Genetic diversity for grain Zn concentration in finger millet genotypes：Potential for improving human Zn nutrition

Rmegowd Ymunrni，Geeth Govind，Venktegowd Rmegowd，，Hrshvrdhn Vokklig Thmmegowd，Shnkr Amrhlli Guligowd，*Deprtment of Crop Physiology，University of Agriculturl Sciences，Gndhi Krishi Vignn Kendr，Bnglore 560 065，Krntk，IndiDeprtment of Crop，Soil nd Environmentl Sciences，University of Arknss，Fyetteville，AR 72701，USA

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Genetic diversity for grain Zn concentration in finger millet genotypes：Potential for improving human Zn nutrition

Ramegowda Yamunarania，1，Geetha Govinda，Venkategowda Ramegowdaa，b，
Harshavardhan Vokkaliga Thammegowdaa，Shankar Ambarahalli Guligowdaa，*
aDepartment of Crop Physiology，University of Agricultural Sciences，Gandhi Krishi Vignana Kendra，Bangalore 560 065，Karnataka，IndiabDepartment of Crop，Soil and Environmental Sciences，University of Arkansas，Fayetteville，AR 72701，USA

A R T I C L E I N F OA B S T R A C T

Article history：

Received in revised form

30 December 2015

Accepted 2 February 2016

Available online 11 February 2016

Nearly half of the world population suffers from micronutrient malnutrition，particularly Zn deficiency.It is important to understand genetic variation for uptake and translocation behaviors of Zn in relevant crop species to increase Zn concentration in edible parts.In the present study，genetic variation in grain Zn concentration of 319 finger millet genotypes was assessed.Large genetic variation was found among the genotypes，with concentrations ranging from 10 to 86 μg g-1grain.Uptake and translocation studies with Zn/65Zn application in 12 selected low-Zn genotypes showed wide variation in root uptake and shoot translocation，with genotypes GEC331 and GEC164 showing greater uptake and translocation.Genotypes GEC164 and GEC543 showed increased grain Zn concentration. Genotypes GEC331 and GEC164 also showed improved yield under Zn treatment. Appreciable variation in grain Zn concentration among finger millet genotypes found in this study offers opportunities to improve Zn nutrition through breeding.

?2016 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license （http://creativecommons.org/licenses/by-nc-nd/4.0/）.

Finger millet

Genetic variation

Human nutrition

Micronutrient

Zinc

1.Introduction

Micronutrients play vital roles in the biochemical and physiological functions of biological systems［1］.It is reported that the diets of over two thirds of the world's population lack one or more essential micronutrients［2］.Among micronutrients，zinc （Zn）deficiency accounts for many severe health complications ［3］.Zn deficiency is one of the major risk factors in human health and cause of death globally.Plants play a vital role in human nutrition by providing all essential nutrients required forhumanhealth［4］.Zndeficiencyinsoilsandplantsisaglobal micronutrient deficiency problem reported in many countries. Notably，50%of cultivated soils in India and Turkey，a third of cultivated soils in China，and most soils in Western Australia are classified as Zn-deficient［5］.

Conventional approaches such as Zn supplementation or fortification and dietary diversification adapted to ameliorate Zn deficiency in humans are neither practical nor cost-effective in thedevelopingworld.GeneticstrategiesforZnbiofortificationare more practical，sustainable，and cost-effective［6，7］.It is possibleto improve Zn concentration in crops grown on deficient soils by exploiting genotypic differences in Zn uptake and tissue-use efficiency that are present in crop species［8，9］.Strategies to increase Zn concentrations in edible portions seek to exploit genetic variation in acquisition of Zn from the soil，accumulation in edible portions，and tolerance of high tissue Zn concentrations ［2］.There is considerable species and varietal variation in Zn uptake，translocation，and storage in edible parts of crop plants，despite the very low Zn concentrations in edible parts of all food crops［10］.It is important to identify staple food crops specific to the Zn deficient regions of the world and address Zn deficiency issues.This activity requires a comprehensive exploration of potential genetic resources in regional crops and an in-depth understanding of the physiological and genetic basis of nutrient accumulation processes in seeds.In this study，we assessed the diversity in grain Zn concentration of 319 genotypes of finger millet［Eleusine coracana（L.）Gaertn.］，the predominant millet food crop of India and Africa.We also measured uptake and translocation differences in 12 selected low-Zn genotypes.

