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Research ArticleVariability in Prolificacy Total Carotenoids Lutein andZeaxanthin of Yellow Small-Ear Waxy Corn Germplasm

Supaporn Sukto 12 Khomsorn Lomthaisong3 Jirawat Sanitchon14

Sompong Chankaew14 Marvin P Scott5 Thomas Lubberstedt6 Kamol Lertrat4

and Bhalang Suriharn 14

1Department of Agronomy Faculty of Agriculture Khon Kaen University Khon Kaen 40002 ailand2Uthai ani Agricultural Research and Development Center Department of Agriculture (DOA) Uthai ani 61110 ailand3Department of Biochemistry Faculty of Science Khon Kaen University Khon Kaen 40002 ailand4Plant Breeding Research Center for Sustainable Agriculture Khon Kaen University Khon Kaen 40002 ailand5USDA-ARS Corn Insects and Crop Genetics Research Unit USDA-ARS Ames IA 50011 USA6Department of Agronomy Iowa State University Ames IA 50011 USA

Correspondence should be addressed to Bhalang Suriharn sphalakkuacth

Received 10 August 2020 Revised 2 November 2020 Accepted 6 November 2020 Published 19 November 2020

Academic Editor Maria Serrano

Copyright copy 2020 Supaporn Sukto et al ampis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Waxy corn is a popular alternative staple food in most Asian countries including ampailand ampe availability of small-ear waxy corngenotypes with prolific ears and a high level of carotenoids is expected to benefit growers and consumers Integrated evaluation amongsource germplasm is essential before performing further breeding efforts for enhancing prolific ears and high-carotenoid contentampusthe present study explored the variability of ear prolificacy total carotenoids lutein zeaxanthin and beta-carotene among yellow small-ear waxy corn accessions About 44 corn accessions and 4 check varieties were evaluated for agronomic traits and yield componentsunder multienvironment trials in a randomized complete block design (RCBD) ampe immature seed sample of these genotypes wasanalyzed to quantify the content of total carotenoids and some carotenoid fractions All traits showed that low GXE interaction andsignificant genotypic diversity existed among all tested accessions with the predominant contribution of genotype to total phenotypicvariation and beta-carotene Accessions were clustered into four major groups based on the similarity of multiple carotenoids profilesampree selected accessions (UT121001 KKU-WX112087 and KKU-WX212001) had higher values of total carotenoids lutein zeax-anthin and beta-carotene than those of all check varieties High and positive correlations among second-emerged ears marketablesecond-emerged ears and total ear number indicate that a higher chance of secondary ears becomesmarketable ears with an increase oftotal ears per plant per hectare Lutein and zeaxanthin had positive strong correlations with total carotenoids ampe implications andbreeding strategies are discussed prior to promoting yellow small-ear waxy corn as a biofortified crop

1 Introduction

Carotenoids are of interest to human health Conse-quently their consumption is correlated with safeguardfrom numerous chronic diseases such as reducing the riskof cancer cardiovascular diseases and night blindness [1]Carotenoids are plant pigments that normally can befound in plants fruits and vegetables accounting for redorange and yellow pigmentations [2] ampey consist of two

major groups namely xanthophylls (lutein zeaxanthinand β-cryptoxanthin) and carotene (β-carotene andα-carotene) [3] ampe major fractions lutein and zeaxanthinare largely found in corn kernels [4] and play a primaryrole in human health associated with eyes [5] especially asantioxidants or blue light filters that can protect oculartissues from phototoxic damage [6] ampus corn is a goodsource of non-provitamin A carotenoids with lutein andzeaxanthin as major components [7]

HindawiInternational Journal of AgronomyVolume 2020 Article ID 8818768 12 pageshttpsdoiorg10115520208818768

Waxy corn (Zea mays L var ceratina) is popular and iscommonly consumed in most Asian countries particularlyChina Korea Laos Myanmar ampailand and Vietnam [8]In Northeastern ampailand people enjoy cooked fresh waxycorn as an alternative staple food to rice Its unique textureand sticky kernels due to high amylopectin content [9]appeal to consumers ampe kernel coloration of fresh waxycorn is normally either white or bicolor purple and whitewith a poor expression of carotenoids including lutein andzeaxanthin [10] Since the presence of orange corn kernelspositively correlates with higher carotenoid concentrationsand is a heritable trait [11] exploring corn accessions thatpossess a significant content of carotenoids is possibleMoreover promoting small-ear waxy corn as a biofortifiedcrop is promising because the carotenoid contents especiallylutein and zeaxanthin are increased after steaming [12] andconsumer acceptability of provitamin A biofortifiedmaize inSouth Africa is high [13] ampe content of carotenoids isquantitative traits controlled by a few major genes [14]While additive gene effects are important for lutein zeax-anthin and beta-cryptoxanthin nonadditive effects arepredominant for beta-carotene and provitamin A [14]

Inampailand waxy corn is consumed fresh and commonlypaid for on a per ear basis instead of the kernel weight ampuswaxy corn with prolific ears will benefit growers by boostingtheir incomes Criteria of prolificacy are at least two mar-ketable ears per plant and short period for ear emergence ofboth ears Small-ear waxy corn is one of the prolific waxy cornraces plants with a smaller ear size because there is a closecorrelation between ear number per plant and marketableyield [15] Since ear prolificacy is heritable and is controlled byadditive gene effects [16] improvement of prolificacy throughmass selection has been reported [15]

One of the keys to success in breeding programs issufficiently diverse germplasm Evaluation of germplasm isthe first step in population improvement to producepromising lines with favorable traits Previous evaluation ofgermplasm for phytochemical attributes has been conductednamely for lutein zeaxanthin and beta-cryptoxanthin [17] infield corn and anthocyanin content in purple waxy corn [18]Screening for both ear prolificacy and several fractions ofcarotenoids in small-ear waxy corn has not yet been reportedampis study aimed to explore the variability of ear prolificacytotal carotenoids lutein zeaxanthin and beta-caroteneamong yellow small-ear waxy corn accessions ampis infor-mation will be useful in a practical breeding program thatemphasizes increased carotenoid levels and prolificacy insmall-ear waxy corn

2 Materials and Methods

21 Plant Materials Forty-four small-ear waxy corn ac-cessions were used in this study (Table 1) ampese accessionspossess a wide range of kernel coloration from light yellow todark orange ampese materials originated from ampailand andother Asian countries and were collected by Chai Nat FieldCrops Research Center Department of Agriculture ampai-land and Plant Breeding Research Center for SustainableAgriculture of Khon Kaen University ampailand

22 Field Experiment Forty-four accessions and fourcommercial small-ear waxy corn varieties (Tein Leang KhonKaen Tien Lai-52 Tien Salabsai Tien Khaow) were sub-jected to randomized complete block design (RCBD) withthree replications under field conditions In total there were144 plots with a plot size of two rows that were 50m longwith a spacing of 08m between rows and 025m betweenplants to produce 40 plants per plot

ampese genotypes were evaluated at two locations and twoseasons in ampailand namely Uthai ampani Agricultural Re-search and Development Center Uthaiampani province (UT)(1524prime303Prime N 9942prime501Prime E 84m above sea level (masl))and Field Crop Research Station Khon Kaen UniversityKhon Kaen province (KK) (1628prime277Prime N 10248prime365Prime E190 masl) in the dry season 20182019 (rainfall rate 680mm(UT) and 985mm (KK) minimum air temperature 229 oC(UT) and 204 oC (KK) maximum air temperature 337 oC(UT) and 326 oC (KK) relative humidity 73 (UTand KK)and rainy season 2019 (rainfall rate 590mm (UT) and695mm (KK) minimum air temperature 256 oC (UT) and247 oC (KK) maximum air temperature 343 oC (UT) and336 oC (KK) relative humidity 752 (UT) and 766(KK)

