A major quantitative trait locus controlling phosphorus utilization efficiency under different phytate-P conditions at vegetative stage in barley

GAO Shang-qing, CHEN Guang-deng, HU De-yi, ZHANG Xi-zhou, Ll Ting-xuan, LlU Shi-hang, LIU Chun-ji

1 College of Resources, Sichuan Agricultural University, Chengdu 611130, P.R.China

2 Triticeae Research Institute, Sichuan Agricultural University, Chengdu 611130, P.R.China

3 Commonwealth Scientific and Industrial Research Organization (CSIRO) Agriculture Flagship, St Lucia, Queensland 4067,Australia

1. lntroduction

Phosphorus (P) is an essential nutrient for plant growth and development. Globally, application of P fertilizer was greater than removal of P by harvested crops in 2000. In addition, deficits of P occurred in 30% of cropland areas around the globe (MacDonaldet al. 2011). In soils, P has low mobility and a high rate of fixation (Schachtmanet al.1998; Tianet al. 2012). Because of these characteristics, P limits crop productivity around the world (Vanceet al. 2003).At present, applying phosphate fertilizer is still an effective way to improve yield and quality of crops (Cordellet al.2009). Statistics from the International Fertilizer Industry Association (IFA) (http://www.fertilizer.org/ifa/) in 2006 showed that the annual input of phosphate fertilizer (P2O5)was more than 30 million tons, with the average input into wheat crops being 20 kg or more P2O5per ha (FAO 2006).However, the extensive application of phosphate fertilizer has fundamentally altered the global P cycle, and caused eutrophication and other environmental problems (Maet al.2012). Organic P constitutes 50–80% of total P in soils(Turneret al. 2002). Phytate-P is an important component of soil organic P, which can be absorbed and used by plantsviaacid phosphatase and phytase hydrolysis (Sharmaet al.2007; Starneset al. 2008; Yeet al. 2015). Therefore, it is desirable to develop cultivars with enhanced efficiency of phytate-P use because they may offer a sustainable solution for managing P supplementation in crop production.

Phosphorus utilization efficiency (PUE) is mainly controlled by complex polygenic regulation, and significant differences in regulation exist between crop varieties and genotypes(Suet al. 2009; Wanget al. 2010). Previous studies have shown that plant tolerance to low P is regulated by multiple genes (Suet al. 2006; Oonoet al. 2013). Tolerance to low P and quantitative trait locus (QTL) analysis of related traits has been conducted in rice (Wissuwaet al. 1998), corn (Zhuet al. 2005), rapeseed (Yanget al. 2011), common bean (Liaoet al. 2004), and soybean (Liet al. 2005). In a rapeseed recombinant inbred line (RIL), Yanget al. (2011) conducted a QTL analysis and assessed six phenotypic traits at the vegetative stage under high P and low P conditions. They detected a total of 71 QTLs on 13 linkage groups, including 28 in low P conditions, 22 in high P conditions and 21 for relative traits. Kinget al. (2013) analyzed QTL for P accumulation in soybean, and identified three candidate genes on chromosomes 7, 12 and 17. In common bean, 19 related root morphology and P uptake traits were detected in eight linkage groups (Yanet al. 2004). Most of the phenotypic variance explained by these QTLs are low, and the identified markers could be difficult to use in breeding.A low P tolerance gene named asPSTOL1was detected in rice using a population of RILs under P deficiency (Wissuwaet al. 1998). Expression analysis in low P soil showed that this gene significantly enhanced grain yield. Further analysis showed that the gene significantly enhanced seedling root length and improved ability of P uptake (Gamuyaoet al.2012).