Fig.1-Frequency distribution of grain Zn concentration in 319 finger millet genotypes.The grain of 319 finger millet genotypes grown in three different seasons on red soil was analyzed for Zn concentration using Atomic absorption spectroscopy（AAS）.

2.Materials and methods

2.1.Finger millet grain material

Grain of 319 finger millet genotypes，grown in three different seasons on red soil with Zn concentration of 7 g kg-1soil，was procured from All India Coordinated Research Project on millets（AICRP），Gandhi Krishi Vignana Kendra，University of Agricultural Sciences，Bangalore，India.

2.2.Zn treatment and Zn estimation

Twelve finger millet genotypes（GEC265，460，331，164，543，392，329，440，61，236，403，and 202）were grown in pots（21 cm height and 21 cm diameter）filled with red soil.Plants were providedwithrecommendedlevelsoffertilizers（NPK 60:30:30 kg ha-1）.Fifteen days after sowing，plants were supplied with two concentrations of Zn in the form of ZnSO4.Three sets of plants were maintained at different Zn fertilization:a control（normal red soil containing 7 mg Zn kg-1soil），T1（moderate Zn fertilization with 5 mg kg-1soil in addition to 7 mg kg-1present in the soil sample）and T2（high Zn fertilization of 10 mg kg-1in addition to 7 mg kg-1present in the soil sample）.One set of plants were harvested after 20 days of treatment to determine Zn concentrations in root and shoot，and grain was harvested from the second set of plants at physiological maturity.Zinc was estimated as described［11］on dry weight（DW）basis.

65Zn treatment was given to 12 selected low-Zn genotypes，whichwereraisedin perforatedStyrofoamcups（10 cmlongand 5 cm diameter）filled with soil mixture（soil，sand and farmyard manure at the ratio 5:4:1）.Cups were placed on a sand bed to facilitate growth of roots.Twenty-one-day old seedlings in the cups were transferred to plastic trays containing 2.5 L of half-strength Hoagland's medium［12］，such that hanging roots werecompletelyimmersedinthenutrientmedium.After4 days of acclimation，the regular Hoagland's solution was exchanged with 2.5 L of Hoagland's solution containing65Zn（obtained from Board of Radiation and Isotope Technology，Department of Atomic Energy，Mumbai，India；40 mL of65Zn stock with specific activity of 196 μCi diluted with 60 L of Hoagland's solution）.The experiment was performed in a radioisotope containment facility for 48 h.Plants were harvested and washed in calcium sulfatesolution.65Znactivityinthefreshsampleswasmeasured with a liquid scintillation counter（WALLAC 1409，Perkin Elmer，California，USA）.Radioactive disintegrations per minute were converted into pmol g-1fresh weight.

2.3.Statistical analysis

Data were analyzed for significant differences by ANOVA （generalized linear model procedure）using SAS software version 9.3（SAS Institute Inc.，Cary，NC，USA）.Differences in short-term uptake of65Zn，transportation of65Zn，and grain Zn concentration under external Zn application in finger millet genotypes were tested by one-way ANOVA at P＜0.05. Differences in grain yield in finger millet genotypes under Zn application was tested by two-way ANOVA at P＜0.05.

3.Results

3.1.Genetic diversity for grain Zn concentration among finger millet genotypes

Genetic diversity for grain Zn concentration was studied among a large collection of 319 finger millet genotypes including core germplasm，locally adapted cultivars，and released varieties of India.Frequency distributions of grain Zn concentration are presented in Fig.1（for detailed data see Table S1）.There was large variation among the genotypes，with Zn concentrations ranging from 10 to 86 μg g-1.In nearly 50%of the genotypes（155）the Zn concentration was 21-30 μg g-1and only six genotypes showed＞63 μg Zn g-1（Table S1）.Genotypes with extreme Zn concentrations were called low and high grain Zn types and the 12 genotypes with less than 20 μg Zn g-1grain were selected for study of their uptake and translocation characteristics.The grain Zn concentrations in these genotypes ranged from 10 to 17 μg g-1.

