Using leaf sodium concentration for screening sodicity tolerance in cotton (Gossypium hirsutum L.)

Field Crops Research 246 (2020) 107678

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Using leaf sodium concentration for screening sodicity tolerance in cotton (Gossypium hirsutum L.)

T

Shiming Liu*, Greg Constable, Warwick Stiller CSIRO Agriculture and Food, Narrabri, NSW 2390, Australia

ARTICLE INFO

ABSTRACT

Keywords: Cotton G. hirsutum Low leaf Na trait Nutrient use efficiency Sodicity tolerance

The ability of a plant to maintain low Na concentration is a critical component for its tolerance to salt affected soil stresses. Leaf Na concentration was investigated to understand its usefulness in screening of cotton for tolerance to soil sodicity and to assess its effect on agronomic performance. A set of family lines and high yield lines with low and high leaf Na concentration were tested in field experiments with sodic soils. The youngest fully expanded leaves at early flowering and whole plant biomass samples at the cut-out stage were collected for nutrient analysis. Plots were harvested for yield and fibre quality. Leaf Na concentration was found to be a reliable measure for differentiating test lines with low and high leaf Na trait. Leaf K/Na and P/Na ratio acted similarly. Low leaf Na concentration was due to higher Na accumulation in plant stems and roots. It was associated with higher K/Na and P/Na ratio in leaves and bolls, and responsible for improved K and P use efficiency. High performing lines with the low leaf Na trait had yield and fibre properties competitive with the control. It is concluded that the low leaf Na trait at flowering provided a reliable phenotypic indicator for cotton tolerance to soil sodicity. Breeding for the trait can improve sodicity tolerance, K and P use efficiency, while maintaining agronomic performance.

1. Introduction Soil deterioration with accumulated water-soluble salts in the root zone is one of the major environmental constraints for modern crop production (Tester and Davenport, 2003; Rengasamy, 2006). Globally about 1.1 billion hectares of land are affected by increasing salts. Saline soils account for 60 % of that area, followed by sodic soils (26 %), while the remainder is saline-sodic (14 %) (Wicke et al., 2011). In Australia, soils are classified as sodic when having an exchangeable sodium (Na) percentage (ESP) ≥6 and the Na impairs soil physical structure (Isbell, 1996; Rengasamy, 2002). Sodic soils are dominant in sub-humid and/or humid parts of eastern and south-western Australia, where irrigated agriculture has been set up for producing high value crops, such as cotton, rice and horticultural crops (Rengasamy and Olsson, 1993; Rengasamy, 2002). The majority are heavy clay soils with high levels of sodicity in the subsoil as well as high pH (> 8.0) (Rengasamy, 2002; Rochester, 2010), which impose a significant effect on cotton productivity (Rochester and Constable, 2003; Dodd et al., 2009; Rochester, 2010). Plants experience two kinds of stress when grown in salt-affected soils (Tester and Davenport, 2003; Munns and Tester, 2008). The first is osmotic stress due to an increased salt concentration in the soil solution ⁎

that makes it difficult for plants to extract water from soils. The second is caused by excessive Na in plant tissues, which can interfere with influx and transport of other important nutrients, resulting in nutrient imbalance, deficiency, and even cytotoxic effects (Tester and Davenport, 2003; Munns and Tester, 2008; Shabala and Cuin, 2008; Gorham et al., 2010). High Na concentration in the root zone can also compete with plant uptake for essential nutrients, such as K and P (Rochester and Constable, 2003; Shabala and Cuin, 2008; Rochester, 2010). Sodic clay soils are prone to compaction and waterlogging which restrict plant root growth and hence affect nutrient availability and balance in plant root zones. Milroy et al. (2009) reported waterlogging can result in an increased Na uptake and reduced uptake of K and P in cotton when grown in sodic soils. Plant tolerance to salt stresses is a complex trait and research has been concentrated on identifying and characterising heritable tolerant variation and understanding trait genetics (e.g. Ahmad et al., 2002; Munns and James, 2003; Flowers, 2004; Genc et al., 2010, 2013; Shahbaz and Ashraf, 2013; Ashraf, 2014; Genc et al., 2016). The ability of a plant to exclude Na accumulation in leaves and/shoots has been identified in wheat and other species. It has attracted significant attention, because of its critical role in plant salt tolerance (e.g. Tester and Davenport, 2003; Munns and James, 2003; Genc et al., 2010, 2016).

Corresponding author. E-mail address: [email protected] (S. Liu).

https://doi.org/10.1016/j.fcr.2019.107678 Received 23 July 2019; Received in revised form 26 October 2019; Accepted 7 November 2019 0378-4290/ Crown Copyright © 2019 Published by Elsevier B.V. All rights reserved.

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The trait was found to be inherited either quantitatively (Gregorio and Senadhira, 1993; Garcia et al., 1997; Genc et al., 2010; Liu et al., 2015a) or monogenically (Lindsay et al., 2004; James et al., 2006). The best example for the latter is in durum wheat, two independent alleles, Nax1 and Nax2, were identified as being responsible for low Na in sheath and leaf blades. After introgression into a commercial durum wheat background, the derived near-isogenic line with Nax2 allele outyielded its counterpart without the allele, by 25 % when grown in a high saline field (mean ECe = 14.8 dS/m) (Munns et al., 2012). Breeding for crop tolerance is continuing, but the release of tolerant cultivars at a commercial scale is slow and rare (Flowers, 2004; Genc et al., 2016). Two tetraploid species of cotton are grown commercially for fibre. Gossypium hirsutum L. accounts for > 90 % of total growing area (Campbell et al., 2010), and is ranked as a moderate salt-tolerant crop species and yield decline only occurs at salinity above 7.7 dS/m (Grieve et al., 2012). The G. barbadense L. species, originated in coastal Peru (Percy and Wendel, 1990), was reported more salt-tolerant than G. hirsutum (Abul-Naas and Omran, 1974; Tiwari et al., 2013a, 2013b). Heritability and QTL mapping of cotton tolerance to salt stresses were investigated in structured genetic populations from crosses where G. barbadense was used as donor for tolerance (Tiwari et al., 2013a, 2013b; Liu et al., 2015a). Janardhan et al. (1976) may have been the first to report Na exclusion and high K/Na ratio importance in cotton salt tolerance. In an inheritance study of leaf Na concentration of a recombinant inbred line (RIL) population derived from a G. hirsutum × G. barbadense cross, Liu et al. (2015a) revealed low leaf Na trait in a G. barbadense parent was associated with high Na concentration in the roots. In the population, leaf Na concentration measured at flowering was moderately heritable with two significant QTLs explaining 31 % of the observed phenotypic variation. Selection for the trait was effective to shift the mean of the selected population to a lower value as well as to identify the RILs with low Na concentration, i.e. Na exclusion. The above change also led to higher leaf K concentration and K/Na ratio concurrently. Rochester and Constable (2003) reported the strong and negative association of both leaf K and P concentrations with leaf Na

