Soil water capture trends over 50 years of single-cross maize (Zea mays L.) breeding in the US corn-belt

Highlight Soil water capture potential of maize has not changed over 50 years of single-cross breeding. Changes in resource use efficiency and allocation to reproductive organs must underpin yield improvement.


Introduction
Maize yield in the US corn-belt has increased steadily for over 80+ years and doubled between 1965 and 2010 (Smith et al., 2014). Improvements in germplasm, agronomic practices, and cropping systems intensification contributed to these sustained yield gains. The long-term genetic gain in maize yield for the conditions of the US corn-belt over the past 80 years was demonstrated by quantifying the productivity of successful hybrids commercialized over eight decades; considered together and analysed in the context of the negative association between grain yield and ASI (Campos et al., 2004)-which is an indicator of increased ear growth rate and resource availability per ovule -they lead to the hypothesis that genetic gain was determined by an increased stress tolerance and resource allocation to reproductive organs (Duvick, 2005). This hypothesis was evaluated with tropical germplasm selected for improved drought tolerance Edmeades et al., 1999), and Argentinian flint and semi-dent hybrids (Echarte et al., 2000;Luque et al., 2006), but still remains to be evaluated for US temperate maize.
The correlated response of genetic gain for yield in the USA between well-watered and drought environments, and the selection in a target population of environments where biotic and abiotic challenges including drought are frequent, suggest that multiple physiological mechanisms contributed to the observed genetic improvement of yield (Duvick, 2005;Cooper et al., 2014). It is plausible that the reduction in ASI and the apparent increase in carbohydrate allocation to reproductive organs could result at least in part from improved plant, kernel, and ovule water status. The reduction in ASI as determined by early silk emergence rather than late shedding (Welcker et al., 2007), the high sensitivity of silks to water deficit (Westgate and Boyer, 1985), the high correlation between silk emergence and kernel set (Schussler and Westgate, 1994), and the absence of changes with selection in total biomass in tropical germplasm (Edmeades et al., 1999), provide evidence to support this interpretation. Results from a simulation study, conducted to consider the roles of leaf and root architecture in light and water capture, helped formulate the hypothesis that, changes in root architecture associated with improved soil occupancy and water capture could be a process underpinning the observed hybrid by density interactions in historical maize yield improvement in the USA . The onset of accelerated yield improvement under drought stress conditions that coincides with the initiation of singlecross (SX) hybrids breeding in the 1960s , changes in root architecture (York et al., 2015), and reduction in canopy temperature (Barker et al., 2005) with the year of commercialization, and the contrast between water uptake patterns between single and double-cross hybrids (Campos et al., 2004), supports the hypothesis that the observed trend in yield during SX breeding could be associated, at least in part, with an increased water capture.
The objective of this study was to test the hypothesis that water capture increased as the result of selection for yield in SX hybrids in the US corn-belt and that this process underpinned at least in part the genetic improvement in grain yield under drought stress. The results from this study can inform the design of breeding strategies that are aimed at developing germplasm that fully utilizes available water resources.

Field experiments
Two field experiments were conducted in 2010 and 2011 at a DuPont Pioneer experiment station located 6 km west of Woodland, CA, USA. Eighteen ERA hybrids commercialized between 1963 and 2009 (Table 1) were planted in a Yolo Silt Loam (Fine-silty, mixed, superactive, nonacid, thermic Mollic Xerofluvents) (Andrews, 1972). The hybrids included in this study represented the SX hybrids that have previously been included in long-term genetic gain experiments for maize in the US corn-belt (Duvick et al., 2004;Smith et al., 2014). They represent a historical sequence of hybrids that were widely adopted by growers following their commercial release. Experimental evaluations under a range of water-limited environments, through use of managed drought environments at low rainfall locations, have demonstrated that this set of hybrids demonstrated genetic gain for both water-limited environments and high-input environments .
Weather variables were from the Esparto weather station located near the experiment site and maintained by the California irrigation management information system (www.cimis.water.ca.gov/ WSNReportCriteria.aspx). Cumulative precipitation between April and September was 97.1 mm and 67.3 mm in 2010 and 2011, respectively (Table 2), whereas total potential evapotranspiration for the same period was 1069 mm in 2010 and 984 mm in 2011. Supplemental irrigation was applied through a buried drip tape to promote good stand establishment, incorporation of fertilizer, and to mitigate heat stress at flowering time (Table 2). Temperatures greater than 40 °C during one month bracketing flowering were seven in 2010 and zero in 2011.