3.2.Uptake and distribution of Zn between plant organs in finger millet genotypes

The effect of external Zn application on Zn distribution in severalorganswasmeasuredintheselected12genotypes.Most of the genotypes responded positively to Zn treatment with increased root uptake.Genotypes GEC331，GEC164，GEC543，GEC329，GEC61，GEC236，GEC403，and GEC202 showed significant increase in root uptake under moderate Zn application （T1）.However，there was no further increase in root Zn concentration with increase in external Zn concentration（T2）. The relative increase was very high in genotypes GEC331，GEC164，and GEC329，which accumulated approximately 30，20，and 17 μg g-1respectively，more than control plants（Table 1）.Shoot translocation increased in all genotypes except GEC329，GEC440，GEC236，and GEC202.Genotypes GEC331 and GEC164 which showed highest root uptake，also showed higher translocation to shoots.The increase in shoot Zn concentration under moderate Zn treatment in these two genotypes was greater than 20 μg g-1，suggesting that these genotypes were able to take up and translocate higher Zn than other genotypes when it was available in the soil.As observed for root uptake，there was no further increase in shoot Zn with increased Zn application（Table 1）.These results suggest that genotypes differ in their uptake and translocation and that some genotypes respond positively to external Zn application. GEC329，which showed higher root uptake under moderate Zn application，did not show significant change in shoot Zn compared to control plants，suggesting that this genotype may not be capable of translocating Zn from root to shoot.Genotype GEC440 did not show an increase in either root uptake or shoot translocation，suggesting a genetic limitation of this genotype with respect to Zn uptake and shoot translocation.

To confirm the observed difference in uptake and translocation of Zn among finger millet genotypes in the soil Zn application experiment，15-day-old finger millet seedlings were supplied with radiolabeled65Zn via roots.After 48 h of treatment，Zn uptake and translocation were measured in root and shoot tissue.As observed in the soil Zn application experiment，65Zn concentrations were higher in roots than in shoots among all the genotypes.Genotypes GEC403，GEC61，GEC164，and GEC331 showed higher65Zn in roots than the other genotypes（Fig.2-a）.This observation corroborates the results for root Zn concentration in the soil Zn application experiment（Table 1）confirming that these genotypes are efficient in uptake.The65Zn concentration was 4-fold higher in GEC403 than in GEC265，which showed the lowest root65Zn. Similarly，genotypes GEC331 and GEC164，which showed highest shoot Zn under soil application of normal Zn，also accumulated more65Zn in shoots，confirming the greater translocation efficiency of these genotypes（Fig.2-b）.There was a 1.0 to 1.5-fold increase in65Zn in these genotypes relative to the lowest-concentration genotypes GEC202 and GEC236.In summary，among all genotypes studied，GEC331 and GEC164 showed greater uptake and shoot translocation in both experiments.

To determine grain Zn concentration，grain from control and T2 plants with soil application of normal Zn was harvested at physiological maturity and Zn concentration was analyzed.There was a small increase in grain Zn concentration in genotypes GEC164 and GEC543.Surprisingly，there was no increase in grain Zn concentration in other genotypes（Fig.3）.The reason for the absence of increase in grain Zn concentration even with external Zn application could be a dilution effect，as there was a large increase in biomass at maturity，or could be due to the inefficiency of these genotypes intransporting Znto grain.Genotype GEC331，which showed high root uptake and shoot translocation，showedlowgrainZnconcentration.Ithasbeen suggested［13］that in addition to root uptake，remobilization of Zn from root and shoot to grain is also a major source of grain Zn.Thus，genotype GEC331 may have limitations in the remobilization of Zn from vegetative tissue to grain during grain development，resulting in reduced grain Zn.

3.3.Zn application increased grain yield in finger millet genotypes

Table 1-Effect of external Zn application on root and shoot Zn concentration.

