5
Challenges in breeding for yield increase for drought Thomas R. Sinclair Crop Science Department, North Carolina State University, Raleigh, NC, USA Crop genetic improvement for environmental stress at the molecular and physiological level is very complex and challenging. Unlike the example of the current major commercial transgenic crops for which biotic stress tolerance is based on chemicals alien to plants, the complex, redundant and homeostatic molecular and physiological systems existing in plants must be altered for drought tolerance improvement. Sophisticated tools must be developed to monitor phenotype expression at the crop level to characterize variation among genotypes across a range of environments. Once stress-tolerant cultivars are developed, regional probability distribu- tions describing yield response across years will be necessary. This information can then aid in identifying environmental conditions for positive and negative responses to genetic modification to guide farmer selec- tion of stress-tolerant cultivars. Genetic challenge of drought stress There have been tremendous advances in understanding the physiology, biochemistry and molecular genetics of plant response to drought in the past half-century. Increas- ing numbers of papers are being published reporting new insights about the factors contributing to increased plant development and growth rates [1]. Yet, this information has had little or no impact to date in improving the intrinsic behavior of plants resulting in increased crop yield [25]. For example, the two highly visible, successful transgenic crops glyphosate tolerance and Bacillus thur- ingiensis (Bt) insect tolerance ‘defend’ against pest-me- diated yield loss, but do not address fundamental, intrinsic plant performance that would lead to improved crop re- sponse to yield-limiting abiotic factors. Indeed, these two commercial transgenes do not rely on acceleration or sup- pression of fundamental biochemical or physiological path- ways altering plant development or growth they are based on specific chemicals that are alien to plants and reside outside the contributory mechanisms leading to crop yield. These two cases do not offer a model for improving crop performance when subjected to environmental stres- ses, including drought. Alteration of an entire synthetic process in plants to get adequate changes at whole plant and field levels is ex- tremely difficult, as proved in nearly a half century of physiological studies [5,6]. Many individual processes, each influenced by a myriad of reactions and molecular controls, contribute and integrate to affect whole plant performance. This is certainly true for drought-adaptive traits [7]. The ‘bottomup’ approach, starting at the molec- ular level, is often promoted as a simple and direct means for achieving major yield gains. However, plant modifica- tions via transformation of an existing molecular pathway will likely flounder, given the vast number of interacting biochemical networks and the damping of any simple molecular change when scaling-up the complex physiologi- cal hierarchy leading to yield. The route to yield-change expressed by crops is different for each geographical loca- tion and new growing season, reflecting new combinations and temporal dynamics in the biotic and abiotic environ- ments [1]. Certainly the DNA code may carry information that influences alternate plant responses to each new environment, but how that information is integrated throughout the growing season at the physiological level and across the community of plants are the critical deter- minants of crop yield. An alternative to the bottomup perspective is a ‘topdown’ approach that starts with a whole-crop perspective to consider how the plant community may perform in the field across a range of environments. What traits can be altered in individual plants to result in an increased yield by a community of such altered plants that is a crop? The advantage of the topdown approach is that the starting point is the performance of intact crop plants that can be studied for expression of a desired behavior, instead of an abstract extrapolation of what a specific molecular-level transformation might contribute at the whole crop level. The topdown view may sound a bit old fashioned, but it must be remembered that it is the use of field experiments and careful observations that have enabled successful selection and genetic gain for increased crop yield. Indeed, numerous studies have demonstrated yield to be geneti- cally complex and under control of many genes of small impact. The challenge is to integrate the modern tools of molecular genetics and physiology to bear on a practical approach for yield advances in the field [8]. How can these new insights be used to improve plant phenotype, thereby increasing crop yield? Can this new information be used in genotype selection to alter the expression of specific traits and achieve yield increase? The answers to these critical questions are not resolved. There are only a few examples where the topdown approach has been successfully ap- plied to intrinsically alter biochemical or physiological activity to increase crop yield [2,3]. The few examples resulting in cultivar releases with increased yield based on this approach include heat tolerance in cowpea (Vigna unguiculata) [9], improved water use in wheat (Triticum Opinion Corresponding author: Sinclair, T.R. ([email protected]). 1360-1385/$ see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2011.02.008 Trends in Plant Science, June 2011, Vol. 16, No. 6 289

