Genetic loci for Na+ exclusion and K+/Na+ ratio were identified in a durum mapping population, produced by a cross between the elite Australian cultivar, Jandaroi and an Afghani exotic durum landrace, AUS-14740, which accumulates in leaves half the Na+ concentration compared to other durum wheats.
In the current study, the distributions of all traits (third leaf Na+ concentration, third leaf K+ concentration, and the K+/Na+ ratio) in both populations was suggestive of a bimodal indicating a possible monogenic segregation. These findings suggest that single major genes are associated with Na+ exclusion in both the Afghani durum landraces studied. Heritability for the traits studied was high in both populations, with the exception of third leaf K+ concentration. A moderate to high heritability for shoot Na+ concentration has previously been estimated in two F2 populations of durum wheat [19]; two major QTL, Nax1 for HKT1;4 and Nax2 for HKT1;5, had been then identified in the same populations [13, 15]. Markers flanking these putative QTLs were used to develop salt tolerant durum wheat cultivars [17].
In order to define the genetic regions and identify potentially useful markers associated with third leaf Na+ concentration in the durum wheat of Afghani landraces, bulked segregant analysis (BSA) was conducted using SNP markers in the F2 population originating from the cross Jandaroi × AUS-14740.
In the Jandaroi × AUS-14740 F2 population, two SNP markers were found to be strongly associated with Na+ concentration in the leaves. The first marker, Xm5511, located on the long arm of chromosome 3B, was associated only with third leaf Na+ concentration and with neither third leaf K+ concentration nor the K+ /Na+ ratio. In contrast, a second marker, Xm564, was identified in the distal region of the long arm of chromosome 4B, and found to have a strong association with all three traits studied (Table 2).
Other QTL associated with these traits have been detected in other studies. The Kna1 locus was mapped to the distal region of chromosome 4DL in bread wheat [20] and this locus is associated with the selective accumulation of K+ over Na+ in the shoot. In durum wheat, the Nax2 locus was mapped to a region of the long arm of the chromosome 5A and this is associated with Na+ concentration and the ratio of K+/Na+ in the leaf blade [19]. It was found that Nax2 encoded a similar gene as Kna1 [15]. In rice (Oryza sativa), a QTL for shoot K+ content was mapped onto chromosome 1 in an F2 population derived from a cross between a salt tolerant indica (Nona Bokra) and an intolerant elite Japonica variety (Koshihikari). This QTL was named SKC1 [21]. Low Na+ concentration in the leaves, improved selection of K+ over Na+ transported from roots to shoots, and a high ratio of K+/Na+ in the leaves are common phenotypic characteristics between Nax2, Kna1 and SKC1 [15]. As mentioned above, these characteristics were seen in the F2 population derived from Jandaroi and AUS-14740. A candidate gene of the SKC1 locus is OsHKT1;5, which reduces the amount of Na+ transported from the root to the shoot [21]. An HKT-like gene was also cloned in Kna1 and Nax2, and HKT1;5 is a candidate for both loci; named as TaHKT1;5-D and TmHKT1;5-A, respectively [15]. Kna1 is found in bread wheat (T. aestivum L.) whereas Nax2 was derived from T. monococcum. It is possible that the Xm564 locus identified in this study corresponds to the Kna1 and Nax2 loci and the candidate gene for the locus on the long arm of chromosome 4B is an HKT-like gene. Huang, et al. [22] found four bands of HKT1;5-like genes in bread wheat using a HKT1;5 probe in Southern blots. Three hybridisation bands were located on the long arm of chromosome 4B and one band on the long arm of chromosome 4D. The HKT1;5 gene has only been found in bread wheat and T. monococcum, and has not been identified in durum wheat [17]. The results suggest that Na+ exclusion in AUS-14740 may be due to the gene HKT1;5.
