1. Introduction
Durum wheat (
Triticum turgidum var.
durum Desf.) represents about 5% of the global wheat production and is mainly grown in three principal areas: the Mediterranean basin, the Northern Plains between USA and Canada, and within the desert areas of South West USA and Northern Mexico, with a global production which exceeded 38 million tons in the last cropping seasons. Among the Mediterranean countries, Italy is the major durum wheat producer with an annual average of almost 4.0 MMT (million metric tons) (International Grain Council,
https://www.igc.int/en/default.aspx). Despite several local food products are obtained from durum wheat semolina (the coarse, purified durum wheat middlings), such as typical breads, couscous, or bulgur, this cereal crop is mainly used for high-quality pasta production. One of the most important characteristics determining semolina’s high end-use quality is grain protein content (GPC), as directly related to both nutritional and technological values of final food products [
1,
2]. The development of high GPC new varieties has been a constant priority in breeding programs, although it has been difficult to pursue, as GPC is a quantitative trait, regulated by a complex genetic system and affected by environmental factors, as well as genotype and management practices [
3]. Moreover, one relevant aspect to be considered in GPC breeding programs is the well-known strong negative correlation with grain yield, which makes the simultaneous increase of both traits challenging to achieve [
4,
5]. Indeed, at a genetic level, both GPC and grain yield-related traits are determined by multiple quantitative trait loci (QTLs) interacting with each other and the environment. The negative correlation between GPC and yield-related traits has been observed in both segregating populations and germplasm collections [
6,
7]. Several studies have considered GPC and grain-yield components simultaneously assessed on the same population to identify GPC loci without pleiotropic effects and/or not closely linked to gene for low yield-related traits, and interesting results were reported both for 2A [
8,
9,
10,
11,
12,
13] and 2B chromosomes [
12,
14,
15,
16,
17]. The identification of genetic sources of elevated protein content without negative pleiotropic effects would indeed be useful for improving GPC and GY simultaneously.
Improving GPC could be pursued by considering a candidate gene approach. Taking advantage of the recent advances in both bread and durum wheat genome sequencing and annotations [
18,
19], the identification of genes playing a key role in the Nitrogen Use Efficiency (NUE) process, such as N uptake from the soil, amino acid metabolism, or N transferring to the protein in the grain, has become an effective approach.
The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle represents a bottleneck in the first step of nitrogen assimilation, as these two enzymes work synergistically to incorporate the up-taken ammonium into organic molecules [
20].
QTLs for GPC and NUE have been located on all chromosomes, and several authors reported major ones on 2A and 2B chromosomes of bi-parental mapping population, where
GS2 and
Fd-GOGAT genes have been mapped [
8,
9,
10,
11,
14,
15,
16]. Nevertheless, QTLs validation is of primary importance for further breeding programs development or gene cloning. Single QTL effect on phenotype might be population-specific and/or overestimated, and this has been demonstrated for complex traits such as GPC [
15]. The constitution of near-isogenic lines (NILs) for the two different alleles of a target QTL is a useful and efficient method to validate a putative QTL. NILs are lines segregating only at target QTL but homozygous at the rest of the genome. Principally, NILs can be obtained by two approaches: (a) by backcrossing lines carrying the QTL region from a donor to a receipt line several times [
21,
22]; and (b) by exploiting the residual heterozygous lines (RHLs) from inbred populations and generating heterogeneous inbred family (HIF) at a QTL region [
23,
24], with the latter being more efficient and more rapid for both major and minor QTL [
25].
In previous studies, we identified
GS2 and
Fd-
GOGAT genes as good candidates for GPC QTL with no pleiotropic effect on yield in durum wheat on 2B and 2A chromosomes, respectively [
11,
15,
26,
27]. Functional markers based on polymorphisms detected within the sequences of the two genes in the two parental lines were mapped in the durum wheat Svevo × Ciccio (S×C) RILs population [
28].
GS2 gene co-localized with a GPC-QTL detected on chromosome arm 2BL, in the region flanked by the markers D_304657 and Xwmc332, significant in one environment and across environments with a LOD ≥ 3.0 [
26].
Fd-GOGAT gene co-localized with a GPC QTL detected on chromosome arm 2AS, in the region comprised between the markers Xgwm372c and the EST-SSR TC82001 (including the two closer markers Xgwm339 and Xgwm95), significant in two environments and across environments. The Svevo allele (increasing the trait) had positive additive effects ranging from 0.13 to 0.27 with a mean R
2 value of 0.24, and the percentage of phenotypic variance explained by the additive effects of the mapped QTL ranged from 6% to 19.4% between environments and the mean was 19.4% across environments [
27].