Tillage was done three times with an additional appli-cation of 3125 tons per hectare organic manure Corn seedwas planted 2 seeds per hill and thinned to 1 plant per hill attwo weeks after sowing (WAS) Fertilizer formula 15-15-15of NPK was applied at the rate of 3125 kg per hectare beforeplanting and fertilizer formula 46-0-0 at the rate of 125 kgper hectare was applied at three WAS Corn ears weremanually harvested at the fresh stage approximately 20 daysafter pollination (DAP) for yields and further phytochem-icals analysis

23 Phenotypic Data Collection ampe following list of phe-notypic data was recorded for whole environment trials (twolocations and two seasons) such as anthesis day (days aftersowingDAS) as the number of days from sowing to the timewhen 50 of the plants in a plot shed pollen and ear height(cm) as the distance from ground level to the node bearingthe uppermost ear after milk stage Both plant and earheights were observed after the reproductive stage based onsample data of 10 random plants per plot

Ear yields and yield components were measured at themilk stage approximately 20 DAP ampe first DAP wascounted when silks were at least 1 cm emerged Ear yieldswere partitioned into first-emerged ear yield (ton haminus1)second-emerged ear yield (ton haminus1) and third-emergedear yield (ton haminus1) measured in both unhusked andhusked conditions Ear numbers were recorded separatelyfor 1st 2nd and 3rd ear emerged per plant and wereconverted to ears per hectare ampe total ear yield wasderived by adding the ear yield of each emerged ear (1st2nd and 3rd) Yield components were measured on huskedear diameter (cm) by HACHI digital caliper 0ndash150mmand husked ear length (cm) by Electronic Compact DigitalKitchen SF-400 A based on ten representative ears perplot

2 International Journal of Agronomy

24 Sample Preparation Extraction and PhytochemicalAnalysis Corn samples derived from a two-season trial atField Crop Research Station Khon Kaen University KhonKaen were extracted and analyzed Details of the analyticalprocedures are given hereinafter

241 Sample Preparation Ten ears of each plot weremanually harvested 20 DAP ampese samples were derivedfrom sibling hand pollination to ensure genetic purity Onlyundamaged kernels in the middle part of the ears were keptand were packed in a sealed plastic bag as a plot sampleampen the packed samples were dipped in liquid nitrogen for1min and were stored at minus20degC until the day of phyto-chemical analysis to avoid kernel oxidation and decay

242 Carotenoid Extraction Carotenoid extraction fol-lowed Schaub et al [19] with adjustments One gram of freshcorn kernels was crushed and loaded into a test tube ampen6mL ethanol and 1ndash2 flakes of 01 wv Butylated HydroxylToluene (BHT) were added before shaking with a vortexmixer Samples were heated in hot water at 85degC for 3minand then shaken and the process was repeated twice Sa-ponification was performed by adding 120 microL of 80 httpspubchemncbinlmnihgovcompoundpotassium_hydroxide KOH and shaking gently by handampen the samples were heated in hot water at 85degC for 5minand shaken and this procedure was repeated twice Sampleswere placed in ice for 5min and added 4mL of distilledwater Samples were then mixed thoroughly by the vortexmixer 3mL of petroleum ether (PE) diethyl ether (DE) ratio2 1 was added and shaken well until two layers were

separatedampe aqueous layer was loaded into a new test tubeampis solvent fractionation process was repeated twice andthe resulting layers were pooled After that the volume wasadjusted to 10mL with PE DE ampe carotenoid extract wasdivided equally into two parts for different purposes Onefraction was used to determine the total carotenoid con-centration of each genotype Absorbance was measured byspectrophotometer at 450 nm wavelength for determinationof total carotenoid concentration following this formula[20]

total carotenoid μggminus 1FW1113872 1113873 A times V times 104

2500 times W (1)

where A is the light absorbance V is the sample volume andW is the sample weight

ampe 2nd fraction was used to quantify the concentrationof lutein zeaxanthin and beta-carotene ampe extracts wereevaporated with a stream of nitrogen gas and covered withaluminum foil to prevent exposure to light ampen sampleswere stored at minus20degC until further analysis by high-per-formance liquid chromatography (HPLC)

243 Analysis of Carotenoids by HPLC-DAD Frozen ca-rotenoid extracts were dissolved with 1mL of methyl tert-butyl ether (MTBE) methanol (75 25) and passed through a045 microM filter 20 microL of the extracts was injected into HPLCaccording to Gupta et al [20] by using a YMC C30 column(5 microM 46times 250mM) coupled with C18 guard column ampeanalysis was performed on an HPLC system (ShimadzuKyoto Japan) equipped with LC-20AD pumps a SIL-20ACautosampler and an SPD-M20A photodiode array detector

Table 1 Small-ear waxy corn accessions used in this studyaNo Code Accessions bNo Code Accessions1 UT121001 Tein Si ampong 54-1-5 25 KKU-WX221041 No29(T38)2 UT121002 Tak Ngai H3(S)-15-B 26 KKU-WX221057 No31(T40)3 KKU-WX112085 CNW9702BC1(S)38--B-4-B 27 KKU-WX221059 No7(T41)4 KKU-WX112086 CNW9702BC1(S)40--B-1-B 28 KKU-WX211004 Mung Leang Phisanulok5 KKU-WX112087 GD49SKF2 29 KKU-WX211012 Tein Ayutaya6 KKU-WX112088 Tein Yellow (Wichai) (W64) 30 KKU-WX211013 Tein Leang Nakorn Sawan7 KKU-WX112089 Tein Yellow (Wichai) (W65) 31 KKU-WX211014 Tein Ban Kao8 KKU-WX112109 Tein Fancy (Wichai) 32 KKU-WX211015 Tein Leang Udon ampani9 KKU-WX122018 Miang Yang Gia Coi 33 KKU-WX211016 Tein Leang Phisanulok10 KKU-WX111028 Kaow Neaw Sri ampat Leang 34 KKU-WX211017 Saloi Nakhorn Phanom11 KKU-WX111036 Mu Ser 35 KKU-WX211018 Tein Nong Bhau Leang12 KKU-WX112092 GCKrabngoo Nakorn Sawan 36 KKU-WX211019 Tein Leang Tra Peon Yai13 KKU-WX211020 Tein Leang Nam Wang 37 KKU-WX221043 No30(T93)14 KKU-WX211021 Tein Nong Sam Rong Udon ampani 38 KKU-WX221036 GC Saleetien No11 Chiang Mai15 KKU-WX211022 Tein Rom Phan Nam Nan 39 KKU-WX221038 Tein NaKorn Sawan16 KKU-WX211023 Tein Sai Nam Pheong 40 KKU-WX221039 GCTeinluang Nakonsawan17 KKU-WX212001 Tein Leang Pean Maong 41 KKU-WX221040 GCTein No29 Tak18 KKU-WX221045 Tein Yellow (T13) 42 KKU-WX221042 GCTein No30 Tak19 KKU-WX221046 CNW9702BC1(S)36--B-1-B 43 KKU-WX221044 GCNipibangkaChachoengsao20 KKU-WX221047 CNW9702BC1(S)36--B-2-B 44 KKU-WX221054 GCTeinNo8 Chiang Mai21 KKU-WX221048 CNW9702BC1(S)36--B-5-B 45 Check1 Tein Leang Khon Kaen22 KKU-WX221049 CNW9702BC1(S)46--B-B 46 Check2 Tein Lai-5223 KKU-WX221052 Nimitr Bankla 47 Check3 Tein Salabsai24 KKU-WX221037 No11(T37) 48 Check4 Tein KhaowaAccession numbers 1 to 24 are on the left three columns bAccession numbers 25 to 48 are on the right three columns

