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Article

Marker-Trait Associations for Total Carotenoid Content and Individual Carotenoids in Durum Wheat Identified by Genome-Wide Association Analysis

by
María Dolores Requena-Ramírez
1,
Cristina Rodríguez-Suárez
1,
Fernando Flores
2,
Dámaso Hornero-Méndez
3 and
Sergio G. Atienza
1,*
1
Instituto de Agricultura Sostenible (CSIC), Alameda del Obispo, S/N, 14004 Córdoba, Spain
2
Departamento de Ciencias Agroforestales, E.T.S.I. Campus El Carmen, Universidad de Huelva, Avda. Fuerzas Armadas, S/N, 21007 Huelva, Spain
3
Departamento de Fitoquímica de los Alimentos, Instituto de la Grasa (CSIC), Campus Universidad Pablo de Olavide, Edificio 46, Ctra de Utrera, Km 1, 41013 Sevilla, Spain
*
Author to whom correspondence should be addressed.
Plants 2022, 11(15), 2065; https://doi.org/10.3390/plants11152065
Submission received: 20 July 2022 / Revised: 4 August 2022 / Accepted: 5 August 2022 / Published: 7 August 2022
(This article belongs to the Section Plant Molecular Biology)
Browse Figures
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Abstract

:
Yellow pigment content is one of the main traits considered for grain quality in durum wheat (Triticum turgidum L.). The yellow color is mostly determined by carotenoid pigments, lutein being the most abundant in wheat endosperm, although zeaxanthin, α-carotene and β-carotene are present in minor quantities. Due to the importance of carotenoids in human health and grain quality, modifying the carotenoid content and profile has been a classic target. Landraces are then a potential source for the variability needed for wheat breeding. In this work, 158 accessions of the Spanish durum wheat collection were characterized for carotenoid content and profile and genotyped using the DArTSeq platform for association analysis. A total of 28 marker-trait associations were identified and their co-location with previously described QTLs and candidate genes was studied. The results obtained confirm the importance of the widely described QTL in 7B and validate the QTL regions recently identified by haplotype analysis for the semolina pigment. Additionally, copies of the Zds and Psy genes on chromosomes 7B and 5B, respectively, may have a putative role in determining zeaxanthin content. Finally, genes for the methylerythritol 4-phosphate (MEP) and isopentenyl diphosphate (IPPI) carotenoid precursor pathways were revealed as additional sources of untapped variation for carotenoid improvement.