The genotypes were also tested for influence of external Zn applicationongrainyield.GenotypesGEC265，GEC331，GEC164，GEC543，GEC440，and GEC61 showed significant increases in grain yield under Zn treatment relative to control （Fig.4）.Genotypes GEC440 and GEC61 showed greater than 6 g per plant increase in grain yield relative to control-grown plants.Genotypes GEC164 and GEC543 showed more than 3 g per plant increase in grain yield.In two other genotypes，GEC265 and GEC331，the increase was 2 g per plant.Six genotypes，GEC460，GEC392，GEC329，GEC236，GEC403，and GEC202，showed no significant change in yield with external Zn application.Among all the genotypes showing increase in yield with external Zn application，genotypes GEC440 and GEC61 showed the highest increases suggesting the potential for increasing grain yield in these genotypes by Zn fertilization.The lowest yield was found in genotype GEC403 among all the genotypes，even under the control condition.GEC329 showed higher yield than the other genotypes under the control condition.

Fig.2-Short-term uptake and transportation of65Zn in finger millet genotypes.21-day-old finger millet seedlings were provided with65Zn for 48 h.65Zn activity in fresh samples of（a）root and（b）shoot was measured with a liquid scintillation counter.Data represent means±SE of two independent experiments（n=8）.Different letters indicate significant differences between genotypes（one-way ANOVA at P＜0.05）.

Fig.3-Grain Zn concentration of finger millet genotypes under external Zn application.External Zn（T2，10 mg kg-1soil）was supplied to 15-day-old finger millet seedlings grown on red soil.Grain was harvested at physiological maturity and Zn concentration was measured by AAS.Data represents means±SE of two independent experiments（n= 8）.Different letters indicate significant differences between control and treated（T2）grain Zn concentration（one-way ANOVA at P＜0.05）.

The genotypes that maintained high root Zn（uptake）and shoot Zn（translocation），which was translated into high grain Zn and yield，are genotypes likely to be selected as source material for studying the physiological and molecular mechanisms of uptake and translocation of Zn.In the present study，genotype GEC164 showed an increase in grain Zn concentration and could be a source for further studies.

4.Discussion

Studies in various species have shown considerable genotypic variation in Zn concentration.From 2.2 to 11.6-fold variation in seed Zn concentrations in large core collections of cereal germplasm［14-16］and from 1.8 to 6.6-fold variation in seed Zn concentrations in large core collections of legume genotypes［17］have been observed.In our study of 319 finger millet genotypes，we found 8-fold variation in grain Zn concentration.About 50%of the genotypes had 20-30 μg Zn g-1of grain with only 5%having grain Zn greater than 50 μg g-1grain.The genotypeswithhighgrainZnconcentrationarelikely candidates from the human nutritional perspective to supply the recommended daily intake of 15 mg and could also serve as materials for elucidating the physiological and molecular basis of high grain Zn concentration.

To characterize uptake and translocation，the 12 genotypes with lowest grain Zn concentration were selected.Uptake and translocation studies have shown that genotypes respond positively to external Zn application with enhanced uptake under moderate Zn application，but that there is no increase under high soil Zn status，possibly owing to transporter saturation，as low-Zn genotypes could have low-affinity transporters in excess compared to high-affinity transporters.The presence of both low-and high-affinity transport systems in wheat roots has recently been shown［18］.Increased expression of genes encoding Zn transporters can increase root uptake of Zn［19］.Among 12 low-grain Zn genotypes，genotypes GEC331，GEC164，and GEC329 showed more Zn in roots than controls.These genotypes could be good candidates for study of the transporters involved in uptake in low-Zn types.

Fig.4-Grain yield in finger millet genotypes under Zn application.15-day-old finger millet seedlings grown on red soil weresuppliedwithZn（T2，10 mg kg-1soil）intheformofZnSO4. Yield per plant was determined.Data represent means±SE of two independent experiments（n=8）. Uppercase letters indicate significant differences between treatments and lowercase letters indicate significant differences between genotypes（two-way ANOVA at P＜0.05）.