concentration when different cultivars were grown in sodic soils. The relationships remain in cotton grown in sodic soils that suffered waterlogging (Milroy et al., 2009). The evidence suggests that low Na in leaves may hold the key of improved salt tolerance and nutrient status in cotton. Under this assumption, as part of our long-term motive, we have commenced incorporation of the Na exclusion trait from G. barbadense into a high yielding commercial G. hirsutum cultivar through backcross breeding. This has resulted in many high yielding G. hirsutum lines with the low and high Na trait. Despite the success, the questions on how the trait can be screened and how it may affect agronomic performance remain. In addition, the mechanism for low Na concentration in leaves is unclear. As mentioned previously, low Na concentration in leaves is often accompanied by high K/Na and P/Na ratio. Given K and P are critical to activity and function of many enzymes in leaf photosynthesis and sucrose transport pathway and maintain cell membrane integrity (Shabala and Cuin, 2008; Oosterhuis et al., 2013), it is logical to hypothesise the above changes may result in improved K and P use efficiency in cotton and improve yield potential when grown in sodic soils. In this study, we tested breeding lines with low and high leaf Na in field experiments over multiple seasons to examine how season and sampling time would affect Na, K and P concentration. The reliability of using leaf Na measure as a screening tool for Na exclusion was assessed. We also examined Na profiles in different plant tissues of low and high leaf Na lines to understand a possible mechanism for Na uptake, transport and storage as well as how that affected K and P use efficiency in cotton. To the best of our knowledge, this is the first study reported in cotton with clear evidence that the Na exclusion trait can be reliably used in screening cotton tolerance to soil sodicity and that incorporating the trait would not compromise agronomic performance and potentially increase yield in high sodicity soils. 2. Materials and methods 2.1. Development of breeding lines with low and high leaf Na concentration Breeding for incorporating the Na exclusion trait followed a

Fig. 1. Timeline and breeding process to incorporate low and high Na trait into a commercial cultivar and a list of field experiments to test the derived lines. RIL 1-152 and RIL 1-127 are from an interspecific cross between Siokra 1-4 (G. hirsutum, high Na) and 8810 (G. barbadense, low Na) and used as the donor lines for low and high Na trait, respectively. Sicot 71BRF is used a recurrent parent. GH means that the generation and screening were done in glasshouse. 2

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backcross procedure (Fig. 1). Two RIL lines, one with high and the other with low Na concentration in leaves was chosen as the donor parents, each to cross with a commercially released transgenic cultivar, Sicot 71BRF (http://pericles.ipaustralia.gov.au/pbr_db/plant_detail. cfm?AID=28481089), as the recurrent parent. The donor RILs had growth habits adapted to local soils and were from a population originating from the cross between a G. hirsutum cultivar, Siokra 1–4, and a G. barbadense line 8810 from Arizona (Percy and Turcotte, 1998). Siokra 1–4 was released in 1988 by the CSIRO breeding program. It is an okra leaf type, has early to mid-season maturity, and is highly adapted to Australian cotton growing environments (Liu et al., 2013). The recurrent parent, Sicot 71BRF contains two genes, Cry1Ac and Cry2Ab, conferring cotton to synthesise insecticidal proteins of Bacillus thuringiensis against cotton bollworms Helicoverpa spp., marketed as Bollgard II® (https://www.monsantoglobal.com/global/au/products/ Documents/bollgard-ii-technical-manual.pdf) and the two copies of the CP4 EPSPS gene which allow cotton to synthesis a CP4 EPSPS protein tolerance to glyphosate herbicide applications, marketed as Roundup Ready Flex® (https://www.monsantoglobal.com/global/au/ products/Documents/roundup-ready-flex-technical-manual.pdf). Four backcrosses were conducted in a glasshouse (Fig. 1). The screening was conducted for both transgenic traits and leaf Na concentration in the segregating generation after each backcross. The Bollgard II trait components were screened by ELISA and the Roundup Ready Flex trait by directly applying the recommended rate of glyphosate to seedlings. Leaf Na concentration was based on nutrient analysis of ground leaf samples from the youngest fully expanded leaves at early flowering, located at the third or fourth node below the plant terminal. In the family where a low Na RIL was used as a donor, the screening was to keep the segregants with low Na concentration as well as the presence of transgenic traits; and in the other family with the high Na RIL as a donor, plants with high Na concentration and the presence of transgenic traits were kept. During the screening process, other macro- and micro-nutrient status of selected individuals was checked to ensure their concentrations were in optimal ranges (Hearn, 1981), and individuals with G. barbadense or its likely growth habits were eliminated. Plants kept after the screening in the last backcross were self-pollinated for two generations in the glasshouse to derive the families with enriched homozygous individuals for the transgenic traits (Fig. 1). Homozygous plants from each family were identified and retained based on DNA zygosity testing and their seeds were bulked to derive family lines. The lines were tested in replicated field experiments in sodic soils of Australian Cotton Research Institute (ACRI, S30° 11’, E149° 35’) near Narrabri, NSW for two years to identify superior family lines for agronomic performance. The leaf Na, macro- and micro-nutrient concentrations were again checked by nutrient analysis of ground leaf samples collected at flowering to confirm the lines with low and high leaf Na concentration. Two of the low Na elite family lines were chosen to go through within family selection (e.g. Thomson, 1973; Liu et al., 2013) to select higher performing lines with the Na exclusion trait (Fig. 1). In the 2014/15 season, 300 single plants per family were planted in the field at a 50 cm plant spacing, and the individuals were hand-harvested after defoliation. Seed cotton samples harvested were processed with a 20saw gin; and the weight of the sample as well as of resultant lint were recorded to calculate lint percentage. Lint samples were tested with High Volume Instrument (HVI, Uster Technologies AG, Switzerland) of measuring fibre length (mm), uniformity (%), short fibre index (%), fibre strength (g/tex), elongation (%) and micronaire. After culling individuals with poor lint percentage and fibre properties compared with the recurrent parent, Sicot 71BRF, 120 single plants were retained. In the 2015/16 season, single plant derived lines were evaluated in single row plots of a non-replicated progeny row experiment. The experiment was also subject to leaf sample collection and harvest and 23 lines were kept after selection for yield, fibre properties and leaf Na

