Towards carotenoid biofortification in wheat: identification of XAT-7A1, a multicopy tandem gene responsible for carotenoid esterification in durum wheat
BMC Plant Biology volume 23, Article number: 412 (2023)
Yellow pigment content, mainly due to the accumulation of carotenoids, is a quality trait in durum wheat grain as it confers the bright yellow color to pasta preferred by consumers. Also, carotenoids are essential nutrients exerting important biological functions in human health. Consequently, biofortification strategies have been developed in many crops to increase carotenoid content. In this context, carotenoid esterification is emerging as a new breeding target for wheat biofortification, as carotenoid esters have been found to promote both carotenoid accumulation and stability. Until recently, no carotenoid esters have been identified in significant proportions in durum wheat grains, and interspecific breeding programs have been started to transfer esterification ability from common wheat and Hordeum chilense.
In this work, XAT-7A1 is identified as the gene responsible for carotenoid esterification in durum wheat. Sequencing, copy number variation and mapping results show that XAT-7A1 is organized as tandem or proximal GDSL esterase/lipase copies in chromosome 7A. Three XAT-7A1 haplotypes are described: Type 1 copies, associated with high levels of carotenoid esters (diesters and monoesters) production and high expression in grain development; Type 2 copies, present in landraces with low levels of carotenoid esters (monoesters) or no esters; and Type 3 copies, without the signal peptide, resulting in zero-ester phenotypes.
The identification of XAT-7A1 is a necessary step to make the carotenoid esterification ability available for durum and bread wheat breeding, which should be focused on the Type 1 XAT-7A1 haplotype, which may be assessed as a single gene since XAT-7A1 copies are inherited together.
Durum wheat, Triticum turgidum L. subsp. durum (Desf.) is an important crop worldwide, with an annual production of 40 million tons  and with special relevance in the Mediterranean basin, due to its historical origin, distribution and cultural uses . High yellow pigment content (YPC), mainly due to carotenoid accumulation, has been traditionally considered as a quality trait in durum wheat grain, as it confers the bright yellow color to pasta preferred by consumers. Lutein is the main carotenoid in the endosperm of Triticum species [3,4,5]. Carotenoids are lipophilic pigments synthesized by photosynthetic organisms and some fungi and bacteria. They play important roles in plants, contributing to photosynthesis as accessory light-harvesting pigments, preventing the formation of destructive chemical species, quenching the energy excess, and serving as antioxidants . Additionally, they are essential nutrients with important biological functions in human and animal health, being associated with antioxidant and provitamin A activity, enhancement of the immune response, inhibition of carcinogenesis and reduction on the risk of developing cardiovascular and other degenerative or chronic diseases [7, 8]. Consequently, biofortification strategies have been developed in many crops  through transgenic means, such as the well-known Golden Rice , the Carolight® maize  and the tomato enriched for saffron apocarotenoids ; or by using natural variation and traditional breeding such as in vitamin A-biofortified maize , orange sweet potato or cassava (https://www.harvestplus.org/home/crops/).
Also in durum wheat, increasing YPC and carotenoid content has been a breeding target , which has been accompanied by an interest in deciphering the genetic basis of the trait. Carotenoid levels in plants are known to be the result of three interacting variables: biosynthetic rate, degradation rate and storage cell capacity . As a result, abundant information is available about the genetic control of carotenoid synthesis and degradation in wheat [16,17,18]. Conversely, other factors contributing to carotenoid accumulation in wheat grains are not so well understood. In this context, carotenoid esterification is gaining relevance. Carotenoids can be in their free form or esterified with fatty acids. Esterification is known to enhance carotenoid sequestration in plants, providing a metabolic sink acting as a regulatory mechanism leading to higher carotenoid concentrations . Also, carotenoid esters have shown to be more stable than non-esterified carotenoids  and to prevent carotenoid degradation during storage of wheat and tritordeum grain and flour [21,22,23,24]. Furthermore, lutein diesters appeared to be more resistant to lipoxygenase degradation than free and lutein monoesters in noodle sheets . Thus, esterification is emerging as a new breeding target to improve both carotenoid accumulation and stability in durum wheat.
Carotenoid esters had been found in grains of hexaploid and diploid wheats and in tritordeum but not in tetraploid wheats [3,4,5] until the recent characterization of a collection of Spanish durum wheat landraces, which resulted in the identification of four accessions with a high presence of lutein esters (diesters and monoesters), and eleven accessions with lower contents of esters (monoesters only) . The study of the genetic control of lutein esterification had been addressed in the D and Hch genomes [27,28,29]. Further studies in common wheat identified a xanthophyll acyl transferase gene (XAT) belonging to the GDSL esterase/lipase (GELP) family located in chromosome 7D (XAT-7D) as responsible of lutein esterification in this cereal . Shortly after, XAT-7Hch, orthologous of XAT-7D, was reported as the gene responsible of lutein esterification in H. chilense and tritordeum , suggesting a common origin of the mechanism for lutein esterification in the Triticeae species. Thus, carotenoid esterification ability found in durum wheat landraces may be expected to be mediated by the same mechanism, through an orthologue XAT gene in the tetraploid genome. Due to the special importance of carotenoids in durum wheat, where lutein content is a primary selection target for its correlation with pasta color, elucidating the genetic basis of carotenoid esterification in this species would be a milestone in durum wheat breeding. Due to this relevance, and prior to the identification of durum wheat sources , the transfer of XAT-7D and XAT-7Hch genes through interspecific crosses has been proposed and attempted, respectively [32, 33]. However, the availability of durum wheat donors with carotenoid esterification ability eliminates the difficulties associated with interspecific breeding, although both strategies may be complementary.
Thus, the aim of this work is to identify the gene responsible for lutein esterification in durum wheat. For this purpose, several methodologies have been used for answering the questions that arose during the investigation. In this sense, genomic sequencing, expression analysis, copy number variation and genetic and QTL mapping strategies were successfully combined to achieve the objective set forth in this work.
Candidate genes for carotenoid esterification in durum wheat
A common genetic mechanism for carotenoid esterification has been proposed for Triticeae species [29, 31]. Thus, the starting point for identifying the gene responsible for carotenoid ester production in durum wheat grains was that the same mechanism mediated by GDSL esterase/lipase genes described in bread wheat  and in H. chilense genomes , also operated in durum wheat. The hypothesis was that an orthologous and functional copy of XAT-7D, the gene responsible for carotenoid esterification in common wheat , might be present in the durum wheat esterifying genotypes. We searched for the orthologous genes of XAT-7D (TraesCS7D02G094000) in the available tetraploid genomes: T. turgidum (Svevo.v1) and T. dicoccoides (WEWseq_v.1.0) accessible at EnsemblPlants (https://plants.ensembl.org/). The only gene assigned as an orthologue in durum wheat was TRITD4Av1G231510, which had the same exon-intron structure of XAT-7D but shared a sequence identity below 73%. In addition, we performed a BLASTn search of XAT-7D against the durum wheat genomes to identify other putative candidates. Two more genes, TRITD4Av1G231520 and TRITD4Av1G231670, showed similar identity and coverage values with XAT-7D that TRITD4Av1G231510. Additionally, TRITD4Av1G231840 in T. turgidum and TRIDC4AG057350 in T. dicoccoides were identified. Both genes were truncated forms compared to XAT-7D, as they only shared the first exons and introns. Conversely, they were around 95% identical in the region in common. The results of this search are summarized in Additional file 1. None of the genes identified in the two available durum wheat genomes matched what was expected for full-length functional orthologous copy of XAT-7D.