Theselectedgenotypesalsodifferedsignificantlyin translocation of Zn from roots to shoots.Eight of the 12 genotypes showed higher shoot Zn than controls；specifically，GEC331 and GEC164 showed higher translocation to shoots. However，except in GEC331，the relative increase in shoot Zn was low compared to the increase in root Zn concentration under external Zn application，suggesting the inability of some of these genotypes to mobilize Zn from root to shoot. This inability could be one of the reasons for low grain Zn concentration in cereals.Uptake and translocation seemed to vary among genotypes with soil Zn application，suggesting that plants possess different classes（low-and high-affinity）of transporters that operate under different Zn concentrations. There is a report of concentration-dependent kinetics of Zn influx into roots of wheat cultivars［18］.Genotypes able to maintain high root and shoot Zn would be useful，as they might translate root and shoot Zn into grain Zn，resulting in increased grain Zn concentration.In the present study，genotypes GEC331and GEC164，which showed highest root uptake，also showed high translocation to shoots.

Long-term Zn uptake in these studies can reflect and be influenced by several factors including compartmentation in roots as well as translocation and use in shoots［11，18，19］.For this reason，we used65Zn to trace the fate of Zn in 48 h. Radioactive65Zn studies also confirmed these results:the genotypes that showed higher uptake and translocation in the normal Zn treatment experiments also showed higher65Zn uptakeandtranslocation.Theseresultssuggestthat genotypesfollowedthesameuptakeandtranslocation pattern under long-and short-term treatment.

Although there was variation among genotypes in uptake and translocation of Zn to both root and shoot，ultimately it is grain Zn concentration that is important for human nutrition ［11，20］.It is thus desirable to identify genotypes that are able to take up and translocate Zn efficiently when it is available in the soil，and preferably also have high yield.Depending on plant species，soil application of Zn can increase Zn concentration in plants by as much as 2 to 3-fold［21］.Still，even with very high Zn fertilization rates，the Zn concentration in wheat grain did not increase correspondingly［22］.Despite Zn fertilization，under most conditions there appears to be some biological limit that makes it difficult to increase Zn concentration in grain［23］.In support of these findings，also in finger millet，genotypes with high uptake and translocation efficiency did not show increased Zn in grain except for GEC164 and GEC543，which showed marginal increases.

Studies have shown substantial increase in grain yield with Zn in addition to NPK application，indicating the critical importance of this nutrient in crop production［21］.A large increase in grain yield was also demonstrated by Graham et al.［24］.Some low-Zn finger millet types responded positively to external Zn supply.Genotypes GEC264，GEC331，GEC164，GEC543，GEC440，and GEC61 showed significant increases in grain yield under Zn application，suggesting the importance of genotypic variation in Zn use efficiency for improved grain yield.

5.Conclusions

The findings in this study suggest that large genetic variation exists among finger millet genotypes for grain Zn concentration. External Zn application studies in selected genotypes showed wide variation in root uptake，shoot translocation and accumulation in grain.Genotypes，GEC331 and GEC164 showed better uptake and translocation under external Zn application.It is possible that the general homeostatic regulation of tissue Zn concentrations through Zn acquisition and distribution within the plant could be a major constraint to increase Zn concentrations in edible portions.Molecular and biochemical studies for the genetic variability is required by selecting genotypes and also with regard to uptake by root，root to shoot translocation and sequestration in grains.Since there is large variation in grain Zn concentrations in finger millet genotypes，this could serve as resource for breeding programs aimed at improving grain Zn concentration in finger millet.

Acknowledgments

Our work was supported by projects from Department of Science and Technology（DST）（Grant#SR/SO/PS-14/2002）and Department of Biotechnology（DBT）（Grant#BT/01/COE/ 05/03），New Delhi，Government of India.The authors are grateful to All India Coordinated Research Project on millets （AICRP），GKVK，University of Agricultural Sciences，Bangalore，India for providing finger millet genotypes used in this study.

Supplementary data

Supplementary material for this article can be found online at http://dx.doi.org/10.1016/j.cj.2015.12.001.

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29 October 2015

*Corresponding author.Tel.:+91 80 23636713；fax:+91 80 23636713.

E-mail address:ambara8@hotmail.com（A.G.Shankar）.

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science，CAAS.1Present address:Department of Plant Pathology，University of Arkansas，Fayetteville，AR 72701，USA.

http://dx.doi.org/10.1016/j.cj.2015.12.001

2214-5141/?2016 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.