Challenges in breeding for yield increase for drought

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Page 1: Challenges in breeding for yield increase for drought

Challenges in breeding for yieldincrease for droughtThomas R. Sinclair

Crop Science Department, North Carolina State University, Raleigh, NC, USA

Opinion

Crop genetic improvement for environmental stress atthe molecular and physiological level is very complexand challenging. Unlike the example of the current majorcommercial transgenic crops for which biotic stresstolerance is based on chemicals alien to plants, thecomplex, redundant and homeostatic molecular andphysiological systems existing in plants must be alteredfor drought tolerance improvement. Sophisticated toolsmust be developed to monitor phenotype expression atthe crop level to characterize variation among genotypesacross a range of environments. Once stress-tolerantcultivars are developed, regional probability distribu-tions describing yield response across years will benecessary. This information can then aid in identifyingenvironmental conditions for positive and negativeresponses to genetic modification to guide farmer selec-tion of stress-tolerant cultivars.

Genetic challenge of drought stressThere have been tremendous advances in understandingthe physiology, biochemistry and molecular genetics ofplant response to drought in the past half-century. Increas-ing numbers of papers are being published reporting newinsights about the factors contributing to increased plantdevelopment and growth rates [1]. Yet, this informationhas had little or no impact to date in improving theintrinsic behavior of plants resulting in increased cropyield [2–5]. For example, the two highly visible, successfultransgenic crops – glyphosate tolerance and Bacillus thur-ingiensis (Bt) insect tolerance – ‘defend’ against pest-me-diated yield loss, but do not address fundamental, intrinsicplant performance that would lead to improved crop re-sponse to yield-limiting abiotic factors. Indeed, these twocommercial transgenes do not rely on acceleration or sup-pression of fundamental biochemical or physiological path-ways altering plant development or growth – they arebased on specific chemicals that are alien to plants andreside outside the contributorymechanisms leading to cropyield. These two cases do not offer a model for improvingcrop performance when subjected to environmental stres-ses, including drought.

Alteration of an entire synthetic process in plants to getadequate changes at whole plant and field levels is ex-tremely difficult, as proved in nearly a half century ofphysiological studies [5,6]. Many individual processes,each influenced by a myriad of reactions and molecularcontrols, contribute and integrate to affect whole plant

Corresponding author: Sinclair, T.R. ([email protected]).

1360-1385/$ – see front matter � 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.20

performance. This is certainly true for drought-adaptivetraits [7]. The ‘bottom–up’ approach, starting at the molec-ular level, is often promoted as a simple and direct meansfor achieving major yield gains. However, plant modifica-tions via transformation of an existing molecular pathwaywill likely flounder, given the vast number of interactingbiochemical networks and the damping of any simplemolecular change when scaling-up the complex physiologi-cal hierarchy leading to yield. The route to yield-changeexpressed by crops is different for each geographical loca-tion and new growing season, reflecting new combinationsand temporal dynamics in the biotic and abiotic environ-ments [1]. Certainly the DNA code may carry informationthat influences alternate plant responses to each newenvironment, but how that information is integratedthroughout the growing season at the physiological leveland across the community of plants are the critical deter-minants of crop yield.

An alternative to the bottom–up perspective is a ‘top–

down’ approach that starts with a whole-crop perspectiveto consider how the plant community may perform in thefield across a range of environments. What traits can bealtered in individual plants to result in an increased yieldby a community of such altered plants that is a crop? Theadvantage of the top–down approach is that the startingpoint is the performance of intact crop plants that can bestudied for expression of a desired behavior, instead of anabstract extrapolation of what a specific molecular-leveltransformation might contribute at the whole crop level.