Thus, several primer pairs were designed based on the 5’ and 3’ regions of TraesCS7D02G094000 and its homoeologue TraesCS4A02G397900, considering also the sequences at 5’ of genes TRITD4Av1G231840 and TRIDC4AG057350 in order to tag a putative complete and functional copy in durum wheat. The primer pair XAT_dw-Fw / XAT_dw-Rv (Additional file 2) amplified a PCR fragment expected for a complete copy (1.5 Kb approximately) in some genotypes from a durum wheat Spanish landraces collection, previously characterized for their grain carotenoid and xanthophyll esters profile .
Characterization of XAT-like sequences in durum wheat
Out of the 156 durum wheat landraces tested, 28 lines showed amplification for XAT_dw-Fw / XAT_dw-Rv (Additional file 3). All durum wheat lines from the collection producing carotenoid esters showed amplification: the four genotypes producing both diesters and monoesters in significant proportion and the eleven genotypes producing only monoesters in small amounts described in . Additionally, thirteen lines showing no esters in their carotenoid profile showed positive amplification. Accessions from the three phenotypes were selected for cloning and sequencing of the amplified fragment: BGE047520 and BGE047535 producing diesters and monoesters, BGE048494 producing only monoesters and BGE047499 with no esters. The variety ‘Athoris’ was also included as it was the zero-ester parental of the population developed for mapping purposes (see below).
In all cases, a 1,564 bp fragment was successfully sequenced. By comparing all the sequences obtained, they could be classified into three types which were designed as Type 1, Type 2 and Type 3 considering exclusive single nucleotide polymorphism (SNP) positions (Table 1). Type 1 sequences were identified only in the two genotypes producing significant levels of esters, both diesters and monoesters (BGE047520 and BGE047535). Type 2 was the form present in BGE048494, the genotype which produces low amounts of esters (only monoesters), and Type 3 was exclusive to the genotypes that were not able to produce esters BGE047499 and ‘Athoris’. Table 1 summarizes the polymorphic sites among sequence types as well as the expected amino acid substitutions in the predicted proteins.
Two alternative forms at position 705 (T/G) were consistently identified in Type 3 sequences in both genotypes BGE047499 and ‘Athoris’, which were named Type 3.1 and Type 3.2. In Types 2, 3.1 and 3.2 sequences other SNPs were detected in only one clone and, as polymerase amplification or sequencing errors could not be excluded, they were not identified as new subtypes.
Type 1-like sequences revealed a surprising diversity resulting from different combinations of alternative bases at positions 333 (A/G) at intron 1; 644 (T/C), 691 (A/G), 750 (A/G) and 788 (C/A) at exon 3; 1220 (A/T) and 1270 (G/A) at intron 4 and at 1458 (G/T) in exon 5 (Table 1). Types 1.1, 1.2, 1.3 and 1.4 copies were identified in both BGE047520 and BGE047535, being 1.1 and 1.2 the most represented copies (Table 1). Type 1.3 sequences were 1,536 bp – length, as they were 25 bp shorter in the 5’ UTR. Additionally, other two different combinations designed as 1.6 and 1.7 were found in BGE047535, as well as another one in BGE047520 named 1.5. Other SNPs were detected in these sequences and in other clones analyzed but, only those consistent (i.e alternative bases at the positions formerly described were identified in more than one sequence), were considered to define different Type 1 copies. Thus, the existence of more subtypes within this class cannot be ruled out.
Additionally, a small sample of clones from other landraces showing XAT amplification were sequenced in order to identify copy types. Due to the high diversity of XAT sequences shown above, they were only assigned to Type 1, 2 or 3 and were not analyzed at the subtype level. BGE047507 and BGE047536 harbor Type 1 sequences; BGE012301, BGE045645, BGE048496, BGE047498 and BGE047503 Type 2, and BGE045628, BGE045676 and BGE048499 Type 3 (Additional file 3).
All sequences shared about 96.5 % identity with TraesCS7D02G094000 and none of them were represented in the reference genomes of T. turgidum (Svevo.v1) or T. dicoccoides accession 'Zavitan' (WEW_v.1.0) in the Ensembl repository. Conversely, Type 3.2 sequence was 100% identical to gene LOC119344677 available from the new version of T. dicoccoides genome (WEW_v.2.0) at the NCBI, although its chromosomal location has not been established.
The exon-intron structure was estimated by comparing with TraesCS7D02G094000 and the expected proteins were predicted. A 22 amino acid-length signal peptide (cleavage site between positions 21 and 22) was detected in Type 1 and Type 2 sequences predicting an extracellular protein location, and both types encoding for 351 amino acid length peptides (Additional file 4). However, the signal peptide was lacking in Type 3 sequences due to the T / C substitution at position 36 (Table 1) interrupting the ATG start codon and giving rise a to shorter protein of 320 amino acids. All sequences belong to the GELP family and conserve the catalytic triad consisting of the S in block I domain and the D and H residues in block V  (Additional file 4).
XAT-like sequences copy number variation
To further investigate the nature of the great diversity found for XAT-like sequences in durum wheat, a copy number analysis was carried out by qPCR. Amplification of a 131 bp fragment identical in all Type 1, 2 and 3 copies was quantified in BGE047535, BGE047520, BGE048494, BGE047499 and ‘Athoris’. The landrace BGE018318, without XAT copies, was included as negative control. The lowest copy number was detected in BGE048494 with 2.07 copies. In the same range of variation were BGE047535, BGE047520 and ‘Athoris' with 5.54, 5.26 and 5.73 copies, respectively. The landrace BGE047499 showed the highest number of copies with an estimation of 8.68. No copies (0.12) were detected in the landrace BGE018318 used as negative control (Table 2).
Additionally, seven F2 individuals with different phenotypic profiles and characterized as BGE047535, ‘Athoris’ or heterozygous haplotypes (based on the SNP_1220 and SNP_1243 genotyping; see below in Genetic mapping of esterification ability), were also tested for copy number variation. The average number of copies for F2 BGE047535, ‘Athoris’ and heterozygous haplotypes were 5.68, 5.91 and 6.19 copies, respectively, in the same range of variation that the parents of the mapping population (Table 2).
These results show that XAT-like sequences are present in more than one copy in all the genotypes analyzed, accounting for the diversity detected by sequencing. Additionally, copy number is variable among them, ranging from 2.07 to 8.68 copies in the genotypes analyzed.
