u003eFlavanone 3-hydroxylase (F3H)\u003c/p\u003e\n\u003cp\u003eFlavonoid 3\u0026prime;-hydroxylase (F3\u0026prime;H)\u003c/p\u003e\n\u003cp\u003eFlavonol 96 synthase (FLS)\u003c/p\u003e\n\u003cp\u003egenome-wide association study (GWAS)\u003c/p\u003e\n\u003cp\u003eAustralian Grains Genebank (AGG)\u003c/p\u003e\n\u003cp\u003eGenotyping-by-sequencing (GBS)\u003c/p\u003e\n\u003cp\u003eMaximum likelihood (ML)\u003c/p\u003e\n\u003cp\u003eFixed and random model Circulating Probability Unification (FarmCPU)\u003c/p\u003e\n\u003cp\u003eDays after flowering (DAF)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are not publicly available due to intellectual property protection by funding body but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCL, YJ, DS supervised the study. HL, YJ wrote the manuscript. LAM, JB, HL responsible for phenotyping. ZL, YJ, LL, CT for GWAS and variant calling. SK for SNP-chip array genotyping. HL, GR, WW for gene expression, sequencing, and genotyping analyses. All authors have read the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement and Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the chickpea research community for making the genomic data available to the public. The work was funded through Grains Research and Development Corporation (GRDC) project DAW2205-004RTX and UMU2303-003RTX. \u0026nbsp;Seeds were made available through SMTA with the Australian Grains Gene bank.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate, and Consent to Publish declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWallace TC, Murray R, Zelman KM: \u003cstrong\u003eThe Nutritional Value and Health Benefits of Chickpeas and Hummus\u003c/strong\u003e. \u003cem\u003eNutrients \u003c/em\u003e2016, \u003cstrong\u003e8\u003c/strong\u003e(12).\u003c/li\u003e\n\u003cli\u003eYegrem L: \u003cstrong\u003eNutritional Composition, Antinutritional Factors, and Utilization Trends of Ethiopian Chickpea (Cicer arietinum L.)\u003c/strong\u003e. \u003cem\u003eInt J Food Sci \u003c/em\u003e2021, \u003cstrong\u003e2021\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eBegum N, Khan QU, Liu LG, Li W, Liu D, Haq IU: \u003cstrong\u003eNutritional composition, health benefits and bio-active compounds of chickpea (Cicer arietinum L.)\u003c/strong\u003e. \u003cem\u003eFront Nutr \u003c/em\u003e2023, \u003cstrong\u003e10\u003c/strong\u003e:1218468.\u003c/li\u003e\n\u003cli\u003eBrummer Y, Kaviani M, Tosh SM: \u003cstrong\u003eStructural and functional characteristics of dietary fibre in beans, lentils, peas and chickpeas\u003c/strong\u003e. \u003cem\u003eFood Res Int \u003c/em\u003e2015, \u003cstrong\u003e67\u003c/strong\u003e:117-125.\u003c/li\u003e\n\u003cli\u003eJukanti AK, Gaur PM, Gowda CL, Chibbar RN: \u003cstrong\u003eNutritional quality and health benefits of chickpea (Cicer arietinum L.): a review\u003c/strong\u003e. \u003cem\u003eBr J Nutr \u003c/em\u003e2012, \u003cstrong\u003e108 Suppl 1\u003c/strong\u003e:S11-26.\u003c/li\u003e\n\u003cli\u003eMallikarjuna BP, Patil BS, Meena S, Tripathi S, Bhat JS, Vijayakumar AG, Bharadwaj C: \u003cstrong\u003eBreeding Chickpea for Climate Resilience: An Overview\u003c/strong\u003e. 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Appl Genet \u003c/em\u003e2023, \u003cstrong\u003e136\u003c/strong\u003e(12).\u003c/li\u003e\n\u003cli\u003eLi BB, Lu XQ, Gebremeskel H, Zhao SJ, He N, Yuan PL, Gong CS, Mohammed U, Liu WG: \u003cstrong\u003eGenetic Mapping and Discovery of the Candidate Gene for Black Seed Coat Color in Watermelon (Citrullus lanatus)\u003c/strong\u003e. \u003cem\u003eFrontiers in Plant Science \u003c/em\u003e2020, \u003cstrong\u003e10\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eRen YJ, He Q, Ma XM, Zhang LG: \u003cstrong\u003eCharacteristics of Color Development in Seeds of Brown- and Yellow-Seeded Heading Chinese Cabbage and Molecular Analysis of the Candidate Gene Controlling Seed Coat Color\u003c/strong\u003e. \u003cem\u003eFront Plant Sci \u003c/em\u003e2017, \u003cstrong\u003e8\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"anthocyanins, chickpea, black seed coat, genome wide association analyses, MYB transcription factor, molecular markers","lastPublishedDoi":"10.21203/rs.3.rs-7248383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7248383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eGrain colour is an important quality trait affecting the market value and consumer preference. Chickpeas with black-coloured seed coat is known for their beneficial high antioxidant and fibber content, yet the underlying molecular basis remains poorly understood.