1. Introduction

Durum wheat (Triticum turgidum L.) is an important food crop cultivated worldwide. For the 2021–2022 season, the world durum wheat production is estimated to be 30.86 Mt [1]. Durum wheat is used to make pasta and couscous, consumed all over the world, and it is an essential crop for many countries of the Mediterranean basin. Italy is the main producer in the European Union with an average production of 4.4 Mt in the period 2016–2020, followed by France (1.67 Mt), Spain (1.02 Mt) and Greece (0.83 Mt) [2].
Yellow pigment content (YPC) (also referred to as the yellow index, YI) and protein content are the most important quality traits for durum wheat (reviewed by [3]). Carotenoids pigments are responsible for the bright yellow color of pasta and other durum wheat-derived products [4] and the golden color of related cereals such as tritordeum [5]. Lutein is the main carotenoid in wheat endosperm [4,5], but other carotenoids, including zeaxanthin, α-carotene and β-carotene, are also present [6,7,8].
Carotenoids play important roles in both health and product commercialization. On one hand, these pigments are considered essential nutrients in the human diet due to their important functions in health, particularly for their role as antioxidants [9]. For instance, lutein and zeaxanthin have been related with the alleviation of age macular degeneration [10], while the consumption of carotenoid-rich foods reduces the risk of developing certain types of cancer [11]. Furthermore, carotenoids with unsubstituted β-rings, such as β-carotene, have provitamin A activity [12] which has promoted the development of biofortification programs in maize and rice to fight vitamin A deficiency all over the world [13,14].
On the other hand, carotenoids are also important for food commercialization due to their relation to color. This is of paramount importance in the case of durum wheat because consumers expect a bright yellow color of pasta. This demand has encouraged an efficient breeding activity for YPC/YI resulting in new durum wheat varieties with higher carotenoid content in grains [15,16]. The success in breeding has been possible because the genetic component is predominant over environmental. The high heritability reported in durum wheat [17], along with the importance of the YPC in breeding, has promoted the development of many genetic studies for the identification of quantitative trait loci (QTL) or marker-trait associations (MTAs) related to the YPC and/or YI in semolina (reviewed by [3]).
The main QTL for the YPC has been located at the homoeologous group 7 in the Triticeae species [18,19,20,21,22,23], but many other QTL/MTAs related to the YPC have been identified, as reviewed by Colasuonno et al. [3]. The yellow index is strongly related to pigment concentration, but it does not provide information about the carotenoid composition. The profiling of individual carotenoids by using chromatographic techniques, mostly HPLC, is necessary in order to gain information about the nutritional value of grains [15,24], as well as to decipher the genetic control for the biosynthesis of specific carotenoids which has been scarcely studied both in durum [6] and common wheat [25].
Modern breeding has been very successful at fixing numerous beneficial alleles at many loci [26]. However, modern breeding and domestication bottlenecks have left behind many beneficial alleles. This fact has renewed the interest in durum wheat landraces, conserved both in situ and ex situ at germplasm banks, as a source of diversity for many traits of interests that are no longer present in modern varieties.
The potential of landraces in cereal breeding for stress tolerance is widely recognized [27], but they also harbor diversity for quality traits. For instance, carotenoid esterification is a potential target for durum wheat biofortification [28] because carotenoid esters are more stable than free carotenoids. This has promoted the development of breeding programs to transfer the genes responsible for carotenoid esterification from common wheat (XAT-7D) [29] and the wild barley Hordeum chilense Roem. et Schultz. (XAT-7Hch) [30] to durum wheat. These programs were started on the assumption that no lutein esters were present in durum wheat varieties [31].
Interestingly, a recent characterization of the carotenoid profile in a Spanish collection of durum wheat landraces has allowed the identification of some accessions with significant ability to produce lutein esters [7]. In addition, these landraces also showed diversity for other carotenoids, such as zeaxanthin, which unveil the existence of genetic variability useful for the discovery of beneficial untapped MTAs/QTL for specific carotenoids which could be incorporated into new cultivars with enhanced nutritional properties. Despite the nutritional interest of carotenoids, few attempts have been made to investigate the genetic control of individual carotenoids in durum wheat [6] and common wheat [25], likely due to the higher cost of this methodology compared to those used to determine YPC or YI.
Given the importance of carotenoids in durum wheat quality and nutritional value, the aim of this work was to identify MTAs for both the total carotenoid content and individual carotenoids using DArTSeq markers. In addition, this work is also intended to confirm the diversity for the ability of carotenoid esterification in durum wheat landraces.

2. Results and Discussion

2.1. Genotyping

The diversity panel was genotyped using the DArTSeq platform (Diversity Array Technology Pty Ltd. DArT P/L, Canberra, Australia) as described by Ávila et al. [32]. In summary, a set of more than 190,000 markers was obtained, including both the presence/absence variation and SNP markers. The high-confidence and low-confidence gene models from the ‘Svevo’ genome were used as a template for the alignment of the DArTSeq markers using the BLASTn algorithm (E-value < 1.5 × 10−6, sequence identity > 80%) and BLAST+ [33]. A final set of 8025 DArTSeq markers corresponding to genes, with a minor allele frequency above 5% and less than 10% of missing data, were used for the association analyses. The distribution of these markers at each chromosome is shown in Figure 1. The linkage disequilibrium decay for the genotypic panel is 2 Mbp [32].