The top–down viewmay sound a bit old fashioned, but itmust be remembered that it is the use of field experimentsand careful observations that have enabled successfulselection and genetic gain for increased crop yield. Indeed,numerous studies have demonstrated yield to be geneti-cally complex and under control of many genes of smallimpact. The challenge is to integrate the modern tools ofmolecular genetics and physiology to bear on a practicalapproach for yield advances in the field [8]. How can thesenew insights be used to improve plant phenotype, therebyincreasing crop yield? Can this new information be used ingenotype selection to alter the expression of specific traitsand achieve yield increase? The answers to these criticalquestions are not resolved. There are only a few exampleswhere the top–down approach has been successfully ap-plied to intrinsically alter biochemical or physiologicalactivity to increase crop yield [2,3]. The few examplesresulting in cultivar releases with increased yield basedon this approach include heat tolerance in cowpea (Vignaunguiculata) [9], improved water use in wheat (Triticum

11.02.008 Trends in Plant Science, June 2011, Vol. 16, No. 6 289

Page 2: Challenges in breeding for yield increase for drought

Opinion Trends in Plant Science June 2011, Vol. 16, No. 6

aestivum) [10], and drought tolerance of nitrogen fixationin soybean (Glycine max) [11] which is the example consid-ered in more detail later in this paper.

Here I discuss four topics concerning the application ofmolecular and physiological knowledge that may help toresolve these questions for improving adaptation todrought: (i) assessing potential yield benefit and variationwithin a germplasm population for a proposed droughttrait, (ii) developing suitable means of phenotyping thespecific trait of interest, (iii) developing techniques to trackexpression of a desired trait through the breeding process,and (iv) creating new criteria for cultivar selection andmarketing. While the focus is specially on developing cropdrought tolerance, it is anticipated that many of the con-clusions of this discussion apply to other abiotic stresses.

Assessment of benefitIt is necessary to move beyond intuitive anticipation of asignificant benefit from a putative trait before launching amajor research program to develop an improved cultivar.Intuition is not a reliable guide in the face of the complexityof response to soil drying, and it gives no insight about theamount of yield change that might be expected. An exam-ple illustrating the failure of intuition in developingdrought tolerance is the substantial investment in trans-genic lines that display tolerance under severe water defi-cit. Experiments are reported that impose drought stressesresulting in the death or near death of wild-type andsurvival of transgenic sister lines. The problem is thatsimply enhancing survival of grain crops is irrelevant incommercial production. Any situation where water avail-ability is so low to threaten survival means that even if theplants survive, crop yield will necessarily be very low andthe farmer will be economically devastated. Rather thansurvival, realistic yield improvements likely need to ad-dress one of the following traits: (i) increase plant access towater, most likely through deeper rooting, to supportgreater crop growth; (ii) conserve soil water for use duringlate-season water deficits; or (iii) overcome special sensi-tivities in the plant that limit yield formation under water-deficit.

The initiation point to genetically enhance crop yieldwhen subjected to environmental stress should be thecollection of evidence giving a solid basis for anticipatinga relevant benefit under production conditions and forquantifying the expected yield gain. Unfortunately, manycurrent studies started with expensive and sophisticatedmolecular studies without any direct evidence that modifi-cation would have practical benefit. Important sources ofsuch evidence are experimental results in which traitresponse is induced experimentally in high-yield commer-cial cultivars to mimic anticipated genetic modification.How large is the yield-increase observed in such experi-ments? In studies on increasing drought tolerance of nitro-gen fixation, such results were obtained by applying highamounts of nitrogen fertilizer to soybean plots subjected todrought stress [12,13]. In these experiments, removingplant dependence on symbiotically-fixed nitrogen duringwater deficits resulted in yield gains of 15–20%.

A very useful approach to fully understand possibleyield changes as a result of trait modification is to use

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mechanistic crop models to simulate yield response over arange of growing seasons and geographical locations [8,14].We have used a relatively simple model of soybean devel-opment, growth and yield to simulate soybean yieldchanges that could be expected across the USA with modi-fied drought traits ([15], Box 1).

Trait phenotypingThe expression of any particular physiological trait ortransformation event is highly dependent on the environ-ment in which the crop is grown. The existence of desiredgenetic code in a genotype offers no assurance about theexpression of plant phenotype in a given environment. Thechallenge to make progress in developing higher-yieldingcrops is that physiological phenotype other than yieldmustbe documented at some point in the breeding process[14,16].