International Journal of Agronomy 3

ampe carotenoids were separated at a flow rate of1mLminminus1 and with a gradient mobile phase elutioncomposed of (A) methanol deionized water (98 2 vv) (B)methanol deionized water (95 5 vv) and (C) MTBE ampegradient started at 0min with mobile phase A C (80 20)and the concentrations were changed linearly for twominutes to A C (60 40) with a flow rate of 140mLminminus1After 2min the flow rate was changed to 100mLminminus1followed by a change in concentration to B C (60 40) for12min then the concentration was changed to B (100) andafter 1min was changed to A C (60 40) for 7min ampeconcentration of 20 microL of samples was determined by cal-culating the area under the peaks of carotenoids comparedwith that of standard carotenoid solutions at 25degC [21]

244 Preparation of Standard Carotenoids Stock standardsolutions of lutein (CaroteNature Switzerland) zeaxanthin(CaroteNature Switzerland) and beta-carotene (Sigma-Aldrich USA) were prepared with ethanol (HPLC grade)and then adjusted to a volume of 10mL Standard con-centrations were determined by measuring the absorbancewith the UVVis spectrum at 445 450 and 450 nm for luteinzeaxanthin and beta-carotene respectively ampus the con-centration was calculated by the BeerndashLambert Law [21]

A ε middot C middot I (2)

where C is the solution concentration (g mLminus1) A is theabsorbance value I is the distance of the light shiningthrough the sample (cm) (in this case I 1) and ε is theabsorption coefficient defined as A1

1 cm in which A11 cm of

lutein in ethanol is 2550 zeaxanthin in ethanol is 2480 andbeta-carotene in ethanol is 2592

245 Data Analysis ampe error variances of each environ-ment were used for homogeneity tests ampe combinedANOVA in RCBD [22] across two seasons and two locationswas conducted for flowering traits plant architecture yieldtraits and yield components Phytochemical attributes wereanalyzed by combined ANOVA in RCBD of two-season dataderived from Khon Kaen province trials only Broad-senseheritability estimates were calculated using the followingformula [23] by assuming genotype as a random effect andenvironment (location and season) as a fixed effect

VGE MSGE minus MSError

r

VG MSG minus MSError minus rVGE

re

VP VG + VError

h2bs

VG

VP

(3)

whereVGE is the variance of GtimesEMSGE is themean squaresof GtimesE MSError is the mean squares of error r is thenumber of replication VG is the genotypic variance MSG isthe mean squares of genotype e is the number of

environment VP is the phenotypic variance VError is thevariance of error and h2

bs is the broad-sense heritabilityampe least significant differences (LSDs) were used to

compare the genotypemeans at p 005 A dendrogramwasconstructed based on a two-way Wardrsquos cluster analysisderived from a data matrix of all observed traits among 48genotypes Pearsonrsquos linear correlation coefficients werecalculated to estimate the relationship between targetedtraits

Several statistical programs were used for data analysisDendrograms were constructed by JMP Pro software [24]Pearsonrsquos correlation analysis and both single and combinedANOVA were done using Statistix10 [25]

3 Results and Discussion

31 Variabilities in Yields Yield Components andCarotenoids ampe influence of environments (E) was sig-nificant (p 001) for all traits except unhusked yield (UY)husked yield (HY) and ear number at the first ear position(EN1) (Table 2) Genotypes (G) were significantly different(p 001) for all traits Interactions between G and E(GtimesE) were significant for all traits except for the number ofmarketable ears at the second ear position (EN2M) huskedear length (EL) and husked ear diameter (ED) Environmentexplained a large portion for EL (1728lowastlowast) anthesis date(AD) (594lowastlowast) ear height (EH) (41860lowastlowast) and total ca-rotenoids concentration (TC) (2435lowastlowast) Genotypes pre-dominantly contributed to a large portion for yield(110lowastlowastndash449lowastlowast) and some carotenoid fractions(25lowastlowastndash232lowastlowast) However GxE displayed low contributionfor UY and HY (15lowastlowastndash69lowastlowast) ear numbers (EN)(26lowastlowastndash100lowastlowast) and some carotenoid fractions(03lowastlowastndash72lowastlowast) Broad-sense heritability estimates of ob-served traits ranged from low (005) to high (098)

Genotype contributed to the largest proportion of totalphenotypic variation for most traits and often exceeded thecontribution of GtimesE interactions suggesting that totalyields EN and carotenoid content are selectable traits insmall-ear waxy corn ampis corroborates previous studies ongrain yield and carotenoids in maize [17] However whenthe variance of ear number described by GtimesE is greater thanall other terms in the model because the contribution of thegenotype to the total variance in EN was reduced the effectof E was increased In this study heritability estimates of ENwere relatively low Even though the expression of earprolificacy is qualitative the inheritance of this trait iscontrolled by multiple genes and is prone to environmentaleffect [16] For instance the contribution of genotype to thetotal variance in EN was reduced while the effect of E wasincreased A previous study in semiprolific corn reportedthat environmental conditions could determine the size andthe number of second ears that were responsible for anyincreased yields [26] Yields and EN are quantitative traitsand have a polygenic inheritance controlled by multipleminor genes amperefore minimizing experimental errorunder normal conditions with careful attention to selectionwill help breeders to identify true-to-type genotypes withhigh yield and prolific ears Otherwise the phenotypic value


of a genotype will be overestimated or underestimatedmisleading breeders in germplasm selection [27]

In this study both total carotenoids and the fractionslutein (L) zeaxanthin (Z) and beta-carotene (BC) hadminor environmental effects and carotenoid fractionsshowed small but significant G timesE interactions ampisfinding corroborated previous reports on carotenoidcontent among tropical-adapted yellow maize inbreds[28] and F1 maize hybrids [4] ampus most tested geno-types could maintain the level of carotenoids acrossseasons in typical tropical conditions of ampailand Maizegrowers selected the best ears to maintain the germplasmampis finding is helpful for increasing variation of carot-enoid germplasm it makes it beneficial and easier forgenotype improvement for both maize growers andbreeders Since small-ear waxy corn is a cash cropgrowers can optimize the cropping index up to threetimes per year Likewise the low G timesE is desirable forbreeders as they can carry out routine selections up tothree cycles per year to obtain earlier high-carotenoidmaize inbreds In addition genotype was the main sourceof variation in total carotenoids (TC) L and Z ampe resultof our studies corroborated a previous investigation onsimilar traits in provitamin A biofortified maize [29]Both high genotypic variation and broad-sense herita-bility estimates in carotenoids suggest that these traits arehighly heritable and selection on these traits could beperformed at the early generation of population im-provement A comparative study revealed high herita-bility estimates (68 to 80) for TC L and Z in Chinesemaize germplasm [30] ampus phenotypic selection forthese attributes seems feasible