QTL analysis performed with CIM (Composite Interval Mapping) confirmed the presence of these markers in two major QTL for grain protein content. Recently, these data were confirmed on a wider durum wheat collection consisting of 236 durum wheat genotypes [
29,
30]. In addition, we found a significant difference in both
GS gene expression and enzymatic activities between the durum wheat
cvs Ciccio and Svevo, consistently higher in the last one [
31]. Following up these results, here we present the development of two distinct set of heterogeneous inbred family (HIF)-based NILs segregating for
GS2 and
Fd-GOGAT genes from heterozygous lines at those loci (previously identified in the S×C RIL population), and their genotypic and phenotypic characterizations, aimed to validate the previously identified GPC QTL on 2A and 2B chromosomes. Furthermore, we investigated the genomic characterization of the promoting regions of
GS2 and
Fd-GOGAT genes, as regulatory elements involved in the transcription processes might be the ones responsible for the different level of gene expression and might contribute in assessing the role of these key genes in GPC control.
3. Discussion
Here, we report the development of two distinct sets of HIF-based NILs, segregating for GS2 and Fd-GOGAT genes, from heterozygous lines at those loci, previously identified in a durum wheat RIL population, as well as their genotypic and phenotypic characterization.
The HIF method [
24] used in this work allowed us to generate useful NILs at a single locus from a single cross (RIL Svevo × Ciccio [
28]) instead of using markers flanking a QTL [
39]. Single functional markers, for
GS2 and
Fd-GOGAT genes, respectively, both linked with the GPC QTL of interest, were used for generating the two NILs sets. Compared to the traditional method, which is based on the selection of two flanking markers delimiting a QTL of interest, the HIF method allows the production of NILs with reduced sizes of the “non-desirable” chromosomal segment discriminating the isolines.
Traditionally, each NIL set has its own genetic composition, which determines unique morphological and phenological characteristics influencing the expression of a quantitative trait [
38].
The assessment of the effect of a particular gene on a specific trait should be more accurate by using a NIL pair obtained by the HIF method, as fewer isolines would be needed for investigating the effect of a QTL/gene.
Thus far, HIF-based NILs have been intensively used for investigating the effects of various QTL and traits of interest in wheat, such as dormancy QTL [
40], pre-harvest sprouting [
41], and spikelet number per spike [
42].
During the past decades, the increase in GPC has mainly been achieved by intensifying nitrogen (N) fertilization. Considering the high costs of N fertilizers and the harmful environmental impacts of nitrate loss from the soil, decreasing the amount of N applied to cereal cropping systems while maintaining high productivity of modern cultivars has therefore become a breeding priority. The identification of candidate genes and their allelic variants affecting this trait is an effective method to develop new wheat varieties with high GPC. Nitrogen remobilization efficiency is an important factor increasing grain protein content (GPC) and consequently, the nutritional and technological properties of flour and semolina [
43,
44]. Several authors have focused on deciphering GPC and NUE QTLs, and genetic diversity at candidate genes have recently been considered for this purpose. Indeed, in our previous studies, we identified both
GS2 and
Fd-GOGAT genes as good candidates in GPC study in the S×C RIL population [
26,
27]. Specifically, these two genes work synergistically in the GS/GOGAT cycle, which has a key role in nitrogen assimilation and recycling in young leaves within the chloroplast, where nitrite reduction occurs, and ammonium is assimilated [
45]. Both genes are located on the chromosome 2 homoeologous group, whose influence on GPC control was reported in different genetic materials, thus suggesting its key role in the control of the character [
11,
15,
46,
47,
48].
Interestingly, Kichey et al. (2005) and Habash et al. (2007) [
9,
49] showed that QTLs for GS activity co-localized with QTL for grain N on chromosome 2A and hinted this may be coincident with QTL on 2B and 2D homeologs for soluble protein content, and that increased activity was associated with higher grain N. This finding was confirmed on different genetic material [
50]. In addition, in maize, the
GS2 locus was found to be coincident with a leaf senescence QTL [
51], but, unlike in maize, Fontaine et al. (2009) [
50] did not find a correlation between GS activity and yield components in wheat, this being in agreement with our data. Indeed, as previously reported [
11,
15], QTL for GPC were found on 2A and 2B chromosomes, in the same region where
Fd-
GOGAT and
GS2 gene were mapped in Svevo × Ciccio RIL population, respectively [
26,
27]. Both authors reported that these QTLs showed no negative effects on grain yield related traits, making them good candidate for marker-assisted selection to improve GPC and grain yield simultaneously. However, considering that a QTL usually spans several cM, it is necessary to perform more detailed genetic analyses on larger populations or specific genetic material, such as NILs, in order to assess whether a single gene with pleiotropic effects or different loci within the linkage group are responsible for the different traits. The near-isogenic lines we developed at both
GS2 and
Fd-GOGAT loci showed that, despite having significant differences in GPC, no significant ones were observed in GYS. This could be either explained with both
GS2 and
Fd-GOGAT having no negative pleiotropic effects on yield components, or that a potential effect is actually masked by environment. Despite in both HIF-NILs families it was noticed that lines having the Svevo allele showed higher GPC, it was also outlined that the differences observed within NILs were highly statistically significant especially for
GS HIF-derived NILs, as
Fd-GOGAT HIF-derived ones showed a lower value of significant difference, as shown in
Table 2 and
Table 3. In both cases, we could assume that the Svevo allele is the one increasing the GPC trait.