2.2. Phenotypic Assessment

The carotenoid content and profile were determined in the diversity panel. The following traits were analyzed: free lutein = (all-E)-lutein + (Z)-lutein isomers (including both (9Z)- and (13Z)-lutein); total lutein = free lutein + lutein monoesters (including both lutein monolinoleate and lutein monopalmitate) + lutein diesters (including lutein linoleate-palmitate, lutein dipalmitate and lutein dilinoleate); (all-E)-zeaxanthin (hereinafter referred as zeaxanthin); (all-E)-α-carotene (hereinafter referred as α-carotene) and (all-E)-β-carotene (hereinafter referred as β-carotene). The total carotenoid content was calculated as the sum of the total lutein, zeaxanthin, α-carotene and β-carotene. The proportion (%) of the carotenoids derived from the β,β-branch of the carotenoid pathway relative to the total carotenoid content (hereinafter referred as Pββ) was also considered as an additional trait for the association analysis.
The carotenoid content and profile for the first season were reported in a previous work [7] aimed to identify the esterification ability in durum wheat (Supplementary Table S1). As expected, lutein was the main carotenoid, representing around 90% of the total carotenoid content in agreement with previous results [6,8,31,34,35]. The carotenoid profile also included zeaxanthin with a 10.5% mean contribution to the carotenoid pool in accordance with previous reports [35]. Minor quantities of β-carotene and α-carotene were also detected. Both the total carotenoid content and individual carotenoids showed high broad-sense heritability values: 0.97 for total carotenoid and for zeaxanthin, 0.96 for free lutein, 0.95 for total lutein and 0.85 for α–carotene. This is in agreement with the high values of heritability reported in previous studies: 0.48–0.99 [19]; 0.91–0.94 [15]; and 0.78–0.96 [6]. The only exception was β–carotene with a broad-sense heritability of 0.25.
The carotenoid content and profile were analyzed in a second season with similar results (Supplementary Table S1). The esterification ability of the accessions BGE047507, BGE047535 and BGE047536, reported by Requena-Ramírez et al. [7], was confirmed with the results obtained in the second season. The accession BGE047520 was lost in this field trial, but its esterification ability was also confirmed with an individual grown at a greenhouse (data not shown).
The Pearson correlation between the seasons for the total carotenoid content was 0.782 (Figure 2). Similar values were obtained for the total lutein (r = 0.769) and free lutein (r = 0.767). Significant correlations were also found for zeaxanthin (r = 0.597), β-carotene (r = 0.575) and α-carotene (r = 0.920) (Figure 2).
In addition, moderate to high correlations among the traits were also detected (Figure 3). The total carotenoid content was highly correlated with both total lutein and free lutein as expected because lutein accounts for around 90% of the total carotenoids. Zeaxanthin, β-carotene and α-carotene showed high correlations among them with r-values above 0.879 (Figure 3), while they showed moderate correlations with the total carotenoid content (r-values of 0.673, 0.680 and 0.670, respectively).