Transcript profiling of XAT-like sequences during grain development
An expression analysis was carried out in three distinct phenotypic classes considering the carotenoid esters profile, and whose XAT-like copies have been sequenced: two genotypes (BGE047535 and BGE047520) with a relevant presence of lutein esters (both mono- and diesters), one genotype (BGE048494) with lower content of esters (only monoesters) and one genotype (BGE047499) with no esters, corresponding to the defined sequence Types 1, 2 and 3, respectively. The primer pair for RT-qPCR was designed at the end of exon 4 and the beginning of exon 5, where no polymorphisms were detected among all sequences, to avoid a preferential amplification of any copy. Grain samples from three developing stages (St) were used for transcript profiling: St1 (Zadoks 77, 18 dpa), St2 (Zadoks 83-85, 25 dpa) and St3 (Zadoks 87, 30 dpa), corresponding to the caryopsis late milk, early-soft dough and hard dough developmental stages, respectively.
A similar transcriptional profile was observed in the four genotypes (Fig. 1), with maximum expression at St2 (early-soft dough). Conversely, expression values were dramatically higher in the landraces BGE047535 and BGE047520, producing carotenoid monoesters and diesters in a high percentage, than in the other two genotypes producing only monoesters in a low percentage or no esters (BGE048494 and BGE047499, respectively).
Genetic mapping of esterification ability
An F2 population derived from the cross between the esterifying genotype BGE047535 and the zero-ester variety ‘Athoris’ was constructed for gene mapping purposes and grown under field conditions. Carotenoid profile was evaluated in the parental lines and in the progeny consisting of 120 individuals. A subset of 90 F2 plants and the parent lines were genotyped by using DArTseq markers, which generated the data set for the linkage map construction spanning 2,277.4 cM (Additional file 5). The detailed carotenoid content and profile (including esterification) of the grain harvested in each F2 plant is shown in Additional file 6. Lutein was the major carotenoid followed by zeaxanthin and minor amounts of α- and β-carotene. As expected, the population segregated for esterification ability. The observed segregation for carotenoid esters presence/ absence showed a good fit to the expected ratio for a single dominant gene (χ2 = 1.11; p = 0.292). Total carotenoid content and degree of esterification (%) were used for QTL identification. A QTL for esterification ability (QTL_Est) was detected on chromosome 7A explaining 90% of the phenotypic variation. In addition, a QTL for total carotenoid content (QTL_TCar) was detected in the distal part of chromosome 7A explaining 24% of the total variation (Table 3, Fig. 2).
A diagnostic marker was designed to detect the SNP at position 1243 bp between BGE047535 and ‘Athoris’ XAT-like copies (Table 1). All Type 1 variants detected in the parental line BGE047535 harbor a T at this position, whereas the two Type 3 variants identified in the parental line ‘Athoris’ harbor a C (Table 1). The SNP was scored in the total mapping population (Additional file 6) and showed a good fit to the 1:2:1 ratio expected for a single co-dominant locus (χ2 = 1.2; p = 0.545). The SNP_1243 marker mapped to chromosome 7A (60.7 cM) and co-localized with QTL_Est, indicating that XAT-like sequences are responsible for carotenoid esterification in durum wheat and thus this locus was named XAT-7A1.
A second SNP marker was designed to locate different copies of the BGE047535 haplotype in the mapping population. Type 1.4 and 1.6 copies harbor a T at position 1220 bp, whereas the remaining copies identified in BGE047535 and in the other parental line ‘Athoris’ harbor an A (Table 1). The two specific fragments of 357 bp and 457 bp (corresponding to the ‘A’ and ‘T’ copies, respectively) amplified in BGE047535 and, only the 357 bp fragment (‘A’-copies) was obtained in ‘Athoris’ as expected. The SNP_1220 marker was scored in the F2 mapping population (Additional file 6), where only two phenotypes were identified: A+T (homozygous for BGE047535 or heterozygous) and A (homozygous for ‘Athoris’), indicating that BGE047535 copies were inherited together or they were tightly linked. The segregation showed a good fit to the 3:1 ratio expected for a dominant marker (χ2 = 0.41; p = 0.52), co-segregated with SNP_1243 and then, co-located with QTL_Est for carotenoid esters content.
Figure 3 shows the collinearity between chromosome 7A (genetic vs. physical). The expected physical position of XAT-7A1 would approximately correspond to 54,2 Mbp.
In this work, the gene responsible for carotenoid esterification in durum wheat is identified and named XAT-7A1. A candidate gene approach based on XAT-7D, the gene underlying carotenoid esterification in bread wheat , revealed that orthologous copies were present in some durum wheat lines from a Spanish landrace collection. Sequences of XAT-7A1 gene were obtained from fifteen genotypes (fourteen landraces and ‘Athoris’) with different carotenoid esterification phenotypes. All sequences shared a high identity with XAT genes previously described in Triticeae as being responsible for carotenoid esterification (84 % with XAT-7Hch; 96.5 % with XAT-7D) and contain the conserved GDSL motif near the N- terminal  and the catalytic triad consisting of the active-site S in block I and the D and H amino acids in block V .
Within the landraces harboring XAT-7A1 copies, different esterification phenotypes were observed. In the genotypes analyzed in this work, Type 1 sequences are present in the four accessions (BGE047507, BGE047520, BGE047535 and BGE047536; Additional file 3) producing carotenoid esters (diesters and monoesters) at a remarkable high percentage. Type 2 sequences can be associated with those accessions presenting low levels of carotenoid esters, mostly monoesters, (BGE012301, BGE045645 and BGE048496) or no esters (BGE047498 and BGE047503). In this case, due to the low amounts of monoesters shown, it is technically difficult to discern between monoester traces or no esters at all, which could explain both phenotypes found in accessions with Type 2 XAT-7A1 sequences.
Type 3 sequences are characteristic of the genotypes with no esterification ability such as BGE045628, BGE045676, BGE047499 and BGE048499 (Additional file 3). These type 3 XAT-7A1 copies are lacking the signal peptide sequence directing the protein to the apoplastic region, where carotenoid esterification has been suggested to occur in common wheat grain . An inappropriate location may explain the absence of enzymatic reaction, regardless of protein functionality.
Furthermore, a surprising variability of XAT-7A1 sequences was detected within each genotype, which could not be explained by the hypothesis of two alternative alleles at a single locus. In our analyses, subtypes were only considered if a sequence variant was detected more than once, or if the polymorphic positions were consistent between them (Table 1). This conservative criterion underestimated the number of different XAT-7A1 copies, as shown by copy number analysis carried out afterwards. The XAT-7A1 gene was detected in more than one copy in all the genotypes analyzed, and the copy number was variable among them, ranging from 2.07 to 8.68 copies per haploid genome.