The genotypic differences of PUE in wheat under P deficiency have been confirmed (Batten 1992). Hayeset al.(2004) showed that the P-efficient wheat accumulated 32%more P at a similar dry weight than P-inefficient wheat under low P conditions. At present, a variety of methods have been used in studying resistance to low P in wheat. These methods include the use of Chinese Spring nullisomics and substitution lines for a given chromosome and as well as QTL mapping(Liet al. 1999a, b; Suet al. 2006, 2009). In Chinese Spring nullisomic-tetrasomic lines, genes conferring resistance to low P stress have been found on chromosomes 1A, 4A, 7A, 3B,5B, and 7D. In addition, suppressor genes were also detected on chromosomes 1B, 4B, 7B, 3A, and 6D (Liet al. 1999a, b).QTL analysis using hybrids of Chinese Spring and the low P tolerant variety Lovrin 10 indicated that the main effect QTL controlling P efficiency was found on chromosomes 3B, 4B,and 5A (Suet al. 2006). A further study found seven QTLs for P absorption and six QTLs controlled PUE (Suet al. 2009).

Barley is one of the most important cereal crops and is grown all over the world; it is widely used in feed and in the food industry, and is the main raw material for brewing beer(Feuillet and Muehlbauer 2009). It has a planting area of about 560 000 km2worldwide, with a total output of about 120 000 million kg (FAO, http://www.fao.org). Widely used in feed and food industry, barley is the main raw material for brewing beer (Feuillet and Muehlbauer 2009). Genotype differences also exist in PUE in barley (Asmaret al. 1995;Georgeet al. 2011). There are few studies on QTL mapping and genetic analysis of genes associated with PUE in barley,however, three QTLs for PUE have been identified on chromosomes 2H and 5H under different P conditions (Gonget al. 2016). Górny and Ratajczak (2008) successfully imported an exogenous gene to improve P uptake and utilization efficiency in barley and thus showed that improvement of PUE in barley is feasible. In this study, a RIL population derived from the cultivated barley variety Baudin and wild barley CN4027 was used to map QTLs for PUE, and to analyze the correlation of QTLs for PUE and P efficiency related traits at the vegetative stage under normal organic P (+P, 0.5 mmol L–1) and low organic P (–P, 0.05 mmol L–1)conditions using phytate-P as an organic P source.

2. Materials and methods

2.1. Plant materials

A population of RILs were generated using the embryo culture procedure described in previous studies (Chenet al.2013; Zhenget al. 2013). The RIL population contained 128 lines derived from a hybrid between a P-efficient wild barley (Hordeum spontaneum) genotype CN4027 and a P-inefficient cultivated barley (H.vulgare) variety Baudin.

2.2. Experimental design

Hydroponic culture experiments were conducted to investigate the phenotypic traits of the two parents and the RIL population. Uniform seeds selected from the 128 RILs and the two parents were surface-sterilized for 15 min in 3% H2O2and then washed three times with deionized water.The surface-sterilized seeds were germinated on filter paper that was placed in Petri dishes covered with deionized water,followed by growth in an illuminated culture room until the cotyledons were fully developed after 6 days at 22–24°C.Uniform seedlings were then transferred carefully to a container containing modified Hoagland’s solution (Hoagland and Arnon 1950) without monopotassium phosphate. The P deficient solution consisted of 5.0 mmol L–1KNO3,2.0 mmol L–1MgSO4·7H2O, 4.0 mmol L–1Ca(NO3)2·4H2O,1.0 μmol L–1H3BO3, 0.5 μmol L–1CuSO4·5H2O,1.0 μmol L–1ZnSO4·7H2O, 1.0 μmol L–1MnCl2·4H2O,0.5 μmol L–1NaMoO4·2H2O, and 50 μmol L–1EDTA-Fe.The pH value of the nutrient solution was adjusted to 5.7±0.2 using 2 mol L–1NaOH or HCl every 2 days.

A low phytate-P condition (0.05 mmol L–1) and a normal phytate-P condition (0.5 mmol L–1) added as phytate isolated from rice (myo-inositol hexaphosphoric acid dodecasodium salt, Sigma, USA) (Yeet al. 2015)were used in the RIL population and three replicates were conducted in two independent trials. Experiments were conducted in a split plot design, with P conditions as the main plot and genotype as the subplot. The two trials in the RIL population were conducted in the same greenhouse at Sichuan Agricultural University, Chengdu,China. Trial 1 was carried out from September 1 to November 6, 2014 and trial 2 was carried out from February 27 to April 25, 2015. The minimum and maximum temperatures in trial 1 ranged from 6 to 10°C and from 21 to 25°C and those in trial 2 ranged from 5 to10°C and from 16 to 21°C, respectively.