2.3. Marker-Trait Associations

The total carotenoid, total lutein, free lutein, zeaxanthin, α-carotene and β-carotene contents were considered as primary traits for the association analysis. In addition to them, the relative contribution of the β,β-carotenoids to the total carotenoid pool (Pββ) was also considered as a secondary trait. The Manhattan plots obtained for each trait are shown in Figure 4. A total of 28 MTAs were identified (Table 1): 4 for total carotenoids (2 on 2B and 2 on 7B), 4 for total lutein (2 on 2B and 2 on 7B), 6 for free lutein (1 on 2B, 1 on 3B and 4 on 7B), 9 for zeaxanthin (2 on 2B, 1 on 3A, 1 on 4A, 3 on 5A, 1 on 5B and 1 on 7B) and 5 for the relative contribution of the β, β-branch carotenoids to the total carotenoid pool (1 on 2B, 1 on 3A, 2 on 5B and 1 on 6B). No MTA was identified for β-carotene or α-carotene, but these carotenoids account for less than 1% of the total carotenoid content in durum wheat, and thus, the potential of any MTA for these traits in breeding is very limited.
The position of the MTAs identified in this work were compared with the regions for the YPC and YI previously reported. In a first round, the QTL track of the ‘Svevo’ genome browser was considered [37], which provides the position of the known QTL curated by the International Durum Wheat Genome Sequencing Consortium. The overlapping confidence intervals of QTLs for the YPC or YI were used to define eight QTL regions co-locating or in the vicinity of the MTAs identified in this work (Figure 5) as follows: QTL-2B includes QTL0090 (YI) [38] and QTL0057 (SY) [39]; QTL-3A represents QTL0992 (YPC) [40]; QTL-3B is QTL0954 (YPC and YI) [6]; QTL4A1 is composed of QTL1799 (YPC), QTL1800 (YI) [41] and QTL0061 (YI) [39]; QTL4A2 includes QTL1801 (YI) [41] and QTL2086 (YI) [23]; QTL5A1 with QTL0955 (YPC) [6]; QTL5A2 with QTL1802 (YPC) [41]; QTL5B with QTL0069 (YI) [39]; and QTL7B is composed of QTL1810, QTL1811, QTL1812, QTL1813, (YPC, YI) [41], QTL0996 (YPC) [40], QTL0079 (YI) [39], QTL2088 (YPC, YI) [23] and QTL0176 (YI) [42].
The main QTL controlling the YPC variation in durum wheat is represented by QTL7B (Figure 5). Thus, the identification of MTAs for the total carotenoids, total lutein and free lutein co-locating with QTL7B is in agreement with previous reports in wheat and related species [19,21,22,43]. The Phytoene synthase 1 gene is known to be responsible for the variation at this QTL in durum wheat [22,43] and other Triticeae species [3,5].
Plants 11 02065 g005 550
Figure 5. Co-localization of marker-trait associations identified in this work with previous QTLs for yellow pigment content (YPC) and semolina yellowness (YI). Regions identified as ‘QTL’ were depicted according to their position at the ‘Svevo’ genome browser [37]. Haplotype regions associated to semolina pigment [44] were identified as ‘hap’ regions. The MTA between wPt-2724-2B and Yellow index was reported by [45]. Trait abbreviations: Tlut: Total lutein; Flut: Free lutein; Tcar: Total carotenoids; Zeax: Zeaxanthin; Pbb: Relative proportion of carotenoids from the β,β-branch relative to the total carotenoid pool.
Figure 5. Co-localization of marker-trait associations identified in this work with previous QTLs for yellow pigment content (YPC) and semolina yellowness (YI). Regions identified as ‘QTL’ were depicted according to their position at the ‘Svevo’ genome browser [37]. Haplotype regions associated to semolina pigment [44] were identified as ‘hap’ regions. The MTA between wPt-2724-2B and Yellow index was reported by [45]. Trait abbreviations: Tlut: Total lutein; Flut: Free lutein; Tcar: Total carotenoids; Zeax: Zeaxanthin; Pbb: Relative proportion of carotenoids from the β,β-branch relative to the total carotenoid pool.
Plants 11 02065 g005
The MTAs identified in chromosomes 2BS and 3BL co-localized with the QTL-2B and QTL-3B regions, respectively, (Figure 5), and thus, these regions can be also considered validated. The same happens with the MTA 1126970-Zeaxanthin in chromosome 3A because it is within the confidence interval of QTL-3A (Figure 5). In this case, the confidence interval of QTL-3A almost spans the complete chromosome and its utility is limited. Nevertheless, this MTA also co-localizes with the haplotype hap-3A-5 (see below) which is located in a narrow interval. The QTL regions QTL-4A-1, QTL-4A-2, QTL-5A-1, QTL-5A-2 and QTL-5B are in the vicinity of some MTAs, but they cannot be associated with those described in this work.
Thus, in a second round, we also considered the haplotype loci reported by N’Diaye et al. [44], described to be under selection for the semolina pigment in Canadian durum wheat. Interestingly, many of the MTAs identified in this study co-localized with some of these regions (reported as hap-regions in Figure 5). Indeed, seven hap-regions, hap_2B_6, hap_2B_7, hap_3A_5, hap_3A_6, hap_5B_5, hap_5B_3 and hap_6B_2, co-localized with MTAs for the carotenoid content or profile. Thus, the MTAs identified in this study that are co-localizing with these haplotype regions are validating them.