To locate the gene responsible for carotenoid esterification in durum wheat, a mapping population was developed by crossing the landrace BGE047535 and the zero-ester variety ‘Athoris’. The carotenoid ester profile was phenotyped in the F2 progeny, showing that the carotenoid esters presence/absence fit well with a single dominant gene segregation. The QTL analysis allowed the location of the carotenoid esterification ability in the short arm of chromosome 7A. The QTL_Est explained 90% of variation, indicating that carotenoid esterification in durum wheat was controlled by a single locus. Two SNP markers, SNP_1243 and SNP_1220, were designed in XAT-7A1 sequences and scored in the F2 population, also showing segregations expected for single dominant loci. The co-localization of SNP_1243, polymorphic between BGE047535 and ‘Athoris’ copies (Table 1), with QTL_Est demonstrated that XAT-7A1 was responsible for carotenoid esterification in durum wheat (Fig. 2). Marker SNP_1220, targeting a different polymorphism among BGE047535 copies (Table 1), co-segregated both with QTL_Est and with marker SNP_1243, showing that XAT-7A1 copies were inherited together and suggesting a tandem organization. This fits with the hypothesis that XAT-7A1 is actually a multicopy locus of linked copies rather than a single copy gene. The results of copy number analysis support this model, as all genotypes tested harbor more than one copy. Specifically, BGE047535 and ‘Athoris’ harbor between five and six XAT-7A1 copies (Table 2). The same range of variation was observed in the F2 individuals analyzed, showing that XAT-7A1 is located in the same chromosomal region (chromosome 7AS) in both parents and also that XAT-7A1 copies segregate as a block, as expected for a multicopy tandem locus and in agreement with the SNP genotyping results discussed above.
Additionally, a QTL for total carotenoid content (QTL_TCar) was identified in the distal part of chromosome 7A (Fig. 2), co-locating with QTLs described in other works for semolina color and YPC [17, 36, 37] and in agreement with previous knowledge of grain pigment content inheritance in durum wheat .
To date, the two genes identified as responsible for carotenoid esterification in cereal grains [30, 31], and XAT-7A1 described herein, belong to the GELP family. This protein family has been extensively studied in many plant species due to its important roles in pathogen defense, growth and development and stress responses . At genomic level, it has been demonstrated that GELP genes are abundant and not evenly distributed on chromosomes, as shown in soybean , tomato  or rice . It has also been proposed that segmental duplication and differences in evolutionary rates are the main causes of the increase in the number and diversity of GELP genes in cotton, resulting in gene and functional diversity . Furthermore, in a recent genome-wide analysis of GELP gene family in bread wheat , three genes, TaGELP452, TaGELP454 and TaGELP455 were mapped to the distal part of the short arm of chromosome 7D, co-locating with XAT-7D (TaGELP453). Then, the model proposed for XAT-7A1 as tandem copies of GELP genes is coherent with the genomic organization of this gene family described in other plant species, although the results presented herein do not make possible to distinguish between tandem or proximal copy duplications.
Transcriptional analysis results are also in agreement with XAT-7A1 being responsible for carotenoid esterification in durum wheat. Expression is detected during grain development, with maximum values at 25 dpa (early-soft dough). Up-regulation was also reported at the same physiological stage for the XAT-7D and XAT-7Hch genes [30, 31]. For XAT-7D gene, transcript levels decrease from this stage on through ripening as happens with XAT-7A1 expression (Fig. 1). XAT-7A1 copies in the landraces BGE047535 and BGE047520, with Type 1 copies and producing carotenoid esters (diesters and monoesters), are highly expressed during grain development. BGE048494 and BGE047499 share the same trend and low expression levels. In the case of BGE048494, with Type 2 sequences, low expression levels correlate to the scarce presence of carotenoid esters, which are detected in a low percentage compared to BGE047535 and BGE047520. Regarding BGE047499, although expression is detected during grain development, the absence of carotenoid esters in this landrace (an in ‘Athoris’ also with Type 3 XAT-7A1 sequences) seems to be related to the lack of signal peptide directing the protein to the apoplastic region, where carotenoid esterification has been suggested to occur in wheat grain . Landraces with the maximum copy number (BGE047499 with 8.68) and the minimum (BGE048494 with 2.07) share low expression levels all through grain development. Therefore, expression levels are not associated with gene copy number but with gene copy type, as the highly expressed Type 1 haplotypes showed higher carotenoid esterification activity (Additional file 3). However, it cannot be excluded that an effect on expression and on phenotype is related to the number of copies within the Type 1 sequences, since the BGE047535 and BGE047520 genotypes used in this study had the same number of copies (Table 2).
In the landraces studied in this work, all XAT-7A1 copies identified within a genotype belong to the same sequence type. In the case of Type 1 sequences, where more subtypes were detected, the possibility cannot be excluded of different functionalities among copies. However, for practical purposes, the Type 1 XAT-7A1 copies have shown to be inherited as a block. Thus, transferring the esterifying ability through a breeding program may be assessed as for a single-gene strategy. The availability of durum wheat XAT donor sources facilitates intraspecific introgression into elite durum wheat lines. Nevertheless, the value of interspecific XAT sources for durum wheat breeding should be evaluated, such as the use of XAT-7Hch  or XAT-7D , since new substrate specificities or preferences may be added to the carotenoid esterification durum wheat catalog. Indeed, a significant effect on the carotenoid stability due to the fatty acid involved and the acylation position in the lutein molecule has been reported . Moreover, XAT-7A1, located in chromosome 7A, could be considered for common wheat biofortification by pyramiding with the XAT-7D gene.
None of the XAT-7A1 sequences identified in this work were present in the two available tetraploid wheat reference genomes [45, 46]. The lack of representation of XAT-7A1 is not surprising and may be explained by its low frequency in durum wheat. Even in the landrace collection, which by definition is a diversity reservoir, XAT-7A1 was only detected in the 18 % of the accessions. This is also consistent with the lack of previous reports of durum wheat accessions producing carotenoid esters in a significant proportion, especially diesters, until the characterization of the Spanish durum wheat landraces by . Although reference genomes are valuable and essential resources, they do not fully capture intraspecific genomic variation. Thus, pangenomes are needed for a global genomic perspective of variation within a species. Single-nucleotide polymorphisms (SNPs), insertions or deletions (indels), presence/absence variation (PAV) and gene copy number variation (CNV) are sources of genomic variation known to influence agronomic traits. Pangenome studies in bread wheat have revealed that approximately 12% of genes have PAVs and 26% of genes were found in tandem duplications, indicating that CNV is a major contributor to genetic variation . Graph pangenomes, such as the recently published Panache visualization tool for PAVs in bread wheat  are valuable resources to inspect intraspecific genomic variation. Large-scale structural variations have been also identified through bread wheat pangenome studies. For instance, a translocation between chromosomes 5B and 7B was detected in 66% of the 538 common wheat lines analyzed [47, 49]. Unfortunately, a durum wheat pangenome is still in progress. PAV in durum wheat short arm of chromosome 7A may be an explanation for the variability found for XAT-7A1 in the collection. Also, a 4AL/7AS translocation or duplication may be responsible of this variation. Candidate genes initially identified by their identity with XAT-7D (Additional file 1) are all located in a region of the long arm of chromosome 4A between 646,988,559 and 647,955,659 Mbp in ‘Svevo’ genome, being also TRIDC4AG057350 in an equivalent position in ‘Zavitan’ genome (Additional file 1). Conversely, in this work, XAT-7A1 is located in the short arm of chromosome 7A. The construction of a high-density consensus map of durum wheat combining segregation data from six mapping populations , suggested that a translocation event took place between 4AL and 7AS chromosomes considering discordant marker positions. Furthermore, evidences of a large translocation involving the same regions are also reported by  by comparing the ‘Svevo’ and ‘Zavitan’ genomes and through a survey of the genetic diversity of a Global Tetraploid Wheat Collection consisting of 1,856 accessions. Although this hypothesis is plausible, it needs further investigation.