2.3. Evaluation of plant performance

After harvesting, tiller numbers (TN) were calculated and recorded. Then, roots and shoots were separated, and harvested tissues were dried at 105°C for 30 min followed by 75°C to constant weight. Shoot dry weight (SDW) and root dry weight (RDW) were measured for each line. Dried plants were ground to a fine powder and digested with 5 mL of a mixture of concentrated H2SO4and H2O2. Total P content was analyzed with Mo-Sb colorimetry at 700 nm using a spectrophotometer (UV-1200, MAPADA Instrument Co., Ltd., China) (Lu 1999). PUE was expressed as dry weight per unit of P taken up in shoots and roots (Yanget al. 2011).

2.4. Molecular marker analysis and linkage map construction

The method used for DNA isolation was as described by Chenet al. (2012). Diversity array technology (DArT)markers were initially used for linkage map construction and QTL analyses using 128 lines from the Baudin/CN4027 population. DArT genotyping of the parents and the mapping population was carried out by Triticarte Pty. Ltd. (http://www.tritcarte.com.au). Hybridization of genomic DNA to the DArT array, image analysis, and polymorphism scoring were conducted as described by Wenzlet al. (2004). Linkage analysis was carried out using the computer package JoinMap®4.0 (Van Ooijen 2006).

2.5. Data analysis and QTL mapping

Statistical analyses were performed using SPSS v22.0 for Windows (SPSS Inc., Chicago, IL). Pearson correlation coefficients and least significant difference (LSD) tests were estimated between traits and trials. Analysis of variance(ANOVA) was employed to estimate genetic variance (σ2G),environmental variance (σ2E), and subsequently broad-sense heritability (h2b). The effects of replicate and genotype for PUE, TN, and biomass were determined using covariance analysis.

Segregation ratios of assessed markers were tested by Chi-square goodness-of-fit to a 1:1 ratio at the significance level ofP=0.01. LOD thresholds from 3 to 10 were tested,until a threshold with the optimum number of markers in linkage groups maintaining linkage order and distance was obtained. The Kosambi mapping function was used to convert recombination ratios to map distances. MapQTL®5.0 (Van Ooijen 2004) was used for QTL analysis. The Kruskal-Wallis test was used as a preliminary test of associations between markers. Interval mapping (IM)was then used to identify major QTL. Automatic cofactor selection was used to fit the multiple QTL model (MQM) and to select significantly associated-markers as cofactors. A linkage map showing the QTL positions was drawn using Mapchart (Voorrips 2002).

3. Results

3.1. Phenotypic variation under two phytate-P levels

Significant differences in PUE were found between the two parents harvested at the vegetative stage. The PUE of CN4027 for shoots and roots were higher than those of Baudin for both phytate-P conditions (Fig. 1). Under low phytate-P, shoot and root PUE of the parent CN4027 were 534.2 and 426.5 g DW g–1P, respectively, and for the parent Baudin, these values were 366.8 and 328.3 g DW g–1P, respectively. Under normal phytate-P, shoot and root PUE of CN4027 were 156.8 and 141.0 g DW g–1P,respectively and those for the parent Baudin were 132.8 and 120.9 g DW g–1P, respectively. The RIL population exhibited distinct differences from parents in PUE under the two different phytate-P levels. The coefficients of variation ranged from 16.53 to 27.29% in roots and shoots. Lines with better PUE than the parent CN4027 were detected under both of the phytate-P conditions. Transgressive segregation of PUE was observed in the RIL population(Table 1).