Several MTAs did not co-localize with QTLs or hap-regions in chromosomes 2BL, 4AL, 5A (two MTAs), 5B and 7BS. Thus, we inspected the ‘Svevo’ genome in the proximity of MTAs, looking for carotenogenic genes that may be responsible for the detected variation. Interestingly, the MTA for the zeaxanthin content in chromosome 5B is co-locating with TRITD5Bv1G246960 coding for Phytoene synthase (Figure 5). The phytoene synthase (PSY) regulates a rate-limiting step in the carotenoid biosynthesis. Gallagher et al. [46] showed that PSY is essential for the carotenoid accumulation in the endosperm as discussed above. Although Psy1 is mainly responsible for carotenoid accumulation in grains, there are three paralogous Psy genes in grasses [47,48] that may contribute to the determination of the carotenoid content in grain. Indeed, the mRNA levels of Psy2 have been associated with the differences in the total carotenoid content between tritordeum and durum wheat [49]. Thus, the co-localization of the MTA for the zeaxanthin content with TRITD5Bv1G246960 suggests a putative contribution to carotenoid content variation.
Similarly, the MTA 1094075/zeaxanthin is 10Mbp from the gene TRITD7Bv1G017350 coding for a ζ-carotene desaturase (Zds). Although there are many genes in this region, none of them seem related to the carotenoid content. The ‘Svevo’ genome includes four Zds genes located on chromosomes 2A, 2B, 7A and 7B. This enzyme catalyzes the conversion of ζ -carotene to lycopene via the intermediary neurosporene. From lycopene, the carotenoid pathway divides into two branches, the β,ε-branch, leading to the synthesis of α-carotene and lutein, and the β,β-branch for the synthesis of β-carotene and zeaxanthin. Zds genes have received attention in wheat. Indeed, a cDNA sequence encoding a Zds gene was cloned in the hexaploid wheat ‘Chinese Spring’ [50]. Later studies allowed the development of functional markers for TaZDS-D1 [51] and TaZDS-A1 [52]. These markers co-segregated with QTLs for the YPC content on chromosomes 2A [52] and 2D [51], showing the role of ZDS in the determination of the YPC in common wheat. Recent findings by Pasten et al. [53] have demonstrated similar associations of Zds in durum wheat. Indeed, these authors identified the complete sequence of Td-ZDS-A-IWGSC and TD-ZDS-B-IWGSC and confirmed the association between the QTL of grain YPC on chromosome 2A and Td-ZDS-A-IWGSC in durum wheat. Considering our results, the role of TRITD7Bv1G017350 in the determination of zeaxanthin content is worthy of further investigation in the future.
Finally, the distal part of chromosome 2BL seems to be relevant for the determination of the carotenoid content and profile. Indeed, two hap-regions associated with semolina pigment (hap_2B_6 and hap_2B_7) [44], two MTAs for the lutein content and total carotenoid content (this work) and one MTA for the yellow index (wPt-2724-2B) [45] are located in this region. Furthermore, this area is also important for the determination of grain carotenoid content in related Triticeae species. In fact, a QTL for the YPC has been consistently detected in chromosome 2HchL of H. chilense Roem. et Schultz [54,55]. Considering the high degree of collinearity of this wild barley with other Triticeae species [56], it seems that this region corresponds to the candidate region defined in 2BL in this work.
Thus, we inspected the ‘Svevo’ genome at the distal part of chromosome 2BL (green region in Figure 5), searching for candidate genes from the carotenoid precursor pathways because upstream precursors of geranylgeranyl diphosphate (GGPP) and isopentenyl diphosphate (IPP) can affect carotenoid accumulation [57]. The methylerythritol 4-phosphate (MEP) pathway is the source of the isoprenoid precursors isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) [58]. The MEP pathway has seven enzymatic steps [58], including some genes previously investigated in maize [59], such as DXS (1-deoxy-D-xylulose-5-phosphate synthase), DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase), HDS (hydroxy-methylbutenyl diphosphate synthase) and HDR (hydroxymethylbutenyl diphosphate reductase) which catalyzes the reduction of hydroxy-methylbutenyl diphosphate to IDP and DMADP [58]. IDP and DMADP are isomerized by isopentenyl diphosphate isomerase (IDI) [58]. After this, the geranylgeranyl diphosphate synthase (GGPPS) catalyzes the conversion of DMAPP to GGPP which is subsequently used to synthesize phytoene by the phytoene synthase, constituting the first step of the carotenoid pathway [9]. In maize, the mRNA levels for the carotenoid precursor genes during endosperm development correlated with the carotenoid content [59], and thus, it is possible that these genes may contribute to the carotenoid content in durum wheat. Indeed, four upstream genes were detected: TRITD2Bv1G234360 coding for IDI, TRITD2Bv1G241110 coding for GGPPS, TRITD2Bv1G263010 coding for HDS and TRITD2Bv1G265450 coding for HDR. It is necessary to note that this candidate region also contains genes contributing to carotenoid degradation during grain processing, such as lipoxygenases (LOX) [60] and peroxidases (PER) [61]. However, the carotenoid extraction protocol used in this work includes the addition of BHT (butylated hydroxytoluene) as an antioxidant, which prevents the effect of oxidative enzymes, and thus, the association of LOX or PER genes with the MTAs identified in this work can be ruled out.