XAT-7A1 is identified as responsible for carotenoid esterification in durum wheat and it is located in the short arm of chromosome 7A. All genotypes producing carotenoid esters harbor XAT-7A1 gene copies, orthologous to XAT-7D and XAT-7Hch genes responsible for lutein esterification in common wheat and in H. chilense and tritordeum, respectively. XAT-7A1 Type 1 copies are related to relevant production of carotenoid diesters and monoesters and to high levels of expression during grain development. Type 2 copies are present in landraces producing small amounts of carotenoid esters (mostly monoesters) or no esters, whereas Type 3 XAT-7A1 copies, without signal peptide, results in zero ester production as happens in genotypes lacking this gene. Type 2 and 3 copies are both expressed at low levels during grain development. The majority of the durum wheat landraces do not harbor XAT-7A1 and do not show esters in the carotenoid profile of mature grains. Sequencing, CNV and mapping results revealed that XAT-7A1 is organized as tandem or proximal copy duplications and that all sequences within a genotype belong to the same type class. Also, XAT-7A1 expression seems not to be associated to copy number but to gene copy type which is, at the same time, associated to the carotenoid esters phenotype. Consequently, breeding for carotenoid enhancement should then focus on Type 1 XAT-7A1, which is associated with high levels of carotenoid esterification, and can be assessed as for a single dominant gene strategy as XAT-7A1 copies are inherited together. In this context, homozygous individuals from the F2 population constitute an excellent pre-breeding material for the transference of XAT-7A1 to durum wheat elite varieties.
A total of 156 accessions from a Spanish durum wheat landrace collection [26, 51] were used to test for the presence of the XAT candidate gene in durum wheat. This collection was originally provided by the National Centre for Plant Genetic Resources (CRF-INIA-CSIC) as described in . Seedlings from the landraces BGE047520, BGE047535, BGE048494 and BGE047499 from the former collection were selected and grown in field following a completely randomized design with two replicates (blocks) for expression analyses (see below). BGE047520 and BGE047535 show diesters and monoesters in high proportion, BGE048494 monoesters in low concentration and BGE047499 no carotenoid esters in their grain carotenoid profiles.
In addition, an F2 population was developed by crossing the landrace BGE047535 as the maternal parent with the zero-ester commercial cultivar ‘Athoris’ (LG Seeds). The resulting F1 hybrids were checked with molecular markers to ensure true crosses and a F2 population of 120 individuals was obtained by selfing. This population was grown at field conditions at Finca Alameda del Obispo (Córdoba, Spain) using anti-weed nets and anti-bird structure for the determination of carotenoid content and profile in grain.
Genomic DNA was extracted using the CTAB method as described in  with the specifications described by  from young leaves from the parental lines and the BGE047535 × ‘Athoris’ F2 population. Genomic DNA from the landraces collection was isolated from young leaves following the same method in a previous work .
Extraction of carotenoids and HPLC analysis
Carotenoids pigments were extracted from mature grains from the BGE047535 × ‘Athoris’ F2 population as previously described in . The Spanish durum wheat landraces collection of 156 accessions had been characterized for grain carotenoid and xanthophyll esters profile in previous works [26, 51]. Carotenoid extraction and analysis was performed in duplicate under dimmed light to avoid carotenoid isomerization and photo-degradation. All the analyses were carried out on the same day as the extract was prepared. Carotenoids analysis was performed by HPLC as described in previous works . Calibration curves, prepared with pure pigment standards, were used for carotenoid quantification. The concentration of (Z)-isomers of lutein was assessed by using the calibration curve for (all-E)-lutein. Lutein esters were determined as free lutein equivalents. All data were expressed as µg/g fresh weight (µg/g fw).
Candidate gene amplification and sequence analysis
Genomic sequences of genes TraesCS7D02G094000, TraesCS4A02G397900, TRITD4Av1G231840 and TRITD4Av1G231510 were retrieved from Ensembl Plants and aligned using the multiple sequence alignment tool ClustalW . Primers were designed in the conserved 5’ and 3’ regions by using the NCBI Primer-Blast tool . Primer pair XAT_dw-Fw/ XAT_dw-Rv (Additional file 2) was tested in the Spanish durum wheat collection and successfully amplified a 1,564 bp length fragment in some of the accessions. PCR reactions were carried out with Velocity DNA Polymerase (Bioline, London, UK). The amplicons were cloned into pGEMT-Easy vector (Promega, Madison, WI) and transformed into competent Escherichia coli (DH5α) cells. Plasmids were purified using ZyppyTM Plasmid Miniprep Kit (Zymo Research, CA, US) and used as template for sequencing (STAB VIDA, Portugal).
Sequence editing, alignment and assembly were performed with SeqMan Pro Lasergene Software v17 (DNAStar, WI, US). A minimum of 12 clones were sequenced from genotypes BGE047520 and BGE047535, and five from each of BGE048494, BGE047499 and ‘Athoris’. A minimum of three clones of XAT_dw-Fw/ XAT_dw-Rv amplicon was analyzed in accessions BGE047507, BGE047536, BGE012301, BGE045645, BGE047498, BGE047503, BGE048496, BGE045628, BGE045676, BGE048499.
The identity of the clones as GDSL esterase-lipase- like sequences was confirmed by BLASTn at NCBI. The coding sequences were predicted based on the exon-intron structure of TraesCS7D02G094000, and open reading frames were searched by using ORFfinder at NCBI (https://www.ncbi.nlm.nih.gov/orffinder). Signal peptide and cellular location of the expected proteins were predicted by SignalP 6.0 software .
Genotyping, map construction and QTL analyses
A subset of 90 individuals from the BGE047535 × ‘Athoris’ F2 population was selected for map construction. Genotyping by sequencing analysis of the mapping population was performed by means of DArTSeq platform at Diversity Arrays Technology Pty Ltd (Canberra, Australia).
The genetic map was constructed using Joinmap ® v. 5.0 (Kyazma ®, The Netherlands). Only DArTSeq markers with a call rate of above 90% and with consistent segregation in the parental lines of the population were used for mapping. Markers deviating from mendelian segregation were excluded. A minimum LOD of 16.0 was used to assign markers to chromosomes. For each chromosome, several rounds of mapping were performed by excluding markers co-segregating in the same positions and using the EML algorithm (fastest). A final round of mapping was performed using the Regression Mapping algorithm and the Kosambi mapping function to get the final maps. Collinearity between genetic map and physical positions were inspected using CIRCOS  based on DArTSeq markers with a significant match to ‘Svevo’ genome after BLASTn alignment (E-value < 1.5 × 10−6).
Total carotenoid content and degree of lutein esterification were used for QTL analyses with MapQTL ® v. 6.0 (Kyazma ®, The Netherlands). The nonparametric Kruskal-Wallis test was used to identify marker-trait association in a first stage. After this, interval-mapping analyses were carried out [58, 59]. Finally, MQM mapping was performed [60,61,62]. The QTL significance (p-value) was calculated by using a permutation analysis (1,000 permutations) . QTL figures were generated by using MapChart software v2.32 .