32 Alteration of Prolificacy Total Yields and CarotenoidsAffected by Different Environments ampe tested corn acces-sions showed the highest prolificacy regarding ear number atthe first position (EN1) ear number at the second position(EN2) marketable ear number at the second position(EN2M) and total ear number (TEN) at Khon Kaen ex-perimental site in the rainy season (52858 32366 1956688387 ears haminus1 respectively) compared to other envi-ronments (Table 3) However a significant reduction of earnumber affected by different environments was noticed forEN2 (32366 to 7086 ears ha-1) and EN2M (19566 to 3943ears haminus1) leading to a reduction in TEN (88387 to 57007ears haminus1) In contrast different environments slightly al-tered both total unhusked yield (51 to 36 tons haminus1) andtotal husked yield (97 to 76 tons haminus1)

In this study season showed larger effects than the lo-cation in altering EN and UY because our accessions weremore prolific and had better yields in the rainy season testedeither in Khon Kaen (KK) or Uthai ampani (UT) In parallelour weather data showed that average relative humidity anddaily temperatures (both minimum and maximum) in therainy season were higher than those in the dry season Incomparison to a field corn study favorable growing con-ditions such as available soil moisture longer daily solarradiation and higher temperature without exceedingmaximum threshold could promote ear prolificacy and grainyield [31] Since heat will affect maize pollen developmentduring the period between ear differentiation and earemergence [32] these factors became more critical of seedproduction causing a grain filling lag phase [33] Further lessimpact of contrasting environments was noticed on bothEN1 and HY of the first ear ampese findings agree with a

Table 2 Mean squares for yields yield components agronomic traits and carotenoids of 48 small-ear waxy corn genotypes evaluated acrossfour environments in ampailand between 2018 and 2019

SOV Environment (E) Replication Genotype (G) GxE Error CV () h2bsDf 3 8 47 141 376

UY 1st 775lowast 156 110lowastlowast 15lowastlowast 05 188 061Total 992 364 143lowastlowast 22lowastlowast 07 176 059

HY 1st 563 361 362lowastlowast 49lowastlowast 23 217 053Total 1933 1012 449lowastlowast 69lowastlowast 30 197 051

EN

1st 22 60 14lowastlowast 88lowastlowast 36 117 0212nd 18lowastlowast 14 70lowastlowast 100lowastlowast 54 431 0242ndM 72lowastlowast 48 42lowastlowast 67 35 617 036Total 30lowastlowast 37 15lowastlowast 26lowastlowast 12 157 018

EL 1st 1728lowastlowast 08 180lowastlowast 16 13 89 0512nd 704lowastlowast 31 17lowastlowast 103 84 282 006

ED 1st 18lowastlowast 01 11lowastlowast 01 01 81 0452nd 15lowastlowast 01 10lowastlowast 06 06 260 005

AD 594lowastlowast 13 114lowastlowast 73lowastlowast 44 44 067EH 41860lowastlowast 1180 4207lowastlowast 140lowastlowast 903 105 079La 345lowastlowast 09 25lowastlowast 06lowastlowast 001 121 097Za 653lowastlowast 04 80lowastlowast 14lowastlowast 01 162 092BCa 40lowastlowast 00 00lowastlowast 03lowastlowast 00 112 098TCa 2435lowastlowast 90 232lowastlowast 72lowastlowast 09 193 075h2bs broad-sense heritability estimates UY (ton haminus1) unhusked yield HY (ton haminus1) husked yield EN (ear haminus1) ear number EL (cm) husked ear length

ED (cm) husked ear diameter AD (DAS) anthesis date EH (cm) ear height L (microg gminus1 FW) lutein Z (microg gminus1 FW) zeaxanthin BC (microg gminus1 FW) beta-carotene TC (microg gminus1 FW) total carotenoids 1st the first ear position 2nd the second ear position 2nd M the number of marketable ears at the second earposition aData derived from Khon Kaen province across two seasons lowast Significant at p 005 and lowastlowast significant at p 001 respectively


previous investigation in semiprolific corn [26] suggestingthat the first ear yield is relatively stable and any differencesin total yield are mainly due to the degree of development ofthe second ear

Although environmental effects contributed to a minorpercentage (less than 20) of the total variation in carot-enoids (Table 2) further analyses of this effect may beimportant Carotenoid content of grain produced in therainy season had significantly higher levels of all fractions ofcarotenoids (Figure 1) ampe fresh samples of all accessionsgrown in the rainy season had TC L Z and BC more thantwice those in the dry season (386 to 570 microg gminus1 FW 076 to143 microg gminus1 FW 132 to 224 microg gminus1 FW and 035 to059 microg gminus1 FW respectively) ampese results confirmed asimilar report in field corn [28] ampe rainy season provided amore suitable growing condition for plants compared to thedry season such as sufficient water sunlight temperatureand humidity facilitating increased photosynthesis ampusthe plants grown during the rainy season had more plastidsincluding chloroplasts and chromoplasts for carotenoidbiosynthesis than those grown during the dry season [34]

33 Cluster Analysis A dendrogram based on carotenoidconcentration (CT) TC L Z and BC classified 48 small-earwaxy corn genotypes into four major groups (AndashD) (Fig-ure 2) Group A was the smallest group consisting of fivegenotypes namely UT121001 KKU-WX112087 KKU-WX212001 KKU-WX211018 and KKU-WX221040 havingthe highest contents of TC L Z and BC (036ndash1811 microg gminus1

FW) (Table 4) Most of them had higher contents of Z andTC (278ndash1811 microg gminus1 FW) (Table 4) and they had highcontents of L and BC except for KKU-WX221040(037ndash308 microg gminus1 FW) (Table 4) demonstrating the inter-mediate value of BC While group B was the largest groupconsisting of 22 genotypes most maize genotypes com-prising this group had low to medium values of L Z BC and

TC and all members in this group excluding Check 4 hadthe lowest values of L Z BC and TC Among genotypescomprising this group Check 4 showed the lowest values ofL Z TC and a low value of BC Group C consisted of sixgenotypes and all members in this group had mediumvalues of L and TC a low to medium value of Z and amedium to high value of BC Group D contained 15 ge-notypes and all genotypes in this group showed a significantrange from low to high values of L ampese genotypes had amedium to high value of Z a low to medium value of BCand a medium value of TC

ampe carotenoid profiles of the top five corn genotypes(Figure 3) derived from the cluster analysis (Figure 2) arepresented in Table 4ampis result suggested that the utilizationof genotype clustering by considering other important traitsincluding prolific ear and yield could classify all accessionsinto four major groups ampe first group had a high yield andthe highest L (320 and 140 microg gminus1 FW) and Z (726 and288 microg gminus1 FW) concentrations were found in KKU-WX112087 In contrast it has a low number of prolific earsampe second group included UT121001 with the highest levelof L (458 and 258 microg gminus1 FW) and Z (490 and 278 microg gminus1

FW) but it possessed low yield and poor prolific ears ampethird group included KKU-WX212001 KKU-WX211018and KKU-WX221040 with high levels of L (090ndash308 microg gminus1

FW) and Z (231ndash503 microg gminus1 FW) and these accessions hadmoderate yield and prolific ears ampe last group included allcheck varieties with high yield and prolific ear but poor CTampe highest content was observed in KKU-WX112087 andKKU-WX212001 (503 and 360 microg gminus1 FW)

ampe relationships between TC and the carotenoidsfractions (L Z and BC) were revealed in this study (Table 5)L and Z were strongly correlated with TC (r 069 and 081respectively)ampus L and Z were twomajor factions of TC insmall-ear waxy corn genotypes In comparison to previousreports L contributed to 18 to 40 of the TC contentwhereas Z accounted for 40 to 75 of TC of dried kernels