The locations of both markers on chromosomes 3BL and 4BL identified in the current study and known HKT1;5 genes on chromosome 5AL in T. monococcum and on chromosome 4DL in bread wheat are shown in Fig. 2. Both homoeologous genes in the A and D genomes have similar genetic locations along the corresponding chromosomes. HKT1;5-A (Nax2) has been mapped to the distal part of chromosome 5A, with an estimated genetic distance of about 17 cM from the end of chromosome 5AL [15]. Part of chromosome 5AL originated from part of chromosome 4AL due to an ancient reciprocal translocation between the distal ends of chromosomes 4AL and 5AL [23]. HKT1;5-D (Kna1) has been identified in the genetic region about 14% from the end of chromosome 4DL [20].
We used the IWGSC website (http://pgsb.helmholtz-muenchen.de/plant/wheat//iwgsc/index.jsp) and the Gramene website to blast sequences from the Infinium design, where each SNP marker (used in our study) was developed, against the sequence of wheat chromosome 4B. We identified Xm564 markers on the distal part of chromosome 4BL in wheat contig 7,009,377, along with another marker, wsp_BG604404B. The position of Xm564 was 140.39 cM, out of a total length of 157.59 cM for the chromosome 4B genetic map, the marker being located about 17.1 cM from the end of chromosome 4B (Fig. 2). The HKT1;5-B1 like gene is also located on the wheat contig 7,009,377 and is in a very strong candidate for gene underlying this locus. This hypothesis, however, needs to be tested by looking for variation in or around the target gene by genotyping HKT1;5-B1 in the F2 progeny from the cross Jandaroi × AUS-14740. In future, sequencing of the HKT1;5-B1 gene from AUS-14740 and Jandaroi would determine whether there were differences in the protein encoded by the two alleles. If there were differences, heterologous expression of the two alleles of HKT1;5-B1 in yeast and Xenopus oocytes would investigate whether differences in coding sequence resulted in altered function of the protein and its affinity for the Na+ ion. In addition, quantitative RT-PCR would determine whether the differences in the Na+ phenotype observed between AUS-14740 and Jandaroi is due to differences in spatial and/or temporal expression the HKT1;5-B1 gene between the two accessions. It would also be important to make a separate study of the Jandaroi × AUS-14752 segregating population, as the two exotic Afghani landraces may contain different alleles of HKT1;5-B1 gene.
Using genetic similarity between rice and wheat genes, we were able to localise the location of a second HKT1;5 gene on chromosome BL, HKT1;5-B2. This gene, however, is found in the near-centromeric region of the chromosome 4BL and is unlikely to be responsible for the phenotype linked to marker Xm564 (Fig. 2).
The role played by other possible genes remains unclear. SNP marker Xm5511 was located on the chromosome 3BL, and it was found to make a similar, high, contribution to Na+ concentration in the third leaf (Table 2). We mapped the marker Xm5511 to the distal part of chromosome 3BL, with a physical location of about 701.9 Mbp, out of the 774.4 Mbp total length of the chromosome 3B physical map (Fig. 2). However, the favourable allele here originates from the Jandaroi parent rather than the Afghani accession, indicating that Jandaroi possesses other genes that are directly involved in the mechanism of Na+ exclusion.
It should be noted that Jandaroi was developed as the elite durum wheat cultivar adapted to the growth conditions prevalent in Australia; nevertheless, like other existing cultivated durum germplasms, Jandaroi, accumulates very high leaf Na+ concentrations, when compared to bread wheat [6, 8, 18]. This is typically because durum wheat do not contain an active native HKT gene involved in excluding Na+ from the shoot [6, 15, 16]. To date the only durum wheat with significant reductions in shoot Na+ have had HKT genes have had them introduced through selective processes, such as the incorporation of the Kna1 locus from the D genome of bread wheat [7, 15] or the Nax1 and Nax2 locus from T. monococcum [15, 17]. At this stage, there is no published information available that an HKT-like gene or any other potential candidate genes are located in the relevant genetic region of the chromosome 3BL. Therefore, a Jandaroi allele for Na+ exclusion with this region is unique. Consequently, further study is required to shed light on this particular locus, which seems to be very different from those in durum landraces.