Phenotypic measurements
Phenotypic data on silk emergence and pollen shed were collected daily for a period of 3 weeks. Silking and shedding dates were determined when 50% of the plants in a plot exhibited at least one visible silk, or were shedding pollen, respectively. Thermal times to dates of silking and shedding were calculated using daily average temperature, a base temperature of 10 °C, and optimum temperature of 30 °C (Gilmore and Rogers, 1958). ASI was calculated as the difference between the thermal time to shedding and silking. Grain yield was measured using a New Holland automated research plot combine TR series (CNH Global, Burr Ridge, IL, USA) and adjusted to 0.15 g g -1 moisture. Soil moisture measurements were collected using a Time Domain Reflectometer (TDR) sensor model Trime T3-50 TAP (IMKO, Ettlingen, Germany). The instrument was calibrated by IMKO and corrected by bulk density measured in the fields (1.37-1.58 g cm -3 ). The accuracy of the TDR instrument used in this study was 0.02 cm 3 cm -3 for an area of measurement of 180 × 150 mm. One 3 m long access tube per plot was installed between the rows in the centre of each two-row plot when plants reached the two expanded leaves stage (V2). Images of root distribution profiles from excavations conducted to a depth of 1.7 m at physiological maturity and to a depth of 2.0 m at flowering time in experiments conducted at two locations-Viluco, Chile and Woodland, CA, USA, respectivelyindicated that roots for a given genotype did not cross between two-row plots (images not shown). This observed root distribution pattern limits the plausible intermingling of roots close to the access tube, which is placed 1.14 m from the nearest planting row for a different genotype. Similar root architecture in maize, with nodal roots growing at an angle and then turning vertical, was observed in other studies (Tardieu and Pellerin, 1990;Ješko, 1992). Plant stands were uniform and no gaps were observed near the access tubes. Table 1 indicates the hybrids for which soil moisture was measured. Fifteen measurements per tube were taken every 20 cm between 23 June and 20 September in 2010, and from 2 May to 18 August in 2011. Data were collected in 11 d in 2010 and in 10 d in 2011. Water use at each layer was calculated as the difference between the first and the last measurement. Total water use was calculated by integrating soil water use across the soil column and adding irrigation amounts applied during the period of measurement. Rainfall amounts during this period (Table 2) had limited effect on total water use.

Statistical analysis
The experiment was conducted as a randomized incomplete block design, with the factor hybrid randomized within replications, which were blocked to account for field variability. Experiments included four replications in 2010 and three replications in 2011. Formally, the data for flowering, yield, and ASI (Y ijk ) of year (E) i , hybrid (H) j , block (B) k within year, were modelled as a function of an overall mean u, factors for year, hybrid, and two-way way interaction between hybrid and year, block within year and the residual e ijk , where random effects are denoted with underbars and fixed effects without underbars. Variance components of random effects were estimated by residual maximum likelihood method. An F test was used to assess significance for fixed effects. Analyses were conducted with ASREML (Gilmour et al., 2009). Trends over year of release were tested for significance by linear regression (R Core Team, 2014).

Phenology, yield, and ASI
Significant differences among hybrids in thermal time to shedding were observed in both 2010 and 2011 experiments (Table 3). However, trends for thermal time to shedding with respect to year of commercialization were not significant with regression slope [standard error (SE)] values -1.5 (±0.89) and -0.76 (±0.75) °C year -1 in 2010 and 2011, respectively. Drought stress treatments were effective in reducing yields in both the 2010 and 2011 field experiments. Average yield in the experiment conducted in 2011 was significantly higher than that observed in 2010 (677 vs 317 g m -2 ; P<0.001), and both markedly lower than yields close to 1400 g m -2 reported for favourable conditions at this location . Average ASI was 154 °C and 175 °C for experiments conducted in 2011 and 2010, respectively. These positive values for ASI indicate that drought treatments affected reproductive development around flowering time and further demonstrate effective imposition of drought in these experiments. Hybrid variation was observed for both yield and ASI in both experiments (Table 3) and this was associated with year of commercialization (Fig. 1). In agreement with prior studies, yield under stress increased with later year of commercialization, and ASI showed the opposite pattern Edmeades, 1993, 1996;Campos et al., 2006). Yield under stress doubled between hybrids commercialized in 1963 and 2009. Estimated trends with respect to year of commercialization were 4.1(±0.95) (P<0.001) and 8.1(±1.1) (P<0.001) g m -2 year -1 for yield in 2010 and 2011, respectively, and -2.2(±0.6) (P<0.01) and -1.7(±0.8) (P=0.078) °C year -1 for ASI in 2010 and 2011, respectively. Because the high plant population may have been supra-optimal for the first cohort of SX hybrids, the trends in ASI may have been overestimated.