Table 3 Overall genotype means for yields yield components and agronomic characters of 48 small-ear waxy corn genotypes in fourenvironments (location-season combinations) in ampailand between 2018 and 2019

Parameters KKR UTR KKD UTD

UY (tons haminus1) 1st 67 78 63 71Total 97 97 76 79

HY (tons haminus1) 1st 36ab 44a 30b 45aTotal 51a 54a 36b 50ab

EN (ears haminus1)

1st 52858a 53974a 45494b 52496a2nd 32366a 17485b 10981bc 7086c2ndM 19566a 9776b 5307bc 3943cTotal 88387a 72030b 57007bc 59640c

AD (day) 464c 498a 481b 451dEH (cm) 845c 941b 723d 1127a

EL (cm) 1st 126c 136b 115d 139a2nd 107a 108a 93b 105a

ED (cm) 1st 33c 34b 34b 35a2nd 30ab 31a 29b 28c

UY unhusked yield HY husked yield EN ear number AD anthesis date EH ear height EL husked ear length ED husked ear diameter 1ST thefirst ear position 2nd the second ear position 2ndM the number of the marketable ear at the second ear position KKR at Khon Kaen in the rainy season2019 UTR at Uthai ampani in the rainy season 2019 KKD at Khon Kaen in the dry season 20182019 UTD at Uthai ampani in the dry season 20182019Means followed by different letters within the same row are significantly different based on LSD at p 005


among field corn inbreds and hybrids [28 35] ampis studyrevealed that L and Z were also major carotenoid fractions inimmature kernels BC did not significantly relate to TC(r 021) BC is one of the intermediates (provitamin A)whereas L and Z are the terminal product (non-provitaminA) in the carotenoid biosynthesis pathway [36] Positivehigh linear correlations between TC and the two fractions (Land Z) are desired since any increase in the fractions wouldincrease the total carotenoid concentration TC could beused for indirect selection in breeding programs for non-provitamin A carotenoids (L and Z)

Positive close linear correlations between TC and thetwo fractions L and Z are desirable since any increase in thefractions would increase the TC content TC could be usedfor indirect selection in breeding programs to non-pro-vitamin A carotenoids (L and Z) ampe use of a spectro-photometer to quantify TC is relatively cost- and time-saving compared to the use of HPLC for carotenoidsprofiling

Direct value comparison of similar carotenoid fractionsof fresh yellow waxy corn is unavailable However thesevalues were still lower than those reported in dried kernels ofprovitamin A biofortified maize hybrids [35] and tropical-

adapted yellow maize inbred lines [28] ampese differences areunderstandable because mature kernels experience an in-crease in the accumulation of dry matter and a decrease inmoisture contentampe concentrations of L Z and BC in fieldcorn and sweet corn were increased significantly as theharvesting time was delayed from the milking stage tophysiological maturity [37 38]

Linear correlation analysis among fourteen traits ispresented in Figure 4ampe EN1 showed positive and mediumcorrelation coefficients with the EN2 (r 052) and the EN2M(r 048) ampe EN2M was closely correlated with the TEN(r 095) ampe TEN had medium to high correlation coef-ficients with TUY (r 067) and THY (r 067) Hence earnumber per plant had a moderate correlation with totalyields as some accession had a large size of the first earwhich is larger than the second ear as it is sometimesnecessary to harvest only the first ear However some ac-cession had the same size for the first and second ears bothtraits moderate to high compared to other accessions ampusit is reliable to assign prolific ear traits as indirect selection tototal yields Prolificacy illustrates the reproductive plasticityof semiprolific ears in response to different environmentsand plant densities [39]

143a

076b

Rainy Dry

Lute

in (micro

gmiddotgndash1

FW)

00

20

40

60

(a)

Zeax

anth

in (micro

gmiddotgndash1

FW)

224a

132b

Rainy Dry00

20

40

60

(b)

059a 035b

Rainy Dry

Beta

-car

oten

e (microg

middotgndash1

FW)

00

20

40

60

(c)

Tota

l car

oten

oids

(microgmiddot

gndash1 FW

)

570a

386b

Rainy Dry00

20

40

60

(d)

Figure 1 Seasonal effect on the grand means of 48 small-ear waxy corn genotypes for (a) lutein content (b) zeaxanthin content (c) beta-carotene content and (d) total carotenoids content evaluated in the dry season 20182019 and rainy season 2019 in Khon Kaen Meansfollowed by different letters across bars are significantly different based on LSD at p 005


ampe intermediate relationship between EN1 versus EN2

and EN2M indicates that the capacity for prolific ears wasunder the simple genetic control of few genes among in-dividually tested corn accessions All tested accessions aremostly local-adapted races which are open-pollinated duringtheir successive generations ampis study demonstrates thatprolificacy is indirectly connected with total grain yieldPrevious studies of the use of ear prolificacy as selectioncriteria to improve total yield have been conducted in fieldcorn [40] and small-ear waxy corn [15]

34 Implications for Small-Ear Waxy Corn GermplasmAssessment Cluster analysis based on multiple carotenoidprofiles of the diverse yellow-colored-kernel waxy cornaccessions showed that only a few genotypes have com-plementary higher values of TC L Z and BC while othergenotypes contained poor to low values of these carotenoidfractions As shown in Figure 3 the selected genotypes werenot as orange as either field corn or sweet corn [38] Sincetropical corn contains less BC than temperate corn [41] thefive selected accessions from this study could be

A

B

C

D

L Z BC TC

L

00565

UT121001KKU-WX112087KKU-WX212001KKU-WX211018KKU-WX221040UT121002KKU-WX211004KKU-WX221036KKU-WX112089

KKU-WX122018KKU-WX111028KKU-WX112088KKU-WX211022KKU-WX221052KKU-WX221041KKU-WX221059KKU-WX221042

Check 2

KKU-WX211012KKU-WX211019KKU-WX111036KKU-WX211016KKU-WX112109KKU-WX221054KKU-WX112092

Check 3


Check 4

KKU-WX221045KKU-WX221039KKU-WX221047KKU-WX211014KKU-WX211021KKU-WX221046KKU-WX221048KKU-WX221043KKU-WX221049KKU-WX221044KKU-WX221057KKU-WX221013KKU-WX221038

Check 1

02662

04758

06854

0895

11046

15996

20946

25895

30845

35794

00358

03903

07448

10993

14538

18084

24607

3113

37653

44176

50699

Z

B03638

03849

04059

0427

04481

04691

05385

0608

06774

07468

08162

08935

16703

24471

32239

40007

47774

62312

7685

91388

10593

12046

TC

Figure 2 Dendogram of phenotypic relationships among 48 small-ear waxy corn genotypes Four main clusters (AndashD) were formed Two-way Wardrsquos cluster analysis based on lutein (L) zeaxanthin (Z) beta-carotene (BC) and total carotenoids (TC)


intercrossed with temperate corn germplasm that has highlevels of carotenoids Progress for increasing carotenoidcontent could be rapid as indicated by the low environ-ment and GtimesE effects revealed in this study high heri-tability estimates [30] and predominant additive geneticeffects [14]

