1. Introduction
Wheat (
Triticum aestivum L.) contributes one-third of the world’s edible dry matter. It is one of the most important grain crops in the world. Potassium (K) is one of the essential macronutrients for crops, which is not only important for crop growth, development, and fecundity, but also significant for crop yield and quality [
1]. It can increase the salt, drought, and disease tolerance of plants [
2,
3,
4,
5,
6,
7,
8]. The average reserves of K in soil are usually large. However, most of the K in soil is not plant-available, and K deficiency is one of the most common limiting factors for crop production [
9].
Potassium deficiency can significantly affect the use of K or other elements and ultimately affect crop yield [
3,
10]. There are complex interactions among K, calcium (Ca), and magnesium (Mg). Potassium can reduce the uptake of Mg in numerous plant species, such as soybeans, wheat, and rice [
11,
12,
13]. However, the mechanisms for this K-inhibited Mg uptake have not been researched clearly. One possible explanation is the competition for apoplast binding between K
+ and Mg
2+ [
14], while another possible explanation is the competition for the unidentified transporters between them. Tomoaki et al. [
15] suggested that OsHKT2;4 (a K
+-permeable transporter/channel)-mediated currents could also exhibit permeability to both Mg
2+ and Ca
2+, which would be smaller with the competitive inhibition of K
+. Some genes or transporters/channels also showed sensitivity to Mg
2+ and Ca
2+ simultaneously [
16], such as the
PaAlr1 gene in ascospores [
17] and OsHKT2;4 in rice [
15]. Ca can usually promote the uptake of K by plants but competition in absorption at the plasma membrane has also been observed [
18]. In higher plants, some members of the CBL, CaM, and CML genes family of Ca
2+ sensors have been reported to function in plant responses to K
+ deficiency [
19,
20,
21]. Xu et al. [
21] showed that the CBL-CIPK (CBL-CIPK: CBL-interacting protein kinase) complex participates in the regulation of K
+ uptake under K
+ deficiency stress for plants. Obviously, a complex relationship in absorption or transport among K, Ca, and Mg exists widely, and these reports have provided us with some interpretation of it. However, to our knowledge, there have been few similar reports in wheat. Further genetic investigations should be carried out for K, Ca, and Mg nutrition in wheat.
The nutrient-related traits for K, Ca, and Mg are very complicated quantitative traits. Wheat is a very important crop that has a large genome. Until now, few genes related to plant nutrition were cloned in wheat, although the first high-affinity potassium uptake transporter, HKT1 in higher plants, was cloned in wheat [
22]. Quantitative trait loci (QTL) analysis is still an effective way to identify the location of new genes, which dissects complicated traits into component loci and study their relative effects on a specific trait [
23,
24,
25]. In wheat, QTL analysis has been used to study the effects of different nutrient environments [
26,
27,
28,
29], which enables us to understand nutrient use efficiency at the QTL level. However, to this day, few studies have been reported on verified QTLs for the efficient use of K, Ca, and Mg simultaneously in uniform environments.
The main objective of our study was to identify the QTLs related to the absorption and utilization of K, Ca, and Mg at the seedling stage in a hydroponic culture trial and the mature stage in a field trial under different K treatments (LK and CK) using a set of RILs (recombinant inbred lines) derived from two winter wheat varieties of China. The results may help us further understand the effects of K deficiency on K, Ca, and Mg nutrition at the phenotypic and QTL level. They may also provide valuable QTLs for K, Ca, and Mg nutrition in wheat, which deserves further investigation.
2. Materials and Methods
2.1. Plant Materials
The RIL population (F9) used in this investigation was derived from a cross of “Tainong 18 × Linmai 6” using single-seed descent (SSD). A total of 184 lines were randomly selected from the original 305 lines of this population to construct the genetic map and QTL analysis [
30]. The outstanding characteristics of Tainong 18 are high-yield, high quality, and resistance to lodging. It was planted in approximately 300 thousand hectares per year in the Huang-huai Winter Wheat Region of China. Linmai 6 is a high yield wheat variety belonging to the medium to large spike type, and its female parent is a sister line of the famous cultivars “Jimai 22”.
2.2. Experimental Design
2.2.1. Hydroponic Culture Trial
The 184 RILs and their parents were grown in hydroponic culture in the greenhouse at Shandong Agricultural University in February 2013 and March 2013. Optimized Hoagland’s nutrient solution [
31] (
Table S1) was used for the well-balanced growth of wheat seedling. Two treatments with moderate K (CK, 66.47 mg/L) and low K (LK, 6.65 mg/L) concentrations were designed with the consistent concentrations of other elements. A random complete block design was used in our experiments, with three replicates for each treatment.