concentration. 2.2. Field experiment 1: assessing the factors affecting leaf Na, K and P concentration and K and P use efficiency In this experiment, five of the family lines derived immediately after backcrossing were used (Fig. 1). Among them, three were low and two high in leaf Na concentration, and the recurrent parent, Sicot 71BRF, was also included in the experiments. The experiments were conducted for three seasons from the 2015/16 to 2017/18 summer at ACRI. The soil is a self-mulching Vertosol classified as a fine, thermic, montmorillonitic Typic Haplustert (Soil Survey Staff, 1996), and contains high clay with subsoils of moderate sodicity (Rengasamy and Olsson, 1993; Ward et al., 1999; Rengasamy, 2002). As the best crop rotation system was being followed, the 2016/17 experiment was planted in a different field to the one used for 2015/16 and 2017/18 experiments. Although two fields neighbour to each other, according to our previous screening for cotton sodicity tolerance in an interspecific derived recombinant inbred line population and their parents (Liu et al., 2015a), it has found leaf Na concentration was always higher in the field used in 2016/17 than the one used in the other two seasons. We expect the subsoils in the former field naturally has higher ESP and pH but cannot be confirmed as no full profile test for the soil was conducted recently. The experiments consisted of three row plots 12 m long × 3 m wide with 1 m row spacing and had four replicates with each test line being randomly allocated to the plots based on the design generated by CycDesignN software (Whitaker et al., 2002). The experiments were subject to leaf sample, biomass collection and harvest with the detail given below. 2.3. Field experiment 2: identifying and selecting superior performing lines with Na exclusion After within family selection, twenty-three lines were tested in multi-season and location experiments, with the aims of identifying high performing lines with Na exclusion and validating the reliability of leaf Na concentration as a means of screening for Na exclusion in cotton. In the 2016/17 and 2017/18 seasons, the lines were tested in replicated experiments at two sites, ACRI and Hillston, NSW, Australia (S33° 25’, E145°27’) (Fig. 1). Soil types in Hillston site are red Vertosols characterised with high pH in the entire soil profile and very high ESP (≈15) in the subsoils (Onus et al., 2003). In both sites, the experiments were grown in different fields each season. In each season, leaf samples were collected only at ACRI site, and the experiments were machine harvested after defoliation to measure yield and fibre properties. Again, all test lines were grown in three row plots 12 m long × 3 m wide with 1 m row spacing with four replications according to the rowcolumn design generated with CycDesigN software (Whitaker et al., 2002). Sicot 71BRF was included as the commercial control. 2.4. Plant sample preparation, nutrient concentration, yield and fibre property measurements In all field experiments at ACRI, fourteen youngest fully expanded leaves were randomly collected from individual plots during early flowering. In experiment 1, leaf sampling was carried out twice. The first one was at one week after the commencement of flowering (T1) and the second (T2) was at 10 days after T1. During the 2016/17 and 2017/18 season, biomass samples were also taken from the experiment with the detail described as follows. In the experiment 2, leaf sampling was only conducted at T1. All leaf samples were dried in an air forced dehydrator at 70 °C for 48 h and then ground with a plant grinder. When plant reached the cut-out stage of growth in experiment 1 during the 2016/17 and 2017/18 season, 1 m2 whole plant biomass with main roots was harvested from each plot. The samples were first separated into the above- and below-ground parts by sectioning off just 3

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below the cotyledon node. The above ground part was measured for total fresh weight and partitioned into leaves, stems and fruiting bodies including squares, flowers and bolls. To facilitate the dehydration process, stems were cut into small pieces and the bolls were cross-cut at the point before placed into an air forced dehydrator. Root samples were initially cleaned and washed thoroughly with tap water to remove soil debris and followed with triple rinses of rain water. The samples were air-dried at room conditions to remove free water and fresh weight was recorded before cutting into small pieces for drying. Samples were dried at 70 °C for 72 h, and their weight was recorded after drying. The samples were then ground. For boll samples, both mature and immature seeds were separated from the rest of bolls and the weight of each tissue was recorded before grinding. Therefore, bolls in this study refer to boll wall and bracts excluding seeds and lint. In each season, biomass samples of only three test genotypes, namely, CSX601LNa, CSX604HNa and Sicot 71BRF, representing the lines with low, high leaf Na concentration and control, respectively, were ground and analysed for nutrient concentration (Fig. 4). Na, K and P concentration of different samples were determined by ICPAES (Inductively Coupled Plasma Atomic Emission Spectroscopy), which was provided by an accredited analytic laboratory, Nutrient Advantage in Werribee, Victoria, Australia (https://www. nutrientadvantage.com.au/). The ratio of K/Na and P/Na of all samples were calculated for data analysis. All field experiments were chemically defoliated with thidazuron and ethephon when at least 60 % of bolls were open. A single row plot picker was used to harvest the centre row of the three row plots and plot yield recorded. A 250 g seed cotton sample was collected from each plot during harvest and processed with a 20-saw gin and the weight of the sample and resultant lint were recorded to estimate lint percentage. Lint samples were tested using a HVI for fibre properties. Plot seed cotton yield was multiplied with lint percentage to calculate yield as kg lint per hectare.