Temporal patterns of water extraction
The irrigation treatments resulted in a flowering stress followed by a grain filling and terminal stress during both years. In both experiments, soil moisture started to decline around 50 d after planting (Fig. 2), which was around 26 d prior to anthesis. Soil water declined gradually until maturity, when the crop was harvested ( Fig. 2; Table 4).
As the growing season progressed, soil water was extracted from increasingly deeper soil layers (Fig. 3). The patterns of variation with date and depth were significantly different   Experiment year, hybrid, and block were included in the model as random sources of variation. * Indicates variance components that are equal to or greater than 1.5 times their SEs. between years as indicated by the significant date by depth interaction variance component (Table 4). While in 2010 a clear soil water uptake front was observed progressing with depth across dates, in 2011 variation in soil water content was concentrated in the upper soil layers early in the growing season with a shift towards water use from the deeper soil layers post-flowering (Fig. 3). Significant soil water uptake was observed down to 2.4 m in 2010 and 3.0 m in 2011 (Fig. 3). These observations showed water uptake in soil layers deeper than prior reports for maize grown in the US Plains (Norwood, 2001;Payero et al., 2006;Tolk et al., 1998) and in an Argentinian silty loam Haplustoll (Dardanelli et al., 1997). A significant variance component for hybrid by date interaction was observed for volumetric soil water content, but it was small relative to variance component for date (Table 4).

Water uptake did not change during SX breeding
Water uptake was estimated as the difference between the Best Linear Unbiased Predictions for soil moisture content at the first and the last measurements, with the addition of irrigation amounts. Figure 4 shows that total water use varied between years but not among hybrids. This result is consistent with the analysis of variance for soil moisture content ( Table 4) that indicated absence or inability to detect a significant effect of hybrid on soil moisture content. Total soil water use was greater in 2011 than in 2010 (Fig. 4), which was consistent with greater yields observed in 2011 relative to 2010 (Fig. 1). Variation between years in total soil water use was determined by differences in post-flowering water uptake (Fig. 2). At anthesis (approximately 71 and 73 d after planting in 2010 and 2011, respectively), water extraction was observed down to 1.8 m in 2010 and 1.6 m in 2011 (Fig. 3). Total soil moisture estimated at the time of anthesis was 862 and 857 mm in 2010 and 2011, respectively. Higher irrigation applied in 2011 than in 2010 (Table 2) may have contributed to maintenance of leaf area, delayed senescence, and the observed higher post-flowering water use in 2011 than in 2010.
In addition to not differing in their total water use, hybrids did not differ significantly in post-anthesis water use (Fig. 5). Estimated trends with respect to year of commercialization were 0.28 (±0.14) (P=0.078) and 0.09 (±0.27) (P=0.76) mm year -1 for post-anthesis water use in 2010 and 2011, respectively This indicates that differences in timing of water use in relation to crop development were also not associated with year of commercialization during the period of SX breeding.

Discussion
This study tested the hypothesis that yield improvement as evidenced within the ERA hybrid studies was caused at least in part by increased water uptake. The imposed levels of drought stress reduced yields to about 25% or less of yield attainable under favourable conditions. It also enabled Experiment year, date of measurement, hybrid, and block were included in the model as random sources of variation. * Indicates variance components that are equal to or greater than 1.5 times their SEs. measurement of soil water uptake in a terminal drought pattern that forced rooting to express the crop's ability to extract water from deep soil layers (Tinker, 1976). The observed trends for the rate of gain in yield with year of commercialization (4-8 g m -2 year -1 ) compared well with prior estimates for comparable yield levels and drought stress treatment (Campos et al., 2006;Cooper et al., 2014). A negative correlation between ASI and yield was also demonstrated as well as an overall positive ASI, which indicated that drought treatments affected reproductive development around flowering time Edmeades, 1993, 1996;Edmeades et al., 1993;Campos et al., 2004). Soil water extraction, however, remained constant across hybrids with no indication of changes related to year of hybrid commercialization. This fundamental result leads to the conclusion that over the period of SX breeding, total water uptake cannot explain the observed yield increase. Results do not rule out, however, that changes in water uptake could have occurred prior to the SX breeding era. The results therefore imply that the physiological underpinning of genetic improvement of yield during this period of SX breeding could be related to an increased allocation of biomass to the ear (Lafitte and Edmeades, 1995), perhaps at the expense of partitioning of biomass to root systems with the consequential improvement in root system efficiency , maintenance of silk water status (Schoper et al., 1987), sugar metabolism and flux (McLaughlin and Boyer, 2004), or efficiency of conversion of resources (Passioura, 1983).