The differences in GPC between the isolines developed for two key genes involved in nitrogen metabolism Glutamine synthetase and Glutamate synthase, not only further confirm the significance of these two genes in the processes determining the final grain protein content but would also facilitate the exploitation of these HIF NILs families in further characterizing the major GPC QTL on 2A and 2B chromosomes, studying their interaction with other traits of agronomic importance, and developing functional markers that can be reliably used to follow these major loci.
For
GS2 genes, a different gene expression was reported between the two cultivars that could affect the GPC content [
31]. In order to detect eventual polymorphism potentially involved in a differential gene expression, the promoter regions of both
GS2 and
Fd-GOGAT genes were screened in Svevo and Ciccio parental lines.
Among all regulatory elements, Transcription Factors (TFs) regulate cell processes by binding a specific DNA motif on promoter regions and affecting downstream gene expression.
By comparing the promoting regions of the genes considered in this study, the most interesting result was observed in the promoting region of GS-B2 gene, as a 132-bp indel was detected between the two parental lines. The analysis of this region outlined the presence of a 55-bp tandem repeat, as well as a number of TFBSs, such as EIN/EIL1, bZIP, AP2/ERF, SPL, ZF-HD, and NAC; NAM.
A recent review reported the role of EIN3/EIL1 transcription factors in
Arabidospis, highlighting their key role as regulators of ethylene signaling [
52]. Ethylene regulates many different aspects of plant development and stress responses, thus its signaling pathway needs proper modulation depending on the plant conditions. Salih et al. (2020) [
53] reported a very recent study on the possible functions of EIL/EIN3 proteins in cotton. Their GO annotations and KEGG pathway analyses indicated that, besides being involved in the ethylene-activated signaling pathway and a number of cellular macromolecule metabolic process, a great number of EIL/EIN3 genes were also involved in nitrogen compound metabolic process.
In addition, a bZIP binding site was found in the polymorphic indel, which represents one of the largest and most variable families of TFs and are uniquely present in eukaryotes. A genome-wide analysis and gene ontology enrichment analysis of bZIP Transcription Factors performed on 191 bZIP TFs, identified in bread wheat, reported that some of them are involved in cellular metabolic processes related to nitrogen compounds [
54]. More investigations will be needed to define and characterize the interaction between these TFs and
GS2 gene in order to better explain the role of these TF families in nitrogen metabolism pathway.
AP2/ERF transcription factors were classified into five subfamilies [
55], the most known of which are: AP2 (APETALA2), RAV (related to ABI3/VP1), DREB (dehydration-responsive element binding protein), and ERF (ethylene-responsive factor). The AP2/ERF transcription factors were shown to regulate diverse processes of plant development and stress responses, such as vegetative and reproductive development, cell proliferation, abiotic and biotic stress responses, and plant hormone responses [
56,
57,
58].
SQUAMOSA Promoter-Binding Protein-Like (SPL) genes have been shown to play numerous important roles during plant growth and development [
59]. SPLs are known to regulate several biological processes, including leaf development [
60], phase transition [
61], flower and fruit development [
62], plant architecture [
63], sporogenesis [
64], GA signaling [
65], and response to copper and fungal toxin [
66,
67].
Abu-Romman (2014) [
68] reviewed ZF-HD TF family and reported that these classes of homeodomain proteins are involved in regulating intercellular trafficking [
69], inflorescence stem growth [
70], and hormone and stress signaling [
71,
72,
73].
Among all TFBSs identified within the indel in the
GS2 gene promoter in
cv Svevo, the NAC; NAM TFBS was the most interesting and potentially involved in the different expression of
GS2 gene and final GPC between the two analyzed
cvs. Several studies have reported that wheat NAC TFs are involved in several biological processes such as senescence and nutrient remobilization [
35,
74], and stress response, both to biotic (such as stripe rust [
75,
76,
77,
78]) and abiotic stresses including drought and salt tolerance [
79,
80,
81,
82,
83]. Specific studies have been carried out on NAM TFs in relation to GPG and nitrogen metabolism. Waters et al. (2009) [
84] reported a
TaNAM gene which increased protein content in the grain by increasing the remobilization of nitrogen from vegetative tissues. Interestingly, He et al. (2015) [
85] described a
TaNAC2 TF that positively regulated
TaGS2 expression.
In conclusion, the data reported in the present work confirm that GS2 and Fd-GOGAT genes are involved in grain protein accumulation and suggest that the surrounding genomic regions and their promoters could affect gene expression. The identification of new useful superior alleles for both genes could be employed for marker-assisted selection and the constitution of wheat varieties with improved agronomic performance and N-use efficiency.