3. Materials and Methods

3.1. Plant Material, Field Design and Statistical Analysis

A diversity panel composed of 158 Spanish durum wheat landraces was selected for this study, including the core collection development by Ruiz et al. [62] (Supplementary Table S3). The original seeds were obtained from the National Plant Genetic Resources Centre (INIA-CSIC, Alcalá de Henares, Spain). Passport data are available at the Spanish Inventory of Plant Genetic Resources Centre (Inventario Nacional de Recursos Fitogenéticos. Available online: https://bancocrf.inia.es/es/ (accessed on 6 July 2022)).
The diversity panel was characterized for carotenoid content and profile during two seasons at field conditions in Córdoba (Spain). The experimental details and the results for the first season were recently described by (Requena-Ramírez et al., [7]). For the second season, the field trial consisted of non-replicated rows 1 m long with 10 plants per row, arranged using an augmented design with two commercial durum wheat varieties (‘Kiko Nick’ and ‘Olivadur’) as checks. The field trial was cultivated under an anti-bird net structure and using an anti-weed net. Grain samples were harvested at maturity and stored at −80 °C until the extraction and analysis of carotenoids (described below).
The R package ‘AugmentedRCBD’ [63] was used to perform the analysis. This function is designed for analysis of variance of an augmented randomized block design [64,65] and the generation, as well as comparison, of the adjusted means of the treatments/genotypes. Broad-sense heritability was based on the BLUEs of genotypic effects using Formula 19 from [66],
H 2 = σ g 2 σ g 2 + v / 2
where v is the mean variance of the difference of two adjusted treatment means (BLUE). Correlograms were obtained using the BLUEs and GGally packages in RStudio.

3.2. Extraction of Carotenoids and HPLC Analysis

Carotenoids pigments were extracted from durum wheat grains according to the method described in [30,67]. All the steps for carotenoid extraction and analysis were carried out under dimmed light to prevent carotenoid photo-degradation and isomerization.
Analysis of carotenoids was performed by HPLC as described in previous works [67,68]. Carotenoid quantification was performed using calibration curves prepared from pure pigment standards. The concentration of (Z)-isomers of lutein was assessed by using the calibration curve for (all-E)-lutein. Similarly, lutein esters were determined as free lutein equivalents. All the analyses were performed in duplicate and carried out on the same day of the preparation of extracts. Data were expressed as µg/g fresh weight (µg/g fw).

3.3. DNA Isolation, Genotyping and Marker-Trait Associations

The isolation of genomic DNA from two-week-old leaves of seedlings was conducted using the CTAB protocol [69] with the specifications described by Rodríguez-Suárez et al. [30]. Genotyping by sequencing was performed at Diversity Arrays Technology Pty Ltd. (DArTSeq) (Camberra, Australia). DArTSeq markers were processed as described by Ávila et al. [32]. Briefly, DArTSeq markers were aligned to the durum wheat reference genome ‘Svevo’ [37]. A BLASTn search [70] was performed using BLAST+ [33] with the following criteria: E-value of <1.5 × 10−6 and sequence identity of >80%. DArTSeq sequences were used as a query against the durum wheat coding sequences (nucleotides) of annotated high- (HC) and low (LC)-confidence genes. Only DArTSeq markers with a significant match to HC or LC genes were considered for genetic analyses. A principal component analysis (PCoA) was conducted based on genotype data with DArTSeq markers spaced at least 2 Mbp using Tassel 5.2.80 [71] to inspect the existence of structures in the durum wheat collection and depicted using ggplot2 [72]. Marker-trait associations were determined using TASSEL 5.2.80 [71]. Markers with a minimum allele frequency of less than 5 and 10% of missing data points were not included in the association analyses.
Association analyses were performed using a mixed linear model (MLM), including the PCoA as the Q matrix, the kinship matrix calculated with Tassel MLM (Q + K) and the arithmetic mean of both seasons as phenotypic data (considering the adjusted means data for each trait and season). False discovery rate (FDR) for each trait was calculated with the approach developed by Benjamini and Hochberg [36] using the RainbowR package [73] and RStudio v. 1.3.1093 [74]. The significance of each MTA was calculated using the FDR approach [36]. Manhattan plots were obtained using the qqman package [75] in RStudio.