Ben Miflin [17] argued that ‘‘Undue or sole emphasis ongenomics will lead to an ever increasing gap between thegenetic information acquired and an understanding of thephenotype, a ‘phenotype gap’’’. Indeed, the lack of ability tophenotype plants for specific trait performance has been acritical limitation in applying physiological information formore than half a century [6]. The challenge is that it is verydifficult to accurately phenotype plants for anything butthe most obvious trait. Most of the physiological traits thatimpact on response to environmental stress require de-tailed, sophisticated and usually expensive techniques tophenotype plants, and can be applied only to a very limitednumber of genotypes. Not surprisingly, many physiologicalstudies searching for genetic variation have frequentlyinvolved no more than 20 genotypes, and almost nevermore than 50 genotypes. Conversely, there have been a fewattempts to search a large number of genotypes for geneticvariation, and these attempts have failed due to the lack ofsophistication in the screen.

A solution to the dilemma of phenotypic screens eitherbeing too difficult and sophisticated, or too crude and withpoor resolution is amulti-tiered screening sequence involv-ing both types of screens [7]. A simple but less accuratescreen that allows a large number of genotypes to beexamined is a first-tier screen followed by tiers of moresophisticated screens of decreasing numbers of genotypes.Such multi-tiered screens for crop improvement are notnew in classical breeding efforts. ‘Tandem’ selection breed-ing is used regularly in some crops. We developed a three-tiered sequence of physiological screens (Box 2) in ourefforts to identify candidate genotypes for use as parentsin breeding efforts to sustain nitrogen fixation activityduring soil water deficit [18].

Tracking of phenotypic improvementUnfortunately, the approach of using multi-tiered screensis likely only suited for identifying parental lines withdesired physiological traits. This approach is too laboriousfor tracking a trait through each stage of a breeding effort.How can the status of a physiological trait in a breedingeffort be resolved?

Of course, one very appealing approach is to identify agenetic marker or maybe a specific nucleotide sequencecompletely linked with the desired trait. The challenge, as

Page 3: Challenges in breeding for yield increase for drought

Box 1. Model assessment of enhanced nitrogen fixation drought tolerance

A mechanistic soybean growth and yield model was used to simulate

over growing seasons the soil water budget and its impact on crop

development, growth and yield [15]. A Geographical Information

System data base of at least 50 years of weather data for individual

30 km � 30 km grid locations across the U.S. were the input for the

model simulations. To simulate yield for all grid locations and all

years across the U.S., the model was run for>130,000 environments.

The model was used to first simulate a ‘standard’ soybean and then

re-run after modifying the model to simulate the behavior of a

specific drought trait hypothesized to influence crop yield in

response to drought. The probability of yield gain is color coded

for all locations in Figure I a as a result of the trait of nitrogen fixation

tolerance to drought. Yield gain was achieved in >85% of the

growing seasons, and in many regions it was >95%. The absolute

amount of the yield change is color coded in Figure I b, c and d for the

75 percentile, median and 25 percentile years for each grid. The

greatest absolute yield gains were in the drier years (25 percentile).

But the median and 75 percentile years also showed positive gains in

yield, indicating that even in these growing seasons there was

sufficient soil water deficit to impair nitrogen fixation activity.

Almost no penalty in yield was simulated from the nitrogen fixation

drought tolerant trait.

[()TD$FIG]

0.0, 0.05

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0.4, 0.45

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0.6, 0.65

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0, 5

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3035

4045

-105 -100 -95 -90 -85 -80 -75

P(x ≥ 0) g m-2

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(c)

(d)

TRENDS in Plant Science

Figure I. Simulation results for incorporation of the trait for drought tolerance of nitrogen fixation in soybean. (a) Probability of yield gain and (b–d) absolute yield

change for yield percentiles of 75% (wet), median and 25% (dry). Reproduced with permission from [15].

Opinion Trends in Plant Science June 2011, Vol. 16, No. 6

discussed above, is that response to water-deficit is notlikely to be under the control of a single or even a few genes.One possibility is that a collection of gene markers mightbe identified that contribute to physiological phenotype.Nevertheless, geneticmarkers only offer information aboutthe genetic potential related to expression of physiologicaltrait(s) for drought tolerance. While markers offer impor-tant information, they do not provide insight about thelevel of expression of a trait over a range of field environ-ments. Trait expression is likely to be very much depen-dent on the biotic and abiotic environment in which theplant is grown. Within the plant the level of redundancy,location of the gene within the genome, and regulation byother genes may also impact trait expression. Therefore,the genetic tag could be useful in ensuring the possibility ofphenotypic expression, but unfortunately, such tags do not

offer definitive information about phenotypic expressionbeing sought in superior lines [19].