3. Results 3.1. Climate conditions of experiment seasons Monthly average temperature and total rainfall, vapour pressure deficit and solar radiation for the experiment seasons at both the sites are presented in Supplementary material Fig. S1. At ACRI, the summer was hot in 2016/17 and 2017/18 (December to February). This is also indicated by higher vapour pressure deficient in that period of each year. The in-crop rainfall ranged from 285 to 413 mm with less than average in 2017/18, and there was also more rainfall in the early half of the cropping season in 2015/16, unlike 2016/17 where over half of the total rainfall was received later in the cropping season. Solar radiation was similar in 2015/16 and 2016/17, but in 2017/18, a higher radiation was recorded in January, despite a lower than average in October and April. The crop was irrigated 8 times in 2015/16 and 2016/17 and 9 times in 2017/18, and each irrigation provided the amount of water equivalent to about 100 mm rain. At Hillston, the beginning and end of the cropping season was warmer in 2017/18, and otherwise similar between the two seasons. The rainfall received was also similar. When compared with ACRI site, the rainfall was very low (152−168 mm), which agrees with the fact that the sites belong to the different rainfall zones, i.e. summer or winter dominant. Vapour pressure deficit was higher in 2017/18, however, solar radiation was almost identical until a rapid reduction in the end month of 2017/18. The experiments were irrigated for 10–12 times. 3.2. Effect of year, sampling time, genotype and their interactions on leaf Na, P and K concentration In experiment 1, all the main effects were significant for Na, K and P concentration and their ratio (Table 1). Most interactions, either two or three-way, were also significant except year × sample time interaction for Na concentration, K/Na and P/Na ratio, year × genotype and threeway interactions for P concentration. Noticeably, the main effects associated with year and genotype were much larger than any of the interactions. Year effect in this study represented the sum of climatic, soil conditions and management practice, and its large magnitude and significance were understandable. For Na concentration and its involved ratios, genotypic effect was much more important than the main effect of year and leaf sampling time. However, that was opposite for P, but also for K concentration. Na concentration in response to year is illustrated in Fig. 2 for each test family line. Six lines were separated into high and low groups each containing three lines, and genotypic discrepancy was higher within the low Na group. On average, Na concentration was the highest in 2016/ 17, which agrees with our previous results of cotton sodicity screening conducted in two fields that leaf Na concentration of screened population was always higher in the field used in 2016/17 than in the other

2.5. Statistical analysis For experiment 1 all data was pooled for analysis. The analysis was to examine the importance of the effect of test family lines and their interactions with the treatment factors of season and sampling time on phenotypic measurements. Incomplete blocks were used to account for field variability due to soil fertility and management practice, such as irrigation and fertiliser. The effect of such blocking factors was examined and fitted in the analysis, when significant. The analysis obtained best unbiased estimates for the main and interaction terms and the least significant difference at P = 0.05 (l.s.d.0.05) was used for the mean comparison. Rochester (2011) defined internal crop nutrient use efficiency as the amount of dry matter, seed cotton and lint yield produced by the accumulated amount of K or P. The use-efficiency for K and P was calculated based on the mean estimates of total dry matter, seed cotton and lint yield and the amount of K and P accumulated in three test lines in experiment 1 during the 2016/17 and 2017/18 season. The relative improvement in low and high Na lines was measured against the estimates of the control, Sicot 71BRF. For experiment 2, a combined analysis was conducted for the pooled dataset of all test sites. In the analysis, test location and year combinations were considered as an environmental factor; test lines, environment, and their interaction were fitted as fixed. Given the common presence of dimensional spatial variation in cotton breeding experiments (Liu et al., 2015b), such variation was examined in the analysis, and fitted when significant. The analysis obtained the best unbiased estimates for the lines tested and the least significant difference at P = 0.05 (l.s.d.0.05) for the mean comparison. The data analyses were processed using ASreml-R (Butler et al., 2009) and R (http://www.R-project.org).

Table 1 Variance source for leaf Na, K and P concentration and their ratios based on a combined analysis for experiment 1 over three seasons each with six test genotypes and two leaf sampling times. Source

Year (Y) Sampling time (T) Genotype (G) Y×T Y×G T×G Y×T×G

df

2 1 5 2 10 5 10

F value and significance Na

K

P

K/Na

P/Na

32.9** 15.5** 693.5*** 3.4ns 26.8*** 7.3*** 3.8**

115.0*** 8.2* 81.0*** 7.3** 9.8*** 6.8*** 11.1***

40.7*** 93.0*** 4.6** 10.0** 1.9ns 3.2** 1.2ns

44.0*** 16.1** 354.9*** 2.7ns 3.9** 31.5*** 4.1**

63.7*** 30.7*** 249.5*** 2.8ns 3.3* 36.1*** 2.6*

*, **, *** indicate significant at P < 0.05, 0.01 and 0.001, respectively. denotes to non-significant at P = 0.05. 4

ns

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Fig. 2. Change of leaf Na concentration of six test genotypes in response to each experiment season based on a combined analysis of experiment 1 over three seasons. Bar represents l.s.d.0.05 = 84.5 mg/kg.

one (Liu et al., 2015a). Temperature in January across three experiment seasons was also positively correlated with leaf Na concentration of test genotypes (r = 0.54 with mean monthly minimal temperature, P < 0.05; r = 0.62, P < 0.01 with mean monthly maximal temperature, df = 16). Therefore, higher January temperature and higher ESP and pH in subsoils of the field in 2016/17 contributed to higher leaf Na concentration of test genotypes in that season. Nevertheless, the ranking from low to high among the test lines was highly consistent across seasons, suggesting the interaction of year × genotype was caused by the magnitude rather than cross-over change, despite under different seasonal weather conditions (Fig. S1). Na concentration was always higher in leaf samples collected at T1 (one week after the beginning of flowering) than at T2 (10 days after T1). The ranking of test lines did not change largely due to sampling time, but the samples collected at T1 resulted in better discrimination between lines, especially for the three low Na lines (Fig. 3). Na concentration of the test lines in response to the sampling times was similar to the above in each year experiment (Fig. S2), which further implied a magnitude nature of the interaction between genotype, year and