Highly significant differences in P efficiency related traits (TN, RDW, and SDW) were detected between the commercial cultivar Baudin and wild barley CN4027(Table 1). The RIL population exhibited distinct differences in most of the investigated traits in both phytate-P levels.All traits showed continuously and approximately Gaussian distributtion (data not shown), which indicated that the traits were also suitable for QTL mapping. Theh2bfor different traits varied from 66.95 to 87.01%.

Fig. 1 QTL conferring phosphorus utilization efficiency (PUE) with root (A) and shoot (B) detected on chromosome 3H under the low (–P) and normal phytate-P (+P) conditions. The vertical dotted line indicates the average significance threshold (LOD=3.0)derived from permutation tests. RPUE01 and RPUE02, root P utilization efficiency in trail 1 (September 1 to November 6, 2014)and trail 2 (February 27 to April 25, 2015), respectively; SPUE01 and SPUE02, shoot PUE in trails 1 and 2, respectively.

3.2. Genetic linkage map construction

The linkage map constructed in this study consisted of 607 DArT markers that spanned 1 148.70 cM in length with an average interval of 1.89 cM between adjacent markers(Appendix A). The seven linkage groups ranged from 198.11 cM for chromosome 2H to 128.65 cM for chromosome 5H,and the average distances between markers ranged from 1.45 cM for 4H to 2.15 cM for 1H. Linkage distances larger than 20 cM were present only in 3H and 5H. The number of markers ranged from 64 for 4H to 103 for 2H, with an overall average number of markers per chromosome of 87.

3.3. QTL mapping for PUE

A large-effect QTL for PUE in shoots and roots was mapped on 3H (Fig. 1). The LOD values of this QTL varied from 5.08 to 9.16 (Table 2). The QTL was designatedQpue.sau-3H, wherepuestands for P utilization efficiency andsaustands for Sichuan Agricultural University. Based on MQM analysis, markers flanking the locus were bPb3256099 and bPb3931069 under both phytate-P conditions assessed (Table 2). TheQpue.sau-3Hin shoots explains up to 30.2 and 29.5% of the phenotypic variance under low and normal phytate-P conditions, respectively. Favorable alleles from theQpue.sau-3Hin shoots mapped on chromosome 3H, which were from the higher PUE parent CN4027 and contribute to increase PUE. The phenotypic variance in roots explained byQpue.sau-3Hwas 28.9 and 30.7% under the low and normal phytate-P conditions, respectively.

3.4. QTL mapping for P efficiency related traits under two phytate-P levels

A QTL for TN at the vegetative stage was detected under both the low and normal phytate-P conditions (Table 3). The QTL,designatedQtn.sau-5H, was located between bPb6277350 and bPb3265782 on chromosome 5H. This QTL explained 13.8 to 19.7% with an average of 16.8%of the phenotypic variance under the low phytate-P condition and 15.8 to 22.7% with an average of 19.2% of the phenotypic variance under the normal phytate-P condition. LOD values ofQtn.sau-5Hvaried from 4.01 to 6.36 with an average of 5.20, and favorable alleles came from the male parent CN4027.

Two QTLs for DW were identified under the both phytate-P conditions (Table 3).One of them,Qdw.sau-3H, was located on chromosome 3H under both phytate-P conditions (Fig. 2). This QTL explained on an average of 14.2% (–P) and 18.8% (+P)of the phenotypic variance in roots, and an average of 30.9% (–P) and 25.2% (+P)of the phenotypic variance in shoots. The other QTL was only detected under the low P condition. This QTL was localized between bPb6277350 and bPb3265782 on chromosome 5H and it explained 15.1 to 17.8% with an average of 16.4% of the phenotypic variance in roots.

3.5. Effects of TN and biomass on PUE

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Correlation analyses were performed among the traits and their relative values in different phytate-P conditions (Table 4). TN was not correlated with PUE in roots or shoots under either of the phytate-P conditions. DW was significantly correlated with PUE in roots and shoots under both of the phytate-P conditions, and the correlation coefficient varied from 0.27 to 0.46.