4. Conclusions

The identification of Zds and Psy genes co-locating with MTA for zeaxanthin content on chromosomes 7B and 5B, respectively, suggests a putative role of these genes in the determination of the content of this carotenoid in durum wheat. Similarly, genes coding for the MEP and IPPI precursor pathways may constitute an additional source of untapped variation for carotenoid improvement in durum wheat. The co-localization of the MTAs identified in this study with widely reported QTLs such as QTL-7B was expected and supports the findings of this study. Similarly, the co-localization of MTAs for the total carotenoid content with QTL regions for semolina pigment recently identified using haplotype analysis constitute an independent validation of these hap-regions. The MTAs identified in this work will be useful for the pre-breeding and breeding of durum wheat for increasing both the total and specific carotenoid content (lutein and zeaxanthin). In addition, the confirmation of the esterification ability in durum wheat would allow the development of breeding programs aimed at the enhancement of carotenoid esterification in grain.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11152065/s1, Table S1. Carotenoid content and profile. Table S2. ‘Svevo’ gene models matching MTA described in this work. Table S3: List of accessions analyzed in this work.

Author Contributions

Conceptualization, S.G.A. and D.H.-M.; methodology, D.H.-M., S.G.A. and F.F., investigation, M.D.R.-R., C.R.-S., D.H.-M. and S.G.A.; formal analysis, all the authors; writing—original draft preparation, M.D.R.-R. and S.G.A.; writing—review, all the authors; funding acquisition, S.G.A. and D.H.-M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by project AGL2017-85368-P funded by MCIN/AEI/10.13039/501100011033/, by ERDF “ERDF A way of making Europe” and by the Instituto de Investigación y Formación Agraria y Pesquera (IFAPA), Junta de Andalucía, grant number AVA-AVA2019.020 cofounded at 80% by ERDF. M.D.R.-R. was supported by PRE2018-084037 funded by MCIN/AEI/10.13039/501100011033 and ESF “ESF investing in your future”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Centro de Recursos Fitogenéticos (CRF-INIA-CSIC) for providing the original plant material used in this work. We are grateful to Joaquin Ballesteros for his technical assistance in the field experiments.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Plants 11 02065 g001 550
Figure 1. Marker distribution along ‘Svevo’ genome.
Figure 1. Marker distribution along ‘Svevo’ genome.
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Plants 11 02065 g002a 550Plants 11 02065 g002b 550
Figure 2. Pearson correlations between seasons and density functions, showing the distribution of traits in both seasons (BLUEs values). *** p < 0.001.
Figure 2. Pearson correlations between seasons and density functions, showing the distribution of traits in both seasons (BLUEs values). *** p < 0.001.
Plants 11 02065 g002aPlants 11 02065 g002b
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Figure 3. Pearson correlations and density functions showing the distribution of traits (mean values of both seasons). *** p < 0.001.
Figure 3. Pearson correlations and density functions showing the distribution of traits (mean values of both seasons). *** p < 0.001.
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Figure 4. Manhattan plots from the GWAS analyses. For each trait, a suggestive (FDR) threshold by Benjamini and Hochberg [36] at α = 0.2 is shown (blue horizontal line). The significance of each MTA calculated with the same FDR approach is shown in Table 1.