sampling time suggested in Table 1. Unlike Na, the ranking of test lines varied with year as well as the sampling time, when leaf K and P concentration was considered (Figs. S3 and S4). Importantly, the interactions, regardless of two or threeway, all led to the change of genotypic ranking. This means the ability of test lines to maintain K and P nutrient status did interact with seasonal conditions. However, when K/Na and P/Na ratio were considered, the response of test lines to the sampling time in each experiment did not alter their ranking (Figs. S5 and S6), which is similar to the observation for Na concentration mentioned before (Fig. 3 and S2). Higher ratio values at T2 was likely a result of a fast-declining leaf Na concentration (Fig. S2). Nevertheless, the tendency explained why both genotype by time factor interactions were less important for both ratio measurements (Table 1), which contrasted with leaf K and P concentration. Leaf Na concentration ranged from 185 to 611 mg/kg (Table 2). CSX316HNa had the highest concentration comparable to the control, Sicot 71BRF, and both were significantly higher than CSX604HNa in the high Na group. The highest Na concentration in the low Na group was only two third of the CSX604HNa, and a significant difference between low Na test lines was observed with CSX221LNa being the lowest. Mean concentration ranged from 12,514 to 13,788 mg/kg for K and from 3437 to 3752 mg/kg for P among six test lines (Table 2). The concentrations in each test line depended on experiment season and sampling times, but in general, low Na lines had higher leaf K and P Table 2 Mean concentration of Na, K and P, and K/Na and K/P ratio in leaves for five test genotypes and control based on a combined analysis for experiment 1 over three seasons.

Fig. 3. Change of leaf Na concentration in six test genotypes in response to leaf sampling times based on a combined analysis of experiment 1 over three seasons. T1 = 1st leaf sampling taken at one week after flowering; T2 = 2nd leaf sampling at 10 days after T1. Bar represents l.s.d.0.05 = 62.1 mg/kg.

Genotype

Na (mg/kg)

K (mg/kg)

P (mg/kg)

K/Na

P/Na

CSX221LNa CSX601LNa CSX602LNa CSX604HNa CSX316HNa Sicot 71BRF

185 357 388 560 611 591

13788 13164 13007 12514 13243 12834

3752 3656 3520 3437 3675 3662

77.6 49.0 41.5 30.1 27.2 27.5

21.1 a 14.7 b 11.0 c 8.2 d 7.8 d 8.3 d

l.s.d.0.05

27.1

e d c b a a

222

a b bc d b c

159

a ab b b a ab

6.7

a b c d d d

2.0

The values followed with different letters in the same column suggest the difference larger than one l.s.d. at P = 0.05. 5

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Table 3 Variance source for Na, K, P concentration and their ratios in five plant tissues of three test genotypes in experiment 1 over two seasons after a combined analysis. Source

Df

F value and significance Na

Year Genotype (G) Tissue G × Year G × Tissue Year × Tissue G × Year × Tissue

1 2 4 2 8 4 8

ns

2.95 17.97*** 337.40*** 0.44ns 21.04*** 14.99*** 0.31ns

*, **, *** indicate significant at P ≤ 0.05, 0.01 and 0.001, respectively.

ns

K

P

K/Na

P/Na

166.80*** 11.29*** 1373.0*** 1.88ns 2.51* 59.63*** 0.60ns

126.90*** 20.61*** 2234.0*** 7.37** 3.97*** 67.51*** 2.49*

12.42* 0.53ns 2395.0* 0.08ns 2.61* 2.52* 0.012*

27.71*** 8.93*** 920.3*** 1.74ns 7.59*** 99.34*** 2.20*

denotes to non-significant at P = 0.05.

concentration. This resulted in higher K/Na and P/Na ratios in low Na lines, which ranged from 41.5 to 77.6 and 11.0 to 21.1 of K/Na and P/ Na ratio, respectively. In contrast, the ratios ranged from 27.2 to 30.1 and 7.8 to 8.3, respectively in the high Na lines.

Bolls, stems and leaves had higher K concentration, and seeds, bolls and leaves had higher P concentration (Fig. 5). Bolls had the highest K/ Na and P/Na ratios, followed by seeds and leaves, while stems and roots had the smallest ratios. When compared with the control, on average, CSX601LNa had the lowest K concentration in bolls, stems and roots (P < 0.05), and the lowest P concentration in leaves and seeds (P < 0.05), but the highest K/Na and P/Na ratio in leaves and bolls.

3.3. Effect of year, genotype and their interaction on nutrient concentration of different plant tissues

3.4. Lint yield and nutrient use efficiency

In experiment 1, ANOVA for plant biomass samples suggested plant tissue accounted for the largest observed variation, followed by year and year × tissue interaction for K, P concentration and also K/Na and P/Na ratio, however, for Na concentration, genotype and genotype × tissue interaction were also substantial (Table 3). The interaction of genotype × year and genotype × year × tissue was either small or nonsignificant. The Na concentration was lower in leaves but higher in both stems and roots in CSX601LNa, when compared with the other lines (Fig. 4). This evidence is consistent with the result of mature leaf samples in the experiment as illustrated before. The largest genotypic difference was observed in roots, where CSX601LNa had almost double Na concentration than CSX604HNa and Sicot 71BRF. No statistical difference was observed among three test lines for Na concentration in bolls and seeds. The above difference of plant tissues between test lines was also presented in each season experiment (Fig. S7), explaining why year × genotype × plant tissue interaction was non-significant (Table 3). Furthermore, despite a significant and large year effect, it did not affect Na distribution pattern in different plant tissues of high and low Na lines.

In experiment 1, on average, the low Na line, CSX601LNa, had yield potential comparable to the control, Sicot 71BRF (2304 kg/ha) (P < 0.05), but reduced yield potential was observed in the other low and high Na lines (Table 4). All these family lines exhibited fibre length, strength and micronaire at least comparable to the control. Total dry matter production, yield, nutrition accumulation and use efficiency in each year are given in Table 5. Although the results were based on two-year experiments, genotypic ranking based on lint yield of each experiment was similar to that based on three-year results given in Table 4. Similar genotypic ranking was observed according to seed cotton yield. In terms of total dry matter, all three lines were comparable each season, although the absolute amount was generally higher in 2017/18. When the amount of K and P accumulated in plants was considered, no line difference was found in 2017/18 but in 2016/17, CSX601LNa accumulated less K and P. For total accumulated amount of Na, however, CSX601LNa was the highest, while CSX604HNa had the lowest, suggesting lower leaf Na or exclusion did not reduce the overall Na accumulation at whole plant levels.