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eHere, we examined the grain colour trait of a panel of 261 diverse desi chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e) accessions and specially characterized the development of the black seed coat. We showed that the black colouration emerged on embryo tips at 30 days after flowering (DAF) and expanded to whole grain at 35 DAF. Genome-wide association analyses revealed a single major genetic locus \u003cem\u003eCaBlk3-1\u003c/em\u003e on chromosome Ca3 controlling black seed coat. Candidate gene screening within 0.5 Mb upstream and downstream of \u003cem\u003eCaBlk3-1\u003c/em\u003e identified a single MYB-encoding gene \u003cem\u003eCaMYB114\u003c/em\u003e related to anthocyanin biosynthesis. Phylogeny analyses showed that CaMYB114 was clustered with Arabidopsis MYB90, MYB113, MYB114, consistent with their role in anthocyanin production. Subsequent qRT-PCR analyses suggested that \u003cem\u003eCaMYB114\u003c/em\u003e was abundantly transcribed in black genotypes but weakly in the brown genotypes at 35 DAF, closely linked with black colour development. Genetic variation analyses of \u003cem\u003eCaMYB114\u003c/em\u003e identified a 12-bp deletion containing a GAGA motif in the 5UTR region of black chickpea genotype. A gene-specific marker targeting this deletion was developed to validate its link with the black seed coat in a larger chickpea germplasm collection.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eWe identified a single major QTL and the underlying candidate gene CaMYB114 controlling the black seed coat trait in chickpea. Our study has greatly improved our understanding of the genetic basis of chickpea black seed and will unlock the potential for breeding new chickpeas with desired grain colour to meet various market requirements.\u003c/p\u003e","manuscriptTitle":"A transcription factor gene CaMYB114 associated with black seed coat in chickpea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-14 11:17:28","doi":"10.21203/rs.3.rs-7248383/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-28T03:32:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-21T05:45:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266719674435951011107310583834701742323","date":"2025-08-17T04:05:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-15T06:32:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32174229286650253341597307215782064474","date":"2025-08-14T08:07:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308508017375830971081776987223423250431","date":"2025-08-14T04:24:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-10T15:09:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-10T15:03:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-08T12:18:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-08T07:57:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-08-08T07:53:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ce4d6ec-a97a-48c1-810e-98ebdc4342b9","owner":[],"postedDate":"August 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:08:01+00:00","versionOfRecord":{"articleIdentity":"rs-7248383","link":"https://doi.org/10.1186/s12870-025-07544-0","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-11-11 15:57:09","publishedOnDateReadable":"November 11th, 2025"},"versionCreatedAt":"2025-08-14 11:17:28","video":"","vorDoi":"10.1186/s12870-025-07544-0","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07544-0","workflowStages":[]},"version":"v1","identity":"rs-7248383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7248383","identity":"rs-7248383","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}