Development of SNP markers
Two markers were designed for mapping purposes following a Tetra-Primer ARMS (amplification refractory mutation system) strategy for SNPs detection . Primer1 web service (http://primer1.soton.ac.uk/primer1.html) was used for primer design. A SNP between BGE047535 and ‘Athoris’ XAT-like copies at position 1243 bp was scored in the mapping population with primers SNP_1243_C-Fw, SNP_1243_T-Rv, SNP_1243_out-Fw and SNP_1243_out-Rv (Additional file 2). A second SNP at position 1220 bp, polymorphic between BGE047535 copies, was scored with primers SNP_1220_A-Fw, SNP_1220_T-Rv, SNP_1220_out-Fw and SNP_1220_out-Rv (Additional file 2). PCR amplifications were performed following the manufacturer’s instructions with MyTaqTM DNA polymerase (Bioline, London, UK) and resolved in agarose gels stained with SafeviewTM Nucleic Acid Stain (NBS biologicals, Ltd., Cambridgeshire, England).
Durum wheat landraces BGE047520, BGE047535, BGE048494 and BGE047499 were grown following a completely randomized design with two replicates at field conditions. Developing grains at 18 (Zadoks 77), 25 (Zadoks 83-85) and 30 (Zadoks 87) days post-anthesis (dpa) , which correspond to late milk, early-soft dough and hard dough, respectively, were used as samples. Two independent biological replicates were collected from each block and immediately frozen in liquid nitrogen at −80 °C. Four grains were randomly selected from each biological replicate for RNA extraction. Total RNA isolation was carried out in duplicate from whole grains using the TRIzol® Reagent (Invitrogen, CA, US), according to manufacturer’s instructions with minor modifications. cDNA was obtained as previously described .
Real-time qPCR reactions for expression analysis were carried out using SYBR® Green on an Applied Biosystems™ 7500 Real-Time PCR System (Applied Biosystems, CA, USA). Four biological replicates with two technical duplicates (which consisted in 1:4 dilutions of each sample), were used as templates. The primer pair qXAT_dw-Fw / qXAT_dw-Rv (Additional file 2) was designed in a highly conserved region from the end of exon four to the beginning of exon five comprising intron four. The following PCR conditions were used: 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. qPCR reactions were performed by using 5 μl of diluted cDNA (10 ng/μl), 5 μl of iTaqTM Universal SYBR® Green Supermix (Bio-Rad, CA, US), and a primer pair concentration of 0.22 μM each. Control samples with no template were included in the reactions.
LinRegPCR quantitative PCR data analysis program (version 11.0)  was used for determining PCR efficiency, using raw normalized fluorescence as input data. Expression for each sample (N0) was calculated using the equation N0 = 0.2/ECq, being E the PCR efficiency for each primer and Cq the number of cycles needed to reach 0.2 arbitrary units of fluorescence.
Normalization was carried out by using the geometric mean of the reference genes ADP-RF(m), RLI(a) and CDC(a) . Expression stability of these three genes and the normalization factors for each sample were assessed using GeNorm .
Copy number variation (CNV) analysis
The primer pair CNV_XAT-Fw / CNV_XAT-Rv (Additional file 2) was designed in a highly conserved region in exon four for copy number variation analysis of XAT copies in genotypes BGE047520, BGE047535, BGE048494, BGE047499, BGE018318 and ‘Athoris’. Seven F2 individuals from the BGE047535 × ‘Athoris’ population were included. These individuals were assigned as BGE047535, ‘Athoris’ or heterozygous haplotypes based on the SNP_1220 and SNP_1243 genotyping and their phenotypic profile for carotenoid esterification (2, 2 and 3 individuals, respectively). Primer pairs TaFAD6  and RLI(a)  were used as reference genes (Ref 1 and Ref 2, respectively).
The amplification profile was the same as described above for expression analysis. qPCR reactions were performed using 3,5 μl of DNA (6,25 ng/μl), 10 μl of iTaqTM Universal SYBR® Green Supermix (Bio-Rad, CA, US) and a primer pair concentration of 0.22 μM each in a final volume of 20 μl. Five biological replicates were used for landraces and ‘Athoris’, and three technical replicates for F2 individuals. PCR efficiency was determined as described above. The ratio between the target gene and each reference genes was calculated as described in by  including the efficiency correction for copy number estimation with the following formula:
where N is the number of copies per haploid genome for each of the reference genes (N = 2, for RLI(a) and TaFAD6).
Availability of data and materials
All data generated and/ or analysed during the current study are included in this published article, in its supplementary information files or available from the corresponding author on reasonable request. XAT-7A1_Type1.1, XAT-7A1_Type1.2, XAT-7A1_Type1.3, XAT-7A1_Type1.4a, XAT-7A1_Type1.4b, XAT-7A1_Type2, XAT-7A1_Type3.1 and XAT-7A1_Type3.2 have been submitted to the GenBank database under the accession numbers: OR082954, OR082955, OR082956, OR082957, OR082958, OR082959, OR082960 and OR082961, respectively.
Copy number variation
Days post anthesis
Motif consensus amino acid sequence of Gly, Asp, Ser, and Leu around the active site Ser
GDSL-type esterase/lipase protein
Presence/ absence variation
Xanthophyll acyl transferase
Yellow pigment content
Xynias IN, Mylonas I, Korpetis EG, Ninou E, Tsaballa A, Avdikos ID, et al. Durum wheat breeding in the mediterranean region: current status and future prospects. Agronomy. 2020;10(3):432.
Martínez-Moreno F, Solís I, Noguero D, Blanco A, Özberk İ, Nsarellah N, et al. Durum wheat in the Mediterranean Rim: historical evolution and genetic resources. Genet Resour Crop Evol. 2020;67:1415–36.
Ziegler JU, Wahl S, Würschum T, Longin CFH, Carle R, Schweiggert RM. Lutein and lutein esters in whole grain flours made from 75 genotypes of 5 triticum species grown at multiple sites. J Agric Food Chem. 2015;63:5061–71.
Paznocht L, Kotíková Z, Šulc M, Lachman J, Orsák M, Eliášová M, et al. Free and esterified carotenoids in pigmented wheat, tritordeum and barley grains. Food Chem. 2018;240:670–8.
Atienza SG, Ballesteros J, Martin A, Hornero-Mendez D. Genetic variability of carotenoid concentration and degree of esterification among tritordeum (×Tritordeum Ascherson et Graebner) and durum wheat accessions. J Agric Food Chem. 2007;55:4244–51.
Britton G. Functions of Intact Carotenoids. In: Britton G, Liaaen-Jensen S, Pfander H, editors. Carotenoids. Basel · Boston · Berlin: Birkhäuser Verlag; 2008. p. 189–212.
Swapnil P, Meena M, Singh SK, Dhuldhaj UP, Harish, Marwal A. Vital roles of carotenoids in plants and humans to deteriorate stress with its structure, biosynthesis, metabolic engineering and functional aspects. Curr Plant Biol. 2021;26:100203.