Fig. 4. Mean Na concentration in five plant tissues of three test genotypes from a combined analysis of experiment 1 over two seasons. Bar represents l.s.d.0.05 = 163.7 mg/kg. 6

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Fig. 5. Mean K (a), P concentration (b) and K/Na (c) and P/Na ratio (d) in five plant tissues of three test genotypes from a combined analysis of experiment 1 over two seasons. Bars represent l.s.d.0.05 of 920.6 mg/kg for K, 198.5 mg/kg for P, 7.80 for K/Na and 1.81 for P/Na ratio.

2016/17 when based on seed cotton and lint yield. CSX604HNa always showed lower in K and P use efficiency than the control, particularly in 2017/18, regardless of the productivity trait. Therefore, the line with leaf Na exclusion trait can at least maintain high K and P use efficiency, although it took up and accumulated more Na in whole plants during the season, resulting from high Na sequestration in stems and roots.

Table 4 Mean lint yield and three fibre properties for five test genotypes and control based on a combined analysis for experiment 1 over three seasons. Genotype

Lint yield (kg/ha)

Length (mm)

Strength (g/tex)

Micronaire

CSX221LNa CSX601LNa CSX602LNa CSX604HNa CSX316HNa Sicot 71BRF

1962 2168 2064 2123 2122 2304

30.8 31.6 31.2 31.0 31.2 30.9

30.2 31.0 31.1 31.3 30.2 31.4

4.4 4.2 4.4 4.6 4.2 4.5

l.s.d.0.05

138.3

c ab bc b b a

0.37

c a b bc b bc

0.71

b a a a b a

b c b a c ab

3.5. Agronomic performance of breeding lines with Na exclusion trait In experiment 2, leaf Na concentration, K/Na and P/Na ratio obtained across two seasons of testing was strongly correlated, where the fitted linear regression explained 66%–71% of the observed variation (P < 0.001). However, for leaf K and P concentration, that relationship across the seasons was poor or weak (R2 = 0.03 (P > 0.21) and R2 = 0.26 (P < 0.01) for K and P, respectively) (Fig. 6). Clearly, seasonal effect still counted for a good proportion of observed variation for these nutrient variables. Nevertheless, leaf K, P and Na concentration of all test lines was higher in 2017/18, but large genetic variability remained in each season with no test line having Na concentration higher than the recurrent parent, Sicot 71BRF. Leaf K and P concentrations tended to be inversely associated with leaf Na concentration, provided the associations were not statistically significant (r = −0.39 for Na vs. K, P > 0.06, and r=-0.17 for Na vs. P, P > 0.41, df = 22). Twenty out of 23 test lines had leaf Na concentration significantly lower than the recurrent parent, Sicot 71BRF (Fig. 7). Mean leaf Na concentration ranged from 287 mg/kg to 679 mg/kg. Correspondently, 15 lines showed lint yield at least comparable to Sicot 71BRF with five having a yield increase of between 3 % and 5 %. These superior performers had leaf Na concentration between 565 mg/kg and 679 mg/kg,

0.13

The values followed with different letters in the same column suggest the difference larger than one l.s.d. at P = 0.05.

Nutrient use efficiency varied with harvest measure and season (Table 5). Both K and P use efficiency was higher for each test line in 2016/17 compared with 2017/18, suggesting an effect of seasonal conditions on overall plant growth and productivities of test lines. The use efficiency ranged from 55.9 to 68.8 mg per mg K and 375.1 to 576.9 mg per mg P based on total dry matter. The range was from 28.1 to 46.4 mg per mg K and from 188.7 to 389.0 mg per mg P for seed cotton yield, and from 11.7 to 17.5 mg per mg K and 78.6 to 147.1 mg per mg P based on lint yield, suggesting a scaling down effect on use efficiency first by harvest index from total dry matter to seed cotton yield; and finally by lint percentage from seed cotton yield to lint yield. Among three test genotypes, CSX601LNa exhibited comparable or increased K and P use efficiency over the control, when the estimation is based on total dry matter; but only showed increased use efficiency in 7

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Table 5 Mean comparison of total dry matter (TDM), seed cotton yield (SCY), lint yield (LY), K, P and Na accumulation and K and P use efficiency among three test genotypes grown in experiment 1 in two different seasons. Season/genotype

Total nutrient (mg/m2)

TDM

SCY

LY

(kg/m2)

(kg/ha)

(kg/ha)

K

P

Na

2016/17 CSX601LNa CSX604HNa Sicot 71BRF

866.3 a 866.0 a 979.9 a

5842.2 ab 5508.0 b 6215.7 a

2208.9 ab 2097.8 b 2401.4 a

12594.2 b 13662.4 ab 15407.1 a

1501.7 b 1635.7 b 1934.8 a

1201.9 a 978.2 b 1132.6 ab

2017/18 CSX601LNa CSX604HNa Sicot 71BRF

1070.5 a 1051.9 a 1005.8 a

5605.9 a 5291.6 a 5782.2 a

2324.4 a 2203.2 a 2440.5 a

18839.1 a 18801.0 a 17967.2 a

2746.8 a 2804.6 a 2581.2 a

1373.3 a 1131.0 b 1188.6 b

CV (%) l.s.d.0.05

4.33 129.9

3.42 598.6

4.03 268.0

4.7 2345.7

3.61 250.4

5.86 215.7

Season/genotype

K use efficiency (mg/mg)

P use efficiency (mg/mg)

TDM

SCY

LY

TDM

SCY

LY

2016/17 CSX601LNa CSX604HNa Sicot 71BRF

68.8(8.2) 63.4(-0.3) 63.6

46.4(15.0) 40.3(-0.07) 40.3

17.5(12.5) 15.4(-1.5) 15.6

576.9(13.9) 529.4(4.5) 506.5

389.0(21.1) 336.7(4.8) 321.3

147.1(18.5) 128.3(3.3) 124.1

2017/18 CSX601LNa CSX604HNa Sicot 71BRF

56.8(1.5) 55.9(-0.05) 56.0

29.8(-7.5) 28.1(-12.5) 32.2

12.3(-9.2) 11.7(-13.7) 13.6

389.7(0.02) 375.1(-3.8) 389.7

204.1(−8.89) 188.7(−15.8) 224.0

84.6(−10.5) 78.6(−16.9) 94.5

The values followed with different letters in each column under each year suggest the difference larger than one l.s.d. at P = 0.05. The value in bracket represents the increased or decreased percentage of use efficiency against the control.