The ultimate solution is to have ‘tools’ available todocument phenotypic expression at each stage of breedingand in each test environment. However, such comprehen-sive tools seem improbable, if not impossible, and unreal-istic for practical breeding programs. What can be thealternative to tracking phenotypic expression at each stagein the breeding process? One possibility may be to focusefforts on phenotyping for physiological performance onlyat a few critical stages in the breeding cycle. Certainly, onecritical stage would be the initial identification of parentlines, either existing genotypes or transgenic lines thatreadily express the desired trait under a range of condi-tions. This can be accomplished by the multi-tiered ap-proach described above.

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Page 4: Challenges in breeding for yield increase for drought

Box 3. Cultivar selection for enhanced nitrogen fixation

drought tolerance

In the effort to develop cultivars expressing nitrogen fixation

tolerance to water-deficit conditions and high yield, plants were

phenotyped at critical stages in the breeding program. Initial effort

was given to identifying and characterizing the expression of

nitrogen fixation drought tolerance in the soybean cultivar Jackson

[21,22]. Jackson was then crossed with the high-yielding cultivar

KS4895 and the derived population was advanced by single seed

descent to the F3 stage. Dr. Pengyin Chen (University of Arkansas)

eliminated genotypes from the population, based on agronomic

traits for robustness and uniformity in plant maturity in several

environments. Then, 100 selected lines from this population were

tested by Drs. C.A. King and L.C. Purcell (University of Arkansas) in

the greenhouse for nitrogen accumulation by potted plants

subjected to a constant, mild drought stress for two weeks. This

relatively simple physiological screen resulted in the identification

of 17 candidate lines for field yield test in a number of locations and

years. From the field yield tests, two lines were identified as having

higher yield than the commercial checks under moderate drought

conditions. The final step was to test these two lines with controlled

soil drying in the greenhouse to determine directly that both lines

expressed nitrogen fixation drought tolerance [18].

Box 2. Multi-tier selection scheme for nitrogen fixation

drought tolerance

A three-tier selection scheme (outlined in Figure I) was developed to

identify candidate parental soybean lines with high tolerance of

nitrogen fixation under drought conditions [18]. The broadest, and

least accurate, screen was based on the concentration of ureides in

the petioles of well-watered soybean genotypes. High leaf ureide

concentrations had been shown to be associated with sensitivity to

nitrogen fixation to drought. From a screen of about 3500 plant

introduction (PI) lines, about 250 lines or slightly less than 10% with

low petiole ureide concentrations were selected for a field test of

nitrogen accumulation under dry conditions. The lines in the second

tier screen were grown on a sandy soil, which had both a low

nitrogen content and low water holding capacity. The lines were

lightly irrigated approximately every other day to maintain them for

about three weeks at or slightly above a soil water content resulting

in slight leaf wilting. Again about 10% of these field-tested lines (24

lines) were selected for the third tier of screening for intensive

measurement of nitrogen fixation response to soil drying in the

greenhouse. Ultimately, 11 PI lines were identified that had

substantial superiority in nitrogen fixation tolerance to soil drying.[()TD$FIG]

Identification of N2 tolerant germplasm

~ 3 500 plant introductions

Field screen Low petiole ureide

~ 250 PI selections

Field screen N accumulation under drought

24 PI selections

Glasshouse test ARA with drying soil

11 tolerant PIs

TRENDS in Plant Science

Figure I. Diagram of three-tier screen to identify parents with drought

tolerance of nitrogen fixation.