Although biofortification is primarily directed to thebreeding of staple food crops to increase specific nutrientlevels [42] other important keys to success are (i) developingprofitable biofortified varieties and (ii) acceptable to farmersand consumers [43] Developing biofortified small-ear waxy

corn with two marketable ears is an indirect approach toachieve high yielding profitable varieties that fall withinfarmer preferences whereas better cooking and eatingqualities will meet consumer preferences amperefore one ofthe future research directions is performing blind sensorytests to assess consumer preferences such as aromastickiness tenderness and overall-liking We expect that thefuture biofortified orange small-ear waxy corn will be notonly superior at providing carotenoids for daily humanintake but also profitable to farmers and palatable toconsumers

Table 4ampe carotenoid profiles of top five small-ear waxy corn genotypes based on cluster analysis and four check varieties in two differentseasons

GenotypeRainy (microg gminus1 FW) Dry (microg gminus1 FW)

L Z BC TC L Z BC TCTop 5UT121001 458a 490c 119a 1137b 258a 278bc 044b 903aKKU-WX112087 320c 726a 066cndashe 1811a 140 cd 288b 036dndashk 598bcKKU-WX212001 190gh 503b 074bc 765dndashh 151c 360a 043b 809aKKU-WX211018 249de 389de 063dndashg 961bc 122ef 278bc 037cndashj 545cndasheKKU-WX221040 308c 446cd 055fndasho 894 cd 090gh 231ef 037cndashj 543cndashfCheck VarietiesCheck 1 192g 239jndashn 052hndashr 566i-o 108f 283bc 043bc 556bndashdCheck 2 115kl 113sndashv 052hndashr 388ondashr 055nndashq 053pndasht 037cndashj 289pndashvCheck 3 094lndashp 293gndashj 061dndashh 566indasho 044qndashs 072lndashq 033gndashn 244tndashwCheck 4 006 t 002y 048lndashr 101t 005u 005x 035endashl 078yBoth tested accessions and check varieties were ranked on the basis of the overall content of carotenoids fractions L Lutein ZZeaxanthin BCBeta-carotene TCTotal carotenoids FW fresh weight Means followed by different letters within the same column are significantly different based on LSD atple 005

(a) (b) (c) (d) (e)

Figure 3 Appearances of both unhusked and husked corn ears of top five genotypes with a high level of lutein zeaxanthin beta-caroteneand total carotenoids content (a) UT121001 (b) KKU-WX112087 (c) KKU-WX212001 (d) KKU-WX211018 and (e) KKU-WX221040

Table 5 Pearsonrsquos correlation coefficients between total carotenoids and carotenoids fractions of small-ear waxy corn genotypes

Lutein Zeaxanthin Beta-carotene (ns)Zeaxanthin 039lowastBeta-carotene 003ns 011ns

Total carotenoids 069lowastlowast 081lowastlowast 021ns not significant lowastsignificant at ple 005 lowastlowastsignificant at ple 001


4 Conclusions

Forty-eight accessions of small-ear waxy corn could beclustered into four major groups regarding the similarity ofmultiple carotenoids profiles ampree selected accessionsnamely UT121001 KKU-WX112087 and KKU-WX212001 had higher values of total carotenoids luteinzeaxanthin and beta-carotene than those of all check va-rieties ampe strategies for small-ear waxy corn selectionimproved for carotenoids and prolific ears are comprised ofthree steps namely (i) performing visual selection twicefor prolificacy at the early silking stage and during grainfilling in the field (ii) conducting visual selection in thefield against white to pale yellow corn kernels and (iii)assigning total carotenoids as an indirect selection toimprove non-provitamin A carotenoid concentrationssuch as lutein and zeaxanthin

Data Availability

All data analyzed to support the findings of this research areincluded in this research article

Disclosure

ampe funding sponsors had no role in the design of the studyin the collection analyses or interpretation of data in thewriting of the manuscript and in the decision to publish theresults

Conflicts of Interest

ampe authors declare no conflicts of interest

Acknowledgments

ampe research was financially supported by the ampailandResearch Fund through the Royal Golden Jubilee PhDProgram (Grant no PHD02142560) and the Senior Re-search Scholar Project of Prof Dr Sanun Jogloy (Project noRTA6180002) ampis research was supported in part by theUS Department of Agriculture Agricultural ResearchService USDA is an equal opportunity employer Mentionof trade names or commercial products in this report issolely for the purpose of providing specific information anddoes not imply recommendation or endorsement by the USDepartment of Agriculture ampe authors would like to thankthe National Science and Technology Development Agency(NSTDA) ampailand (Grant no P-17-51695) Uthai ampaniAgricultural Research and Development Center Uthaiampani Department of Agriculture (DOA) ampailand and thePlant Breeding Research Center for Sustainable AgricultureFaculty of Agriculture Khon Kaen University ampailand forproviding plant materials and research facilitiesampe authorsare thankful to Mr Abil Dermail for proofreading andcritical discussion

References

[1] G R Dagenais R Marchioli G Tognoni and S Yusuf ldquoBeta-carotene vitamin C and vitamin E and cardiovascular diseasesrdquoCurrent Cardiology Reports vol 2 no 4 pp 293ndash299 2000

[2] H N Basu A J D Vecchio F Flider and F T OrthoeterldquoNutritional and potential disease prevention properties ofcarotenoidsrdquo Journal of the American Oil Chemistsrsquo Societyvol 78 no 7 pp 665ndash675 2001 httpswwwscimagojrcomjournalsearchphpq=25881amptip=sidvol

EN2 052lowastlowast

EN2M 048lowastlowast 096lowastlowast

TEN 073lowastlowast 095lowastlowast 090lowastlowast

UY1 045lowastlowast 017lowast 015ns 026lowastlowast

TUY 062lowastlowast 062lowastlowast 060lowastlowast 067lowastlowast 085lowastlowast

HY1 045lowastlowast 013ns 010ns 022lowast 085lowastlowast 071lowastlowast

THY 064lowastlowast 061lowastlowast 059lowastlowast 067lowastlowast 076lowastlowast 091lowastlowast 084lowastlowast

AD ndash012ns 003ns 007ns ndash004ns 034lowastlowast 028lowastlowast 039lowastlowast 033lowastlowast

EH 009ns 037lowastlowast 039lowastlowast 030lowastlowast 053lowastlowast 060lowastlowast 053lowastlowast 062lowastlowast 073lowastlowast

EL1 ndash002ns ndash001ns 007ns ndash004ns 054lowastlowast 042lowastlowast 052lowastlowast 041lowastlowast 060lowastlowast 061lowastlowast

EL2 021lowast 039lowastlowast 042lowastlowast 035lowastlowast 032lowastlowast 046lowastlowast 028lowastlowast 043lowastlowast 042lowastlowast 047lowastlowast 031lowastlowast

ED1 ndash004ns ndash028ns ndash030ns ndash025ns 058lowastlowast 031lowastlowast 058lowastlowast 030lowastlowast 039lowastlowast 039lowastlowast 041lowastlowast 002ns

ED2 015ns 022lowast 018lowast 022lowast 039lowastlowast 041lowastlowast 032lowastlowast 036lowastlowast 033lowastlowast 039lowastlowast 018lowast 071lowastlowast 032ns

Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1

Figure 4 Triangular heat map representing Pearsonrsquos correlation coefficients between yields yield components and agronomic charactersof small-ear waxy corn genotypes EN1 ear number at the first ear position EN2 ear number at the second ear positionEN2Mmarketable ear number at the second ear position TEN total of ear number UY1 unhusked yield of the first ear positionTUY total of unhusked yield HY1 husked yield of the first ear position THY total of husked yield AD anthesis date EH ear heightEL1 husked ear length of the first ear position EL2 husked ear length of the second ear position ED1 husked ear diameter of the first earposition ED2 husked ear diameter of the second ear position ns not significant lowast and lowastlowast significant at p 005 and p 001respectively