which was substantially less than the control and even less than their three sister lines with high Na concentration. The relationship of Na concentration and lint yield was linear but not statistically significant: lint yield = 2179.7 + 0.564*Na concentration (R2 = 0.088, P > 0.08, df = 23). Fibre quality properties including fibre length, strength and micronaire, of all lines with low leaf Na concentration were comparable to Sicot 71BRF (Table S1). All evidence in the current study suggests that incorporating a Na exclusion trait did not impair cotton productivity and fibre quality.

the variation and genetics of the traits (Liu et al., 2015a). In this study, we further examined how sampling time affected phenotypic expression of nutrient variables. Leaf Na, K and P concentration were highest at T1 (a week after flowering). Sampling 10 days later (T2) resulted in a reduced concentration and the reduced ability to distinguish differences of test lines. Seasonal conditions particularly temperature in January when leaf sample were taken affected the magnitude of leaf Na concentration of test lines, but their rankings were hardly altered. Thus, it is concluded that the appropriate time for leaf sampling for assessing and selecting leaf Na exclusion in cotton is at the beginning of flowering. Excessive Na uptake and accumulation in plant tissues cause both osmotic and metabolic distresses of plants. These stresses reduce plant growth, development and eventually productivity (Ahmad et al., 2002; Munns and Tester, 2008). However, the interrelation of Na exclusion and K/Na ratio with plant tolerance response, such as biomass production and leaf expansion under saline conditions, is often inconclusive in different plant species. For example, in durum wheat, the relationship between leaf Na concentration and plant biomass was negative and strong (Munns and James, 2003); but in bread wheat, this interrelation was often weak (Genc et al., 2010, 2016). In cotton, the relationship reported ranged widely (e.g. no relation (Jafri and Ahmad, 1994; Ashraf and Ahmad, 2000; Leidi and Saiz, 1997); negative relation (Akhtar et al., 2010; Peng et al., 2016)). This may imply the existence of diverse mechanisms in plant species, including cotton, for tolerance to soil salinity or sodicity (Leidi and Saiz, 1997; Niu et al., 2013; Ashraf, 2014). In this study, all field experiments were conducted in soils with moderate sodic subsoils, and test lines were unstressed under management practice, although higher temperature in January increased leaf Na concentration. Large difference for Na exclusion, K/Na ratio and lint yield were observed between test lines (Figs. 6 and 7), most importantly, there was no statistically meaningful relation between yield and Na exclusion trait, and superior lines appeared in leaf Na concentration as low as 455 mg/kg (Fig. 7). Thus, leaf Na exclusion might

4. Discussion 4.1. Screening and breeding for leaf Na exclusion Leaf and shoot Na exclusion is an important component of plant tolerance to the stresses caused by increased salts in soils (Tester and Davenport, 2003; Munns and Tester, 2008; Genc et al., 2010; Munns et al., 2012; Genc et al., 2016). Relying on nutrient analysis results, the Na exclusion trait, i.e. low leaf Na concentration from G. barbadense has been successfully transferred into elite G. hirsutum background (Fig. 1). In this study, we provide evidence that the trait phenotyped at flowering could be reliably used to distinguish breeding lines differing in their ability of reducing Na in mature leaves and even in the whole canopy. The latter was supported by nutrient results of plant biomass samples, from which a low Na line showed significantly lower Na concentration in the leaf canopy, when compared with the other two high Na lines including the recurrent parent, Sicot 71BRF. The K/Na ratio, another trait commonly used to measure plant tolerance (Tester and Davenport, 2003; Munns and Tester, 2008), also showed similar behaviour (Table 1 and Fig. 5). The above evidence explains the reasons behind our breeding success for the Na exclusion trait in cotton (Fig. 1) and addressed the aims of this study. In our previous study, we discussed why nutrient results from fully matured and active leaves at cotton flowering was useful to investigate 8

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Fig. 6. Across season relation of leaf Na, K, P concentration and K/Na and P/Na ratio for 24 genotypes tested in experiment 2. x and y axis represent the mean estimates of the 2016/17 and 2017/18 season, respectively. repesents the control, Sicot 71BRF.

Fig. 7. Mean leaf Na concentration (a) and relative lint yield (b) of 23 lines and the control tested in experiment 2 over two seasons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Blue bars represent sister lines from a breeding population and red bar represents the control, Sicot 71BRF being a recurrent parent. Dash line represents leaf Na concentration of one l.s.d. less than that of the control (a), and its relative yield against the control (b). Leaf Na concentration is based on a combined analysis of experiment 2 conducted over two seasons at ACRI; and lint yield were based on a combined analysis of experiment 2 conducted over two seasons at ACRI and Hillston. l.s.d.0.05 is 148.9 mg/kg for leaf Na concentration, and 6.9 % for relative lint yield when against the control, Sicot 71BRF (2611.2 kg/ha).