The location ofQdw.sau-3Hwas similar to that ofQpue.sau-3H. To quantify the possible effect of DW on PUE, data from the two PUE trials were analysed against data from the two DW trials by covariance analysis. Thisanalysis showed that the LOD value ofQpue.sau-3Hwas reduced when the effect of DW was accounted for by the covariance analysis. However, the position ofQpue.sau-3Hwas unchanged on chromosome 3H (Fig. 3).

Table 2 Quantitative trait locus (QTL) mapping for phosphorus utilization efficiency (PUE) under the low (–P) and normal phytate-P(+P) conditions

Fig. 2 Quantitative trait locus (QTL) conferring dry weight (DW) with root (A) and shoot (B) detected on chromosome 3H under the low (–P) and normal phytate-P (+P) conditions. RDW01 and RDW02, root DW in trail 1 (September 1 to November 6, 2014) and trail 2 (February 27 to April 25, 2015), respectively; SDW01 and SDW02, shoot DW in trails 1 and 2, respectively.

Table 4 Correlation coefficients between investigated traits in two trails1)

4. Discussion

4.1. Variation in PUE of parents and the RlL population at two phytate-P levels

Significant differences in PUE were found between the two parents at the vegetative stage at both phytate-P conditions. The results showed that theH.spontaneumaccession CN4027 was more effective in utilizing organic P for plant growth. Previous studies were focused on variation in P uptake and utilization efficiency using inorganic P sources. However, few studies have explored PUE under organic P treatment. Some researchers found that two types of grass (Duo grass and rye grass) could obtain enough P from phytate to meet their requirements for P, and they had better dry weight growth than plants that were not supplied with P (Priya and Sahi 2009; Sharma and Sahi 2011). The mining ecotype ofPolygonum hydropiperis also capable of assimilating P from Po media supplied as phytate. Compared with the non-mining ecotype ofP.hydropiper, the mining ecotype had significantly higher root and shoot biomass (Yeet al. 2015). Our previous study suggested that P accumulation of P-efficient genotypes was higher than that of P-inefficient genotypes under the various concentrations of phytate-P in barley(Caiet al. 2014).

The RILs in this study also exhibited distinct differences in PUE under both phytate-P levels. RIL lines had significantly higher or lower values than the parents for PUE, indicating potential transgressive variations. In wheat, different fertilization regimes of N and P in 182 RILs led to broad,continuous variation in 22 phenotypic traits, as well as significant transgressive segregation (Xuet al. 2014).Shiet al. (2008) determined that there was a significant separation of P content in grains from 119 lines of a doubled haploid wheat population (Shiet al. 2008). In maize, traits related to P efficiency were found to segregate continuously,and to have an approximately normal distribution with absolute values of less than one for skewness and curtness(Chenet al. 2008). For all of the traits studied, transgressive variation in rice was typically negative with only PUE showing positive transgression (Wissuwaet al. 1998). The phenotypic value of PUE inBrassica napusalso showed large variation among the RIL population under both P conditions (Yanget al. 2011).

Fig. 3 Logarithm of odds (LOD) values of Qpue.sau-3H with root (A) and shoot (B) obtained from combined data of the two trials pre-adjustment by biomass (combined) and post-adjustment by biomass (adjusted). RPUE, root P utilization efficiency; SPUE,shoot P utilization efficiency. –P, low phytate-P condition; +P, normal phytate-P condition.

4.2. QTL for PUE under the two phytate-P levels

P uptake and utilization efficiency is regulated by multiple genes (Suet al. 2006; Oonoet al. 2013). A study on QTL mapping for PUE in barley showed that three loci were mapped on chromosomes 2H and 5H under different P conditions and explained 7.47 to 8.99% in a doubled haploid Commander/Fleet population (Gonget al. 2016). Naturally,breeding barley cultivars with nutrient efficiency require more QTLs. In this study, a novel and major locus controlling PUE, located at the chromosome 3H (Qpue.sau-3H), was detected in P-efficient wild barley (Hordeum spontaneum)genotype CN4027. This QTL explains 28.3 and 30.7%of the phenotypic variation in roots under low and normal phytate-P conditions, respectively.