[3] P Palozza and N I Krinsky ldquoAntioxidant effects of carot-enoids in vivo and in vitro an overviewrdquo Methods in En-zymology vol 213 pp 403ndash420 1992

[4] C O Egesel J C Wong R J Lambert and T R RochefordldquoCombining ability of maize inbreds for carotenoids andtocopherolsrdquo Crop Science vol 43 no 3 pp 818ndash823 2003

[5] G J Handelman E A Dratz C C Reay and F J G M vanKujik ldquoCarotenoids in the human macula and whole retinardquoInvestigative Ophthalmology amp Visual Science vol 29pp 850ndash855 1988

[6] H Gerster ldquoPotential role of beta-carotene in the preventionof cardiovascular diseaserdquo International journal for vitaminand nutrition research Internationale Zeitschrift Fur Vitamin-und Ernahrungsforschung Journal international de vitami-nologie et de nutrition vol 61 no 4 pp 277ndash291 1991

[7] B Buckner P S Miguel D Janick-Buckner andJ L Bennetzen ldquoampe y1 gene of maize codes for phytoenesynthaserdquo Genetics vol 143 pp 479ndash488 1996

[8] K Lertrat and N ampongnarin ldquoNovel approach to eatingquality improvement in local waxy corn improvement ofsweet taste in local waxy corn variety with mixed kernels fromsuper sweet cornrdquo Acta Horticulturae vol 769 no 769pp 145ndash150 2008

[9] V Fergason ldquoHigh amylose and waxy cornsrdquo in SpecialtyCorns A R Hallauer Ed pp 71ndash92 CRC Press New YorkNY USA 2nd edition 2001

[10] Q-p Hu and J-g Xu ldquoProfiles of carotenoids anthocyaninsphenolics and antioxidant activity of selected color waxy corngrains during maturationrdquo Journal of Agricultural and FoodChemistry vol 59 no 5 pp 2026ndash2033 2011

[11] K Chandler A E Lipka B F Owens et al ldquoGenetic analysisof visually scored orange kernel color in maizerdquo Crop Sciencevol 53 no 1 pp 189ndash200 2013

[12] A Junpatiw K Lertrat K Lomthaisong andR Tangwongchai ldquoEffects of steaming boiling and frozenstorage on carotenoid contents of various sweet corn culti-varsrdquo International Food Research Journal vol 20pp 2219ndash2225 2013

[13] K Pillay J Derera M Siwela and F Veldman ldquoConsumeracceptance of yellow provitamin A-biofortified maize inKwaZulu-Natalrdquo South African Journal of Clinical Nutritionvol 24 no 4 pp 186ndash191 2011

[14] A D Halilu S G Ado D A Aba and I S Usman ldquoGeneticsof carotenoids for provitamin A biofortification in tropical-adapted maizerdquo e Crop Journal vol 4 no 4 pp 313ndash3222016

[15] P Kesornkeaw K Lertrat and B Suriharn ldquoResponse to fourcycles of mass selection for prolificacy at low and highpopulation densities in small ear waxy cornrdquo Asian Journal ofPlant Sciences vol 8 no 6 pp 425ndash432 2009

[16] M E Sorrells J H Lonnquist and R E Harris ldquoInheritanceof prolificacy in maize 1rdquo Crop Science vol 19 no 3pp 301ndash306 1979

[17] C T Senete P E d O Guimaratildees M C D Paes andJ C d Souza ldquoDiallel analysis of maize inbred lines forcarotenoids and grain yieldrdquo Euphytica vol 182 no 3pp 395ndash404 2011

[18] B Harakotr B Suriharn M P Scott and K LertratldquoGenotypic variability in anthocyanins total phenolics andantioxidant activity among diverse waxy corn germplasmrdquoEuphytica vol 203 no 2 pp 237ndash248 2015

[19] P Schaub P Beyer S Islam and T Rocheford ldquoMaize quickcarotenoid extraction protocolrdquo 2004 httpwwwcropsci

illinoisedufacultyrochefordquickcarotenoid_analysis20_protocolpdf

[20] P Gupta Y Sreelakshmi and R Sharma ldquoA rapid andsensitive method for determination of carotenoids in planttissues by high performance liquid chromatographyrdquo PlantMethods vol 11 pp 1ndash5 2015

[21] D B Rodriguez-Amaya and M Kimura ldquoHarvest plushandbook for carotenoids analysisrdquo in HarvestPlus TechnicalMonographs IFPRI and CIATInternational Food Policy Re-search Institute Washington DC USA 2004

[22] K A Gomez and A A Gomez Statistical Procedure forAgricultural Research p 680 JohnWiley and Sons Singapore1984

[23] R Bernardo Breeding for Quantitative Traits in Plantspp 175ndash202 Stemma Press Woodbury MN USA 2020

[24] SAS Institute Inc JMP Trial 1500 (403428) SAS InstituteInc 2019 httpwwwjmpcom

[25] Statistix 10 Analytical Software Tallahassee FL USA 2013httpstatistixcom

[26] L F Bauman ldquoRelative yields of first (apical) and second earsof semi-prolific southern corn hybrids 1rdquo Agronomy Journalvol 52 no 4 pp 220ndash222 1960

[27] J Derera P Tongoona B S Vivek and M D Laing ldquoGeneaction controlling grain yield and secondary traits in southernAfrican maize hybrids under drought and non-drought en-vironmentsrdquo Euphytica vol 162 no 3 pp 411ndash422 2008

[28] A Menkir W Liu W S White B Maziya-Dixon andT Rocheford ldquoCarotenoid diversity in tropical-adapted yel-low maize inbred linesrdquo Food Chemistry vol 109 no 3pp 521ndash529 2008

[29] T Muzhingi N Palacios-Rojas A Miranda M L CabreraK-J Yeum and G Tang ldquoGenetic variation of carotenoidsvitamin E and phenolic compounds in provitamin A bio-fortifiedmaizerdquo Journal of the Science of Food and Agriculturevol 97 no 3 pp 793ndash801 2017

[30] S Chander Y Q Guo X H Yang et al ldquoUsing molecularmarkers to identify two major loci controlling carotenoidcontents in maize grainrdquo eoretical and Applied Geneticsvol 116 no 2 pp 223ndash233 2008

[31] M E Otegui M G Nicolini R A Ruiz and P A DoddsldquoSowing date effects on grain yield components for differentmaize genotypesrdquo Agronomy Journal vol 87 no 1 pp 29ndash331995

[32] L M Smith ldquoampe heat is on maize pollen development after aheat waverdquo Plant Physiology vol 181 no 2 pp 387-388 2019

[33] A R Hallauer and A F Troyer ldquoProlific corn hybrids andminimizing risk of stressrdquo in Proceeding of Annual Corn ampSorghum Research Conference 27th ASTA pp 140ndash158Washington DC USA 1972

[34] T Sun H Yuan H Cao M Yazdani Y Tadmor and L LildquoCarotenoid metabolism in plants the role of plastidsrdquoMolecular Plant vol 11 no 1 pp 58ndash74 2017

[35] W B Suwarno K V Pixley N Palacios-RojasS M Kaeppler and R Babu ldquoFormation of heterotic groupsand understanding genetic effects in a provitamin A bio-fortified maize breeding programrdquo Crop Science vol 54 no 1pp 14ndash24 2014