Rodriguez-Concepcion M, Avalos J, Bonet ML, Boronat A, Gomez-Gomez L, Hornero-Mendez D, et al. A global perspective on carotenoids: metabolism, biotechnology, and benefits for nutrition and health. Prog Lipid Res. 2018;70:62–93.
Zheng X, Giuliano G, Al-Babili S. Carotenoid biofortification in crop plants: citius, altius, fortius. Biochim Biophys Acta - Mol Cell Biol Lipids. 2020;1865:158664.
Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, et al. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science (80). 2000;287:303–5.
Zhu C, Farré G, Zanga D, Lloveras J, Michelena A, Ferrio JP, et al. High-carotenoid maize: development of plant biotechnology prototypes for human and animal health and nutrition. Phytochem Rev. 2018;17:195–209.
Ahrazem O, Diretto G, Rambla JL, Rubio-Moraga Á, Lobato-Gómez M, Frusciante S, et al. Engineering high levels of saffron apocarotenoids in tomato. Hortic Res. 2022;9:uhac074.
Menkir A, Palacios-Rojas N, Alamu O, Dias Paes MC, Dhliwayo T, Maziya-Dixon B, et al. Vitamin A-biofortified maize: exploiting native genetic variation for nutrient enrichment. Sci Br Biofortification. 2018;No. 2 (Feb:1–4).
Digesù AM, Platani C, Cattivelli L, Mangini G, Blanco A. Genetic variability in yellow pigment components in cultivated and wild tetraploid wheats. J Cereal Sci. 2009;50:210–8.
Torres-Montilla S, Rodriguez-Concepcion M. Making extra room for carotenoids in plant cells: new opportunities for biofortification. Prog Lipid Res. 2021;84:101128.
Colasuonno P, Marcotuli I, Blanco A, Maccaferri M, Condorelli GE, Tuberosa R, et al. Carotenoid pigment content in durum wheat (Triticum turgidum L. var durum): an overview of quantitative trait loci and candidate genes. Front Plant Sci. 2019;10:1347.
Colasuonno P, Lozito ML, Marcotuli I, Nigro D, Giancaspro A, Mangini G, et al. The carotenoid biosynthetic and catabolic genes in wheat and their association with yellow pigments. BMC Genomics. 2017;18:1–8.
Ficco DBM, Mastrangelo AM, Trono D, Borrelli GM, De Vita P, Fares C, et al. The colours of durum wheat: a review. Crop Pasture Sci. 2014;65:1–15.
Cazzonelli CI, Pogson BJ. Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci. 2010;15:266–74.
Subagio A, Wakaki H, Morita N. Stability of lutein and its myristate esters. Biosci Biotechnol Biochem. 1999;63:1784–6.
Ahmad FT, Asenstorfer RE, Soriano IR, Mares DJ. Effect of temperature on lutein esterification and lutein stability in wheat grain. J Cereal Sci. 2013;58:408–13.
Mellado-Ortega E, Hornero-Méndez D. Carotenoids in cereals: an ancient resource with present and future applications. Phytochem Rev. 2015;14:873–90. https://doi.org/10.1007/s11101-015-9423-3.
Mellado-Ortega E, Hornero-Méndez D. Lutein esterification in wheat flour increases the carotenoid retention and is induced by storage temperatures. Foods. 2017;6:111.
Mellado-Ortega E, Hornero-Méndez D. Carotenoid evolution during short-storage period of durum wheat (Triticum turgidum conv. durum) and tritordeum (×Tritordeum Ascherson et Graebner) whole-grain flours. Food Chem. 2016;192:714–23.
Mares DJ, Cheong J, Goonetilleke SN, Mather DE. Lipoxygenase in wheat: genetic control and impact on stability of lutein and lutein esters. Foods (Basel, Switzerland). 2021;10.
Requena-Ramírez MD, Hornero-Méndez D, Rodríguez-Suárez C, Atienza SG. Durum wheat (Triticum durum L.) landraces reveal potential for the improvement of grain carotenoid esterification in breeding Programs. Foods. 2021;10:757.
Ahmad FT, Mather DE, Law H-Y, Li M, Yousif S-J, Chalmers KJ, et al. Genetic control of lutein esterification in wheat (Triticum aestivum L.) grain. J Cereal Sci. 2015;64:109–15.
Mattera MG, Cabrera A, Hornero-Méndez D, Atienza SG. Lutein esterification in wheat endosperm is controlled by the homoeologous group 7, and is increased by the simultaneous presence of chromosomes 7D and 7Hch from Hordeum chilense. Crop Pasture Sci. 2015;66:912–21.
Avila CM, Mattera MG, Rodríguez-Suárez C, Palomino C, Ramírez MC, Martin A, et al. Diversification of seed carotenoid content and profile in wild barley (Hordeum chilense Roem. et Schultz.) and Hordeum vulgare L.–H. chilense synteny as revealed by DArTSeq markers. Euphytica. 2019;215:45.
Watkins JL, Li M, McQuinn RP, Chan KX, McFarlane HE, Ermakova M, et al. A GDSL esterase/lipase catalyzes the esterification of lutein in bread wheat. Plant Cell. 2019;31:3092–112.
Requena-Ramírez MD, Atienza SG, Hornero-Méndez D, Rodríguez-Suárez C. Mediation of a GDSL esterase/lipase in carotenoid esterification in tritordeum suggests a common mechanism of carotenoid esterification in triticeae species. Front Plant Sci. 2020;11:592515.
Rodríguez-Suárez C, Requena-Ramírez MD, Hornero-Méndez D, Atienza SG. The breeder’s tool-box for enhancing the content of esterified carotenoids in wheat: From extraction and profiling of carotenoids to marker-assisted selection of candidate genes. In: Wurtzel ET, editor. Carotenoids: Carotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology. Academic Press; 2022. 99–125.
Watkins JL, Pogson BJ. Prospects for carotenoid biofortification targeting retention and catabolism. Trends Plant Sci. 2020;25:501–12.
Akoh CC, Lee G-C, Liaw Y-C, Huang T-H, Shaw J-F. GDSL family of serine esterases/lipases. Prog Lipid Res. 2004;43:534–52.
Upton C, Buckley JT. A new family of lipolytic enzymes? Trends Biochem Sci. 1995;20:178–9.
Fiedler JD, Salsman E, Liu Y, Michalak de Jiménez M, Hegstad JB, Chen B, et al. Genome-wide association and prediction of grain and semolina quality traits in durum wheat breeding populations. Plant Genome. 2017;10:plantgenome2017.05.0038.
N’Diaye A, Haile JK, Cory AT, Clarke FR, Clarke JM, Knox RE, et al. Single marker and haplotype-based association analysis of semolina and pasta colour in elite durum wheat breeding lines using a high-density consensus map. PLoS One. 2017;12:e0170941.
Shen G, Sun W, Chen Z, Shi L, Hong J, Shi J. Plant GDSL esterases/lipases: evolutionary, physiological and molecular functions in plant development. Plants. 2022;11(4):468.
Su H-G, Zhang X-H, Wang T-T, Wei W-L, Wang Y-X, Chen J, et al. Genome-wide identification, evolution, and expression of gdsl-type esterase/lipase gene family in soybean. Front Plant Sci. 2020;11:726.