Constant water capture over the period of SX breeding
A fundamental result in this study is the demonstration that total soil water capture remained constant during the ~50 year period of SX breeding and was unrelated to year of hybrid commercialization. This result for US temperate maize complements prior findings for tropical  and Argentinian germplasm (Nagore et al., 2014). No differences were detected among eight cycles of selection of 'Tuxpeño Sequia' in seasonal profiles of water content down to 1.4 m . Similarly, water capture did not differ among three SX Argentinian hybrids commercialized between 1980 and 2004, which was evident from nonsignificant differences in seasonal evapotranspiration, water use efficiency, and biomass production (Nagore et al., 2014). In this last study, significant differences in soil moisture content were detected around flowering time. Genotypic variation in pattern of water use in maize was also shown for temperate maize . Similarly, a significant hybrid by date interaction was observed in the present study (Table 4). However, the magnitude of the variance component for date by hybrid was two orders of magnitude lower than the variance component for date suggesting that differences in the pattern of water use has not played a determinant role on pattern of yield gain as measured by association with year of commercialization. In addition, estimated trends in post-anthesis and total water use with respect to year of commercialization were not significant. The combined results from these studies thus provide strong evidence to reject the hypothesis that the observed long-term yield improvements achieved over the period of SX breeding were driven by increased water capture or the temporal pattern of water use through the crop life cycle.  The hypothesis postulated by Hammer et al. (2009) may still explain a component of the observed yield gain within the period of double-cross breeding. Between 1930 and 1970 there was a significant shift in root system architecture as inferred from root stability scores (Duvick et al., 2004). A negative association between root mass and pulling resistance  suggests that root stability, mass, and water capture may have all increased during this doublecross era of breeding.

Water productivity increased since 1960
The corollary to the lack of association between yield improvement and water use is that genetic improvement for maize yield in the US corn-belt increased water productivity for yield between 0.018 and 0.013 g mm -1 year -1 . A simple identity (Passioura, 1983), Yield = Water use × Transpiration efficiency × Harvest index, can be used to estimate grain yield. In the absence of changes in water use with year of commercialization, and assuming changes in transpiration efficiency within the set of ERA hybrids included in this study were negligible-which is supported by experimental evidence from studies on Argentinean hybrids (Nagore et al., 2014) and sweet corn varieties (Bunce, 2011)-the observed variation in yield must be related to changes in HI under stress. An increase in HI could be due to effects of selection on reproductive resilience and biomass allocation to the ear at expense of root systems, similar to observed changes in tropical maize Lafitte and Edmeades, 1995) and Argentinian semi-flints (Echarte et al., 2000).

Implications for genetic improvement
Breeding trajectories for complex traits in rugged yield-trait landscapes are anything but simple Messina et al., 2011). Trade-offs among adaptive traits, frequency of environment types, structure of breeding programs, and access to genetic diversity among other factors, determine the direction and rate of observed change in genetic gain. Long-term simulation studies demonstrate a diversity of plausible breeding trajectories for a given environment-cropbreeding system with physiological traits changing in overlapping sequences and in a non-linear manner (Messina et al., 2011). When placed in the context of long-term selection (Duvick et al., 2004), this study focused on a relatively short time span of breeding. As such, and in the context of rugged yield-trait landscapes, it is conceivable that current observations on water capture showed a transient plateau. Prior to SX breeding, selection for improved yield stability and reduced root lodging  could have increased the ability of maize to capture water resources, as suggested by the hybrid water use results reported by Campos et al. (2004). During SX breeding, selection operated to realize an intrinsic water capture and yield potential attained during double-cross breeding but limited by the susceptibility of maize reproductive physiology to stress. Further increase in this potential will have to rely on improvements in either resource use efficiencies or in water capture. Thus, it is opportune to evaluate the feasibility to design root systems with improved water capture beyond current levels, which can improve plant water status in the 3 weeks around flowering time. Simulation studies for drought prone environments and for the central US corn-belt indicate that changes in root occupancy can increase yields Messina et al., 2009Messina et al., , 2011. However, meaningful phenotyping of root systems architecture is poorly defined in maize. Despite changes in root architecture in the last 100 years (York et al., 2015), the results of the present study indicated that potential water capture remained constant over the period of breeding represented by the sequence of hybrids, which questions the value of these traits as predictors of increasing water resource capture and their value to inform selection decisions. Modelling approaches are being evaluated to understand root form and function effects on whole plant water status (Draye et al., 2010;Leitner et al., 2014). Application of modelling and meaningful phenotyping proved useful to inform breeding approaches for sorghum and wheat in Australia (Manschadi et al., 2006(Manschadi et al., , 2008Kirkegaard et al., 2007;Singh et al., 2012). It is anticipated that integration of modelling technologies, field, and controlled environment phenotyping, and genetic studies in maize will bring opportunities to develop improved and more stable germplasm that fully utilizes available soil water resources.