Collinearity between genomes of closely related species has been used to predict and locate genes and markers of interest (Chenet al. 2013). In wheat and barley, gene order is highly conserved, however, there are a few translocations between wheat genomes A and B that have been well characterized (Liuet al. 1992; Devos 2005).With the use of Chinese Spring nullisomic-tetrasomic lines genes conferring low P tolerance and suppressor genes were also detected on chromosomes 1B, 4B, 7B,3A, and 6D (Liet al. 1999a, b). Six loci regulating P utilization efficiencies are located on chromosomes 1A,2A, 3A, 3B, 5A, and 6B (Suet al. 2009). The positive and negative linkages between the QTLs mapped to these six loci, suggest that the locus on chromosome 3B is a good candidate for selection to improve PUE. Two QTLs for PUE in shoots were located on chromosomes 1B and 5A,and three QTLs for PUE in the whole plant were located on chromosomes 2B, 5A, and 7A in the RIL population derived from two wheat varieties W7984 and Opata85 (Caoet al.2001). Guoet al. (2012) used QTL mapping in wheat to assess seedling traits under varying concentrations of N,P and K, and found that a locus for PUE in roots is located on chromosome 1B. Thus the QTL controlling PUE on 3A and 3B in wheat is unlikely to be homologous withQpue.sau-3Hin barley.

4.3. Correlation between PUE and P efficiency related traits

PUE is a complex trait, which can be evaluated by biomass,TN, root morphology, and leaf area (Liaoet al. 2004). In this study, DW and TN were selected to evaluate the P efficiency of barley. TN was not correlated with PUE in roots and shoots under either of the phytate-P conditions. However,Suet al. (2006) showed that TN was significantly positively correlated with shoot P uptake, and negatively related with PUE under both of the P conditions. QTL analysis detected a locus for TN from both phytate-P conditions. Different from that ofQpue.sau-3H, this locus for TN was located on 5H (Qtn.sau-5H).

DW was significantly correlated with PUE in roots and shoots of the RIL population. Suet al. (2009) also showed that PUE has a positive correlation with shoot DW in wheat.Two QTLs affecting DW were detected in this study; the location ofQdw.sau-3Hon chromosome 3H was similar to that ofQpue.sau-3H. Similar results have been found for nitrogen-related traits in wheat (Xuet al. 2014). Further analysis showed that the LOD value ofQpue.sau-3Hwas reduced when the effect of DW was accounted for by covariance analysis. But, the position ofQpue.sau-3Hwas unchanged on chromosome 3H. Developing and exploiting near isogenic lines (NILs) and NIL-derived populations(Chenet al. 2012), as carried out for traits related to PUE,could be an option to further investigate the relationship between gene(s) controlling PUE and DW in barley.

5. Conclusion

In summary, enhancing P efficiency is an important target in barley breeding. We report in this paper a QTL study on PUE and PUE related traits under different phytate-P levels (normal phytate-P, 0.5 mmol L–1and low phytate-P,0.05 mmol L–1) using a RIL population. A novel and major locus (Qpue.sau-3H) was detected on chromosome 3H conferring PUE in shoots and roots from the RIL population under two different phytate-P levels. Results from this study also showed that TN was not correlated with PUE, and a QTL controlling TN was detected on chromosome 5H in the RIL population. However, DW was significantly and positively correlated with PUE, and a QTL controlling DW was detected near theQpue.sau-3Hlocus. Existing data by covariance analysis indicated that different genes at this locus are likely involved in controlling these two traits. The existence ofQpue.sau-3Hmay offer valuable clues for fine mapping and map-based cloning in barley.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31401377), the Science and Technology Project of Sichuan Province, China (2017JY0126),and the Key Project of Education Department of Sichuan Province, China (14ZA0002).

Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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