Yao-guang SUN, Yu-qing HE, He-xuan W, Jing-bin J, Huan-huan Y, Xiang-yang XU. Genome-wide identification and expression analysis of GDSL esterase/lipase genes in tomato. J Integr Agric. 2022;21:389–406.
Chepyshko H, Lai C-P, Huang L-M, Liu J-H, Shaw J-F. Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L. japonica) genome: new insights from bioinformatics analysis. BMC Genomics. 2012;13:309.
Wang J, Zhao H, Qu Y, Yang P, Huang J. The binding pocket properties were fundamental to functional diversification of the GDSL-type esterases/lipases gene family in cotton. Front Plant Sci. 2023;13:1099673.
Yang X, Wang K, Bu Y, Niu F, Ge L, Zhang L, et al. Genome-wide analysis of GELP gene family in wheat and validation of TaGELP073 involved in anther and pollen development. Environ Exp Bot. 2022;200:104914.
Mellado-Ortega E, Hornero-Méndez D. Effect of long-term storage on the free and esterified carotenoids in durum wheat (Triticum turgidum conv. durum) and tritordeum (×Tritordeum Ascherson et Graebner) grains. Food Res Int. 2017;99:877–90.
Zhu T, Wang L, Rodriguez JC, Deal KR, Avni R, Distelfeld A, et al. Improved genome sequence of wild emmer wheat zavitan with the aid of optical maps. G3 (Bethesda). 2019;G3(9):619–24.
Maccaferri M, Harris NS, Twardziok SO, Pasam RK, Gundlach H, Spannagl M, et al. Durum wheat genome highlights past domestication signatures and future improvement targets. Nat Genet. 2019;51:885–95.
Walkowiak S, Gao L, Monat C, Haberer G, Kassa MT, Brinton J, et al. Multiple wheat genomes reveal global variation in modern breeding. Nature. 2020;588:277–83.
Bayer PE, Petereit J, Durant É, Monat C, Rouard M, Hu H, et al. Wheat panache: a pangenome graph database representing presence-absence variation across sixteen bread wheat genomes. Plant Genome. 2022;15:e20221.
Zhao J, Zheng X, Qiao L, Yang C, Wu B, He Z, et al. Genome-wide association study reveals structural chromosome variations with phenotypic effects in wheat (Triticum aestivum L.). Plant J. 2022;112:1447–61.
Marone D, Laidò G, Gadaleta A, Colasuonno P, Ficco DBM, Giancaspro A, et al. A high-density consensus map of A and B wheat genomes. Theor Appl Genet. 2012;125:1619–38.
Requena-Ramírez MD, Rodríguez-Suárez C, Flores F, Hornero-Méndez D, Atienza SG. Marker-trait associations for total carotenoid content and individual carotenoids in durum wheat identified by genome-wide association analysis. Plants. 2022;11:2065.
Murray YHG, Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980;8:4321–6.
Ávila CM, Requena-Ramírez MD, Rodríguez-Suárez C, Flores F, Sillero JC, Atienza SG. Genome-wide association analysis for stem cross section properties, height and heading date in a collection of spanish durum wheat landraces. Plants. 2021;10:1123.
Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, et al. The EMBL-EBI search and sequence analysis tools APIs in. Nucleic Acids Res. 2019;47:W636-41.
Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012;13:134.
Teufel F, Almagro Armenteros JJ, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD, et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol. 2022;40:1023–5.
Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: An information aesthetic for comparative genomics. Genome Res. 2009;19:1639–45.
Ooijen JW. Accuracy of mapping quantitative trait loci in autogamous species. Theor Appl Genet. 1992;84:803–11.
Lander ES, Botstein D. Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics. 1989;121:185–99.
Jansen RC. Interval mapping of multiple quantitative trait loci. Genetics. 1993;135:205–11.
Jansen RC. Controlling the type I and type II errors in mapping quantitative trait loci. Genetics. 1994;138:871–81.
Jansen RC, Stam P. High resolution of quantitative traits into multiple loci via interval mapping. Genetics. 1994;136:1447–55.
Churchill GA, Doerge RW. Empirical threshold values for quantitative trait mapping. Genetics. 1994;138:963–71.
Voorrips RE. MapChart: software for the graphical presentation of linkage maps and QTLs. J Hered. 2002;93:77–8.
Collins A, Ke X. Primer1: primer design web service for tetra-primer ARMS-PCR. Open Bioinforma J. 2012;6:55–8.
Zadoks JC, Chang TT, Konzak CF. A decimal code for the growth stages of cereals. Weed Res. 1974;14:415–21.
Rodríguez-Suárez C, Mellado-Ortega E, Hornero-Méndez D, Atienza SG. Increase in transcript accumulation of Psy1 and e-Lcy genes in grain development is associated with differences in seed carotenoid content between durum wheat and tritordeum. Plant Mol Biol. 2014;84:659–73.
Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acid Res. 2009;37:e45.
Giménez MJ, Pistón F, Atienza SG. Identification of suitable reference genes for normalization of qPCR data in comparative transcriptomics analyses in the Triticeae. Planta. 2011;233:163–73.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:research0034.1 – research0034.11.
Guzmán-López MH, Marín-Sanz M, Sánchez-León S, Barro F. A bioinformatic workflow for indel analysis in the wheat multi-copy α-gliadin gene family engineered with CRISPR/Cas9. Int J Mol Sci. 2021;22:13076.
Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 2001;29:e45–e45.
We gratefully thank Dr. M José Giménez (IAS-CSIC) for the technical advice for CNV analysis.
Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was financed by project PID2021-122152NB-I00 funded by MCIN/AEI/10.13039/501100011033/ and by ERDF “ERDF A way of making Europe”. M.D.R.-R. was supported by PRE2018-084037 funded by MCIN/AEI/10.13039/501100011033 and ESF “ESF investing in your future”. DH-M is member of the Spanish Carotenoid Network (CaRed), grant RED2022-134577-T. SA, CR-S and MDR-R are members of CeReS Network, grant RED2022-134922-T. Both networks are funded by MCIN/AEI/10.13039/501100011033.
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Search for XAT candidate genes in durum wheat genomes using XAT-7D (TraesCS7D02G094000) from common wheat as gene model.
List of primers designed in this work.
List of the 28 landraces showing positive amplification for XAT candidate gene. Total carotenoids and percentage of carotenoid esterification is shown. XAT-like Type is indicated for the sequenced accessions.
Alignment of XAT-7A1 and XAT-7D proteins. The predicted active sites are shown with black triangles. Signal peptide in positions 1–22 is highlighted.
Genetic map for the F2 population derived from the cross BGE047535 × 'Athoris' using DArTSeq markers.
Carotenoid content and profile of the F2 population derived from the cross BGE047535 × 'Athoris'.
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Rodríguez-Suárez , C., Requena-Ramírez , M., Hornero-Méndez , D. et al. Towards carotenoid biofortification in wheat: identification of XAT-7A1, a multicopy tandem gene responsible for carotenoid esterification in durum wheat. BMC Plant Biol 23, 412 (2023). https://doi.org/10.1186/s12870-023-04431-4
- GDSL esterase/lipase
- Tandem copies
- Durum wheat