9

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be neutral to agronomic performance. This is similar to the result of no linkage drag of Nax2 allele in durum wheat (Munns et al., 2012). Future studies in cotton should be conducted on highly sodic soils that have previously demonstrated constraints on cotton yield (Rochester, 2010) to verify the true effect of the trait on agronomic performance.

stems and roots. This finding is consistent with the role of Nax1 allele in durum wheat suggesting it controlled Na transfer from root to shoot and from sheaths to leaves while having no influence of overall root or plant Na uptake (Davenport et al., 2005; James et al., 2006). High Na concentration in roots were reported previously in G. barbadense (Liu et al., 2015a) as well as salt-tolerance G. hirsutum cultivars (Khan et al., 1998; Peng et al., 2016), but no difference of root Na concentration was also observed between tolerant and sensitive cotton cultivars (Leidi and Saiz, 1997; Ashraf and Ahmad, 2000; Zhang et al., 2014). In cereal crop species (e.g. rice, barley, maize, wheat), root Na concentration was found to be highly constant (see review, Tester and Davenport, 2003), despite a preferred Na sequestration in leaf sheaths being reported in Nax1 introgressed durum wheat (Davenport et al., 2005; James et al., 2006). It is known that the initial entry of Na into plant roots from soil solution is passive, and Na accumulation in plant and tissues mainly relies on the net outcome of Na influx and efflux (Tester and Davenport, 2003; Munns and Tester, 2008). The Nax1 allele in durum wheat is reported to be responsible for reducing Na loading from root to shoot (Davenport et al., 2005; James et al., 2006), whether a similar genetic control exists in a low Na cotton line for its low Na in leaves or canopy in this study requires further research. In addition, Na concentration in stems and roots in a low Na line in this study may be higher than the threshold tolerable in plant cells, when referring to the estimated range of 230–700 mg/kg suggested by Munns and Tester (2008). This may imply the intracellular Na compartmentation can be involved in cotton tolerance, as suggested by Peng et al. (2016) for cotton seedling salt tolerance and warrants further investigation.

4.2. Effect of leaf Na exclusion trait on K and P use efficiency Despite the significant difference in plant Na accumulation between low and high Na sister lines, there was no difference in their K and P accumulation. Under the test environment used in this study, leaf K and P concentration of test lines were all within optimal ranges (Hearn, 1981), although the concentration of these nutrients varied with seasons as well as sampling time. This contrasted with Na which was primarily determined by the genetic make-up of test lines (Tables 1 and 2). With regard to K/Na and K/P ratios, the low Na line always had much higher values particularly in most active plant photosynthetic tissues such as leaves and bolls. A similar phenomenon was observed in durum wheat isogenic lines with Na exclusion alleles (Davenport et al., 2005; James et al., 2006), and in cotton, where the ability to maintain high K and Na discrimination was reported in salt tolerant cotton (Peng et al., 2016). Higher ratios are desirable for cotton growth and development as well as tolerance to salt stresses (Oosterhuis et al., 2013; Ashraf, 2014; Peng et al., 2016). In this study, in experiment 2 where many test lines were assessed, there was a tendency for increased leaf K and P concentrations in the low Na lines, although not as strong as those studies conducted under similar Australian sodic soils (Rochester and Constable, 2003; Milroy et al., 2009). Nevertheless, the low Na line must exhibit better nutrient status than its high Na sister line. This may explain why a low Na line possessed better K and P use efficiency when compared with its sister line with high Na (Table 5). When compared with the recurrent parent, K and P use efficiencies varied with season, and with productivity traits used for estimation. How seasonal conditions affected K and P uptake and accumulations cannot be interpreted in this study and requires future study. Nevertheless, both K and P use efficiency in the low Na line was comparable to the recurrent parent. The use efficiency observed in this study was within the reported range of 10–20 for K but higher than the range of 60–80 for P based on kg lint yield per kg each nutrient (e.g. Dong et al., 2010; Rochester and Constable, 2015). One long term motive behind this study was to facilitate improvement in cotton yield, particularly to reduce yield limitations from nutrient availability. We have determined that modern locally developed and higher yielding cotton cultivars had reduced leaf Na concentration and increased leaf P and K concentration (Rochester and Constable, 2003). This was indirect selection for improved nutrient status as a result of selection for high yield. There have also been continued improvements in P and K use efficiency with development of new higher yielding cultivars (Rochester and Constable, 2015). Cotton lint yields above 4400 kg/ha require uptake of at least 270 kg/ha of N and K/ha, and 45 kg P/ha (Constable and Bange, 2015). The low leaf Na trait is a more direct approach to optimise nutrient use efficiency while increasing yield potential.

5. Conclusions Leaf Na exclusion or low Na concentration is a critical component of cotton tolerance to the stresses caused by increased salts in soils. The results of this study have provided conclusive evidence that the trait found in G. barbadense cotton can be reliably phenotyped and readily manipulated through breeding. The optimal time for phenotyping is at the beginning of flowering. Low leaf Na concentration was associated with high sequestration of Na in cotton stems and roots instead of reduced Na uptake. This phenomenon indicates net Na transport between stems and leaves and Na intracellular compartments in stems and roots may be involved in controlling low leaf Na. This adaptive change led to better K and P nutrient status in cotton, especially in these most active photosynthetic tissues, i.e. leaves and bolls, as supported with the evidence of higher K/Na and P/Na ratio as well as optimal K and P concentration and resulted in improved use efficiency. Most interestingly, this evidence indicates that leaf Na concentration itself was neutral to agronomic performance, when incorporated into an elite G. hirsutum background. Therefore, manipulating leaf Na concentration would improve cotton tolerance to soil sodicity and also K and P use efficiency while maintaining agronomic performance, particularly under high yielding conditions. The above adaptive significance of the trait should be further verified by testing elite lines in soil conditions with higher levels of socidity.

4.3. Mechanisms of leaf Na exclusion

Declaration of Competing Interest

Regarding the total amount of Na, the low Na line accumulated more than the high Na sister line in both seasons (Table 5). This means that leaf Na exclusion observed in this study is not due to reduced Na uptake and accumulation, instead it is due to Na distribution over different plant tissues, resulting in stems and roots becoming Na sequestration centres (Fig. 4). When the low and high sister lines were compared, the increase for Na concentration was relatively smaller in stems than in roots. This raises the question of whether high Na concentration in the stem of the low Na line is the result of high Na flow from root or of trade-off of demand, transport and sequestration between leaves,

The authors declare no conflict of interest. Acknowledgements The authors acknowledge the assistance of technical support of our past and present team members: Kellie Cooper, Jo Price, Dave Shann, Deon Cameron, Rebecca Warnock, Louise Zemcevicius, Kay Smith and Sandra Magann. This is particularly to Mr Dave Shann for his significant contribution to the development of breeding materials used in this work. The authors also thank Drs. Michael Bange and Warren Conaty 10

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for the critical reading of the manuscript prior to submitting and the referees whose comments have led to an improved manuscript. This work was supported by the Cotton Breeding Australia Joint Venture between CSIRO and Cotton Seed Distributors.

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