A total of 50 seeds of each line and their parents were sterilized for five minutes in 10% H
2O
2. Then, they were germinated at 25 °C for seven days. Eighteen uniform seedlings (3 plants × 2 treatments × 3 replicates) with both the embryogenic primary root and coleoptile were selected and transferred to nutrient solution (20 L with one replication in lightproof container). The distances between various lines were 3 cm × 3 cm. The nutrient solutions were continuously aerated and renewed every 4 days. The 0.1 mmol∙L
−1 HCl or NaOH solution was used to regulate the pH of nutrient solution between 6.0 and 6.2 every day. The plants grew for 28 days in nutrient solution and were harvested. The details referenced the method of Guo et al. [
32].
2.2.2. Field Trial
The field trials were carried out at the agronomy experimental station of Shandong Agricultural University from 2012–2013. The soil type was loamy cinnamon soil (pH 7.8). The average contents of N (available N), P (Olsen P), and K (available K) in the 0 to 20 cm soil profile sampled were 58.2, 21.3, and 86.4 mg·kg−1 without fertilizing. Two K concentration treatments, moderate K (114 kg/hm2) and low K (0 kg/hm2), were designed. In addition, 50% of the total N (97.5 kg/hm2), all the P2O5 (102 kg/hm2), and the corresponding K2O in two treatments were applied as base fertilizer before sowing, and the other 50% of N (97.5 kg/hm2) was applied at the stem elongation stage. Each treatment was replicated twice. Twenty seeds of each line were sown on October 10, and ten seedlings were retained after germination, with a 10 cm spacing between plants and 25 cm between rows. All of the materials were harvested on June 10. Twenty plants of each line in the same K treatment were put together as one sampling during harvest and then threshing for further testing.
2.3. Trait Measurements
All of the investigated traits and their abbreviations are listed in
Table 1. For hydroponic trials, the three replicates for each line (nine plants) of each treatment were pooled together as one mixed sample and separated into roots and shoots. After being dried at 105 °C for two hours and dried at 60 °C for 72 h in an oven, the dry weight and the concentrations of K, Ca, and Mg in roots and shoots were measured. The concentrations of K, Ca, and Mg in roots and shoots were determined using atomic absorption spectroscopy (AA7000) after microwave digestion using HNO
3. K-use efficiency (RKUE) was the ratio between dry weight and the concentration of K in the corresponding part of plant. For example, root K-use efficiency (RKUE) was the ratio between root dry weight per plant and the concentration of K in root (RDW/RKCE). The measurement methods of other nutrient use efficiencies were similar to the root K-use efficiency.
For field trials, the plant height (PH), spike number per plant (SN), and grain number per spike (GN) were determined from five random plants for each replicate of each line. All of the plants for each line of one K treatment were pooled together and then measured for dry weight and K, Ca, and Mg concentrations of the straw and grain, separately. The concentrations of K, Ca, and Mg in grains and straws were determined using atomic absorption spectroscopy (AA7000) after microwave digestion using HNO3. The calculation methods of nutrient use efficiencies were similar to the root K-use efficiency in the hydroponic culture trial. The K harvest index (KHI) is the ratio between grain K content per square meter and aboveground K content per square meter (GKC/AKC). The measurement methods of Ca and Mg harvest indexes were similar to the KHI.
2.4. Data Analysis
The SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was employed to conduct the analyses of variance (ANOVA), the least significant difference (LSD) test, and Spearman’s correlation coefficients (r) between different traits. In a no-repeat trial design, using a two-factor model was adequate for ANOVA. All factors including RILs (n − 1) degrees of freedom, treatments (t − 1), and random error ((n − 1)(t − 1)) were considered sources of random effects. Multiple comparison tests for the traits between “treatments” were calculated by taking all of the RILs as replicates and using the mean value of the same K condition for each trait. The variance of K conditions was excluded when the broad-sense heritability (hB2) was estimated according to the formula: hB2 = σg2/(σg2 + σe2), where σg2 was the genotypic variance and σe2 was the total error variance.
The high-density genetic map for 184 RILs of “TN18 × LM6” [
28] was employed in the QTL analysis. The map comprised of 10739 loci (5399 unique loci) assigned to 21 chromosomes, with a total map length of 3394.47 cM and a density of 0.63 cM/marker. The Windows QTL Cartographer 2.5 software (
Http://statgen.ncsu.edu/qtlcart/wqtlcart.htm) [
33] was used to perform the QTL mapping in this study. The presence of the significant QTL was declared via the threshold that was defined by 1000 permutations at
p ≤ 0.05 [
34]. The identification of QTL cluster and its confidence interval referenced the results of the meta-analysis (Biomercator 2.0 software, AIC = 4 (model 4) in the step Meta-analysis 2/2 (
http://www.genoplante.com)) [
29].
2.5. Naming Method of QTLs
QTLs were named according to the method of “Q + trait name + chromosome name + experimental treatment.” Among them, traits are represented by their English abbreviation, and “−” was added between traits and chromosomes. QTLs for the same trait on the same chromosome were distinguished using an Arabic numeral (1, 2, 3, …). In addition, E1 and E2 stand for the hydroponic trial of February 2013 and March 2013, respectively. T1 and T2 stand for CK and LK treatments, respectively.