Figure 4. Manhattan plots from the GWAS analyses. For each trait, a suggestive (FDR) threshold by Benjamini and Hochberg [36] at α = 0.2 is shown (blue horizontal line). The significance of each MTA calculated with the same FDR approach is shown in Table 1.
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Table
Table 1. Marker-trait associations (MTAs) identified for carotenoid content and profile and DArTseq markers.
Table 1. Marker-trait associations (MTAs) identified for carotenoid content and profile and DArTseq markers.
MarkerTrait 1ChromosomePos (Mbp) 2LODFDR 3R-squareType 4Effect 5Svevo 6
1022270Zeax2B5.333.700.2000.096SNP/T|C0.01C
1699053Flut2B15.65.490.0280.083PAV/T|G0.49T
1699053Tlut2B15.65.040.0730.076PAV/T|G0.47T
1699053Tcar2B15.64.900.1020.075PAV/T|G0.51T
4412035Pββ2B699.63.930.1550.111SNP/A|G3.09A
4910734Zeax2B739.95.860.0060.149SNP/T|C0.01T
2327237Tlut2B765.64.050.1950.065PAV/T|G0.47T
2327237Tcar2B765.64.030.2010.065PAV/T|G0.06T
1126970Zeax3A575.06.090.0060.144SNP/T|C0.02T
1127042Pββ3A679.74.060.1300.133SNP/T|C1.61C
5563469Flut3B796.93.990.1730.060PAV/T|G0.65T
1215093Zeax4A648.05.420.0110.152SNP/T|A0.01A
2346846Zeax5A306.34.480.0600.124SNP/C|T0.10C
4006073Zeax5A567.04.040.1100.102SNP/A|G0.003A
3028544Zeax5A661.44.320.0700.110SNP/T|C0.02T
12772437Pββ5B287.24.730.0510.137SNP/T|C3.56T
1237690Pββ5B601.13.950.1540.108SNP/T|C4.17C
2255960Zeax5B685.83.790.1600.091SNP/G|A0.01A
4408288Pββ6B45.24.930.0510.146SNP/C|G0.69C
1094075Zeax7B37.44.590.0500.078SNP/A|G0.01A
26672068Flut7B692.44.100.1550.055PAV/T|G0.54T
4008170Flut7B697.83.930.1790.040PAV/T|G0.49T
4407472Flut7B697.84.740.0700.044PAV/T|G0.54T
4407472Tlut7B697.84.370.1300.050PAV/T|G0.53T
4407472Tcar7B697.84.440.1450.042PAV/T|G0.56T
4989844Flut7B698.64.520.0950.042PAV/T|G0.67T
4989844Tlut7B698.64.450.1210.0430PAV/T|G0.67T
4989844Tcar7B698.64.110.1900.040PAV/T|G0.69T
1 Trait abbreviations: Tlut: Total lutein; Flut: Free lutein; Tcar: Total carotenoids; Zeax: Zeaxanthin; Pββ: Relative proportion of carotenoids from the β,β-branch relative to the total carotenoid pool; 2 Position in Mbp; 3 Significance level (α) calculated using the False Discovery Rate approach [36]; 4 Type (SNP = Single Nucleotide Polymorphism; PAV = Presence absence variation). Alternative nucleotides are also indicated. In the case of PAV, T means presence, G means absence. The allele favorable to the trait is shown in bold; 5 Difference in the effect between alternative alleles; 6 SNP at ‘Svevo’ for each MTA. ‘Svevo’ genes matching each MTA are shown in Supplementary Table S2.
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Requena-Ramírez, M.D.; Rodríguez-Suárez, C.; Flores, F.; Hornero-Méndez, D.; Atienza, S.G. Marker-Trait Associations for Total Carotenoid Content and Individual Carotenoids in Durum Wheat Identified by Genome-Wide Association Analysis. Plants 2022, 11, 2065. https://doi.org/10.3390/plants11152065

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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(15):2065. https://doi.org/10.3390/plants11152065

Chicago/Turabian Style

Requena-Ramírez, María Dolores, Cristina Rodríguez-Suárez, Fernando Flores, Dámaso Hornero-Méndez, and Sergio G. Atienza. 2022. "Marker-Trait Associations for Total Carotenoid Content and Individual Carotenoids in Durum Wheat Identified by Genome-Wide Association Analysis" Plants 11, no. 15: 2065. https://doi.org/10.3390/plants11152065

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