Opinion Trends in Plant Science June 2011, Vol. 16, No. 6

The second critical stage for physiological phenotypingmight be after the breeding population has been decreasedto a more limited, yet substantial number of candidatelines by using standard agronomic selection criteria. Thatis, initially remove lines from the population that do notcontain the marker for the desired trait, show disease orinsect susceptibility, vulnerability to lodging, or have in-appropriate agronomic characteristics. At this stage, thenumber of candidate lines can be narrowed further usingsecond or third tier screens to a manageable number oflines for yield test.

The final critical stage is to document that those high-yielding lines identified in field tests also readily expressthe desired physiological trait. This information providesassurance that the yield increase might be a result of theputative physiological trait. (Of course, if the high-yieldinglines are shown not to have the desired traits then thebreeder is left with a high yielding line, and additionalstudies will be needed to resolve the reason for the yieldimprovement). We [18] employed such multi-tiered pheno-

292

typing at critical stages with success in efforts to developgenotypes with nitrogen fixation tolerance to soil drying(Box 3).

Cultivar selection and marketingDue to complex interactions between physiological process-es and the abiotic environment in the field, it is unlikelythat small changes in a physiological trait will result in auniform increase in yield across all environments. Physio-logical trait improvement for drought conditions, unlikedisease or insect tolerance for which the presence of thedesired trait itself has virtually no negative consequenceson yield, can have quite variable impact on yield acrossenvironments [15]. Instead of evaluating candidate culti-vars based on grandmean yields over environments, whichis a common approach, the more useful information is aprobability response for yield change in the targeted envir-onments. What are the environmental circumstances andprobability for yield gain and by howmuch, and converselywhat are the environmental circumstances and probabilityfor yield loss and by how much?

Results from field tests with soybean lines identified ashaving drought tolerance of nitrogen fixation providedinformation on yield response across environments [20].Yield tests were made at a number of locations in thesoutheast USA and the yield in each case was comparedwith the average yield of commercial cultivars. The resultsof these tests showed a yield advantage for one genotype(R01-518F) in those environments where average yield ofcommercial cultivars was 250–360 g m–2 (Figure 1). Out-side this commercially important range of yield, there wasno consistent yield advantage or disadvantage for R01-518F. Therefore, if there is a reasonable fraction of growingseasons in the 250–360 g m–2 range, the nitrogen fixationdrought tolerance trait would be advantageous for farmers.

Farmers seem willing to digest probability data on yieldresponse if such information will improve their economicwellbeing. Farmers need to know the fraction of growingseasons a yield gain might be anticipated, the fraction of

Page 5: Challenges in breeding for yield increase for drought

[()TD$FIG]

100 200 300 400

-60

-30

0

30

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90

Consistentyield gain

Check yield (g m-2)

Yie

ld d

iffe

r. (

g m

-2)

[R01

-581

F -

ch

eck]

TRENDS in Plant Science

Figure 1. Graph of differences in yield for individual test environments between

R01-518F, a genotype with drought tolerance of nitrogen fixation, and the average

of commercial cultivars [20]. The developed cultivar R01-581F had consistently

greater yield than commercial cultivars in the yield range of 250–360 g m–2 which is

delineated in the figure with vertical dashed lines. This range of the consistent

yield gain is particularly important in commercial soybean production.

Reproduced with permission from [20].

Opinion Trends in Plant Science June 2011, Vol. 16, No. 6

years when there is no yield benefit, and the fraction ofyears when the stress-tolerant cultivar will result in a yieldloss. Cultivars with stress-tolerant traits might be sold likeinsurance in that the improved cultivar gives increasedyields in low-yield, low-income years but the insurancepremium is ‘paid’ in the very good years when a yielddecrease would result in a small income loss in years ofhigh revenue.

AcknowledgementsThe author is grateful for the input offered by Greg Rebetzke (CSIRO,Canberra, Australia), James Specht (Agronomy & HorticultureDepartment, University of Nebraska, Lincoln, NE, USA) and TommyCarter (USDA-ARS, Raleigh, NC, USA) in their review of the draft of thispaper.

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12 Purcell, L.C. and King, C.A. (1996) Drought and nitrogen source effectson nitrogen nutrition, seed growth, and yield in soybean. J. Plant Nutr.19, 969–993

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fixation tolerance to water deficits. Crop Sci. 40, 1803–180919 Bernardo, R. (2008) Molecular markers and selection for complex traits

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