[36] R Vallabhaneni C E Gallagher N LicciardelloA J Cuttriss R F Quinlan and E T Wurtzel ldquoMetabolitesorting of a germplasm collection reveals the hydroxylase 3locus as a new target for maize provitamin a biofortificationrdquoPlant Physiology vol 151 no 3 pp 1635ndash1645 2009

[37] J-G Xu Q-P Hu X-D Wang J-Y Luo Y Liu andC-R Tian ldquoChanges in the main nutrients phytochemicals


and antioxidant activity in yellow corn grain during matu-rationrdquo Journal of Agricultural and Food Chemistry vol 58no 9 pp 5751ndash5756 2010

[38] T J OrsquoHare K J Fanning and I F Martin ldquoZeaxanthinbiofortification of sweet-corn and factors affecting zeaxanthinaccumulation and colour changerdquo Archives of Biochemistryand Biophysics vol 572 pp 184ndash187 2015

[39] J I Sarquıs H Gonzalez and J R Dunlap ldquoYield response oftwo cycles of selection from a semiprolific early maize (Zeamays L) population to plant density sucrose infusion andpollination controlrdquo Field Crops Research vol 55 no 1-2pp 109ndash116 1998

[40] N Leon and J G Coors ldquoTwenty-four cycles of mass selectionfor prolificacy in the golden glow maize populationrdquo CropScience vol 42 no 2 pp 325ndash333 2002

[41] K Pixley N P Rojas R Babu R Mutale R Surles andE Simpungwe ldquoBiofortification of maize with provitamin Acarotenoidsrdquo in Carotenoids and Human Health Nutritionand Health S A Tanumihardjo Ed pp 271ndash292 SpringerScience + Business Media New York NY USA 2013

[42] W H Pfeiffer and B McClafferty ldquoHarvest plus breedingcrops for better nutritionrdquo Crop Science vol 47 pp Sndash882007

[43] H E Bouis and R M Welch ldquoBiofortification-A sustainableagricultural strategy for reducing micronutrient malnutritionin the global Southrdquo Crop Science vol 50 pp Sndash20 2010


Waxy corn (Zea mays L var ceratina) is popular and iscommonly consumed in most Asian countries particularlyChina Korea Laos Myanmar ampailand and Vietnam [8]In Northeastern ampailand people enjoy cooked fresh waxycorn as an alternative staple food to rice Its unique textureand sticky kernels due to high amylopectin content [9]appeal to consumers ampe kernel coloration of fresh waxycorn is normally either white or bicolor purple and whitewith a poor expression of carotenoids including lutein andzeaxanthin [10] Since the presence of orange corn kernelspositively correlates with higher carotenoid concentrationsand is a heritable trait [11] exploring corn accessions thatpossess a significant content of carotenoids is possibleMoreover promoting small-ear waxy corn as a biofortifiedcrop is promising because the carotenoid contents especiallylutein and zeaxanthin are increased after steaming [12] andconsumer acceptability of provitamin A biofortifiedmaize inSouth Africa is high [13] ampe content of carotenoids isquantitative traits controlled by a few major genes [14]While additive gene effects are important for lutein zeax-anthin and beta-cryptoxanthin nonadditive effects arepredominant for beta-carotene and provitamin A [14]

Inampailand waxy corn is consumed fresh and commonlypaid for on a per ear basis instead of the kernel weight ampuswaxy corn with prolific ears will benefit growers by boostingtheir incomes Criteria of prolificacy are at least two mar-ketable ears per plant and short period for ear emergence ofboth ears Small-ear waxy corn is one of the prolific waxy cornraces plants with a smaller ear size because there is a closecorrelation between ear number per plant and marketableyield [15] Since ear prolificacy is heritable and is controlled byadditive gene effects [16] improvement of prolificacy throughmass selection has been reported [15]

One of the keys to success in breeding programs issufficiently diverse germplasm Evaluation of germplasm isthe first step in population improvement to producepromising lines with favorable traits Previous evaluation ofgermplasm for phytochemical attributes has been conductednamely for lutein zeaxanthin and beta-cryptoxanthin [17] infield corn and anthocyanin content in purple waxy corn [18]Screening for both ear prolificacy and several fractions ofcarotenoids in small-ear waxy corn has not yet been reportedampis study aimed to explore the variability of ear prolificacytotal carotenoids lutein zeaxanthin and beta-caroteneamong yellow small-ear waxy corn accessions ampis infor-mation will be useful in a practical breeding program thatemphasizes increased carotenoid levels and prolificacy insmall-ear waxy corn

2 Materials and Methods

21 Plant Materials Forty-four small-ear waxy corn ac-cessions were used in this study (Table 1) ampese accessionspossess a wide range of kernel coloration from light yellow todark orange ampese materials originated from ampailand andother Asian countries and were collected by Chai Nat FieldCrops Research Center Department of Agriculture ampai-land and Plant Breeding Research Center for SustainableAgriculture of Khon Kaen University ampailand

22 Field Experiment Forty-four accessions and fourcommercial small-ear waxy corn varieties (Tein Leang KhonKaen Tien Lai-52 Tien Salabsai Tien Khaow) were sub-jected to randomized complete block design (RCBD) withthree replications under field conditions In total there were144 plots with a plot size of two rows that were 50m longwith a spacing of 08m between rows and 025m betweenplants to produce 40 plants per plot

ampese genotypes were evaluated at two locations and twoseasons in ampailand namely Uthai ampani Agricultural Re-search and Development Center Uthaiampani province (UT)(1524prime303Prime N 9942prime501Prime E 84m above sea level (masl))and Field Crop Research Station Khon Kaen UniversityKhon Kaen province (KK) (1628prime277Prime N 10248prime365Prime E190 masl) in the dry season 20182019 (rainfall rate 680mm(UT) and 985mm (KK) minimum air temperature 229 oC(UT) and 204 oC (KK) maximum air temperature 337 oC(UT) and 326 oC (KK) relative humidity 73 (UTand KK)and rainy season 2019 (rainfall rate 590mm (UT) and695mm (KK) minimum air temperature 256 oC (UT) and247 oC (KK) maximum air temperature 343 oC (UT) and336 oC (KK) relative humidity 752 (UT) and 766(KK)

Tillage was done three times with an additional appli-cation of 3125 tons per hectare organic manure Corn seedwas planted 2 seeds per hill and thinned to 1 plant per hill attwo weeks after sowing (WAS) Fertilizer formula 15-15-15of NPK was applied at the rate of 3125 kg per hectare beforeplanting and fertilizer formula 46-0-0 at the rate of 125 kgper hectare was applied at three WAS Corn ears weremanually harvested at the fresh stage approximately 20 daysafter pollination (DAP) for yields and further phytochem-icals analysis

23 Phenotypic Data Collection ampe following list of phe-notypic data was recorded for whole environment trials (twolocations and two seasons) such as anthesis day (days aftersowingDAS) as the number of days from sowing to the timewhen 50 of the plants in a plot shed pollen and ear height(cm) as the distance from ground level to the node bearingthe uppermost ear after milk stage Both plant and earheights were observed after the reproductive stage based onsample data of 10 random plants per plot

Ear yields and yield components were measured at themilk stage approximately 20 DAP ampe first DAP wascounted when silks were at least 1 cm emerged Ear yieldswere partitioned into first-emerged ear yield (ton haminus1)second-emerged ear yield (ton haminus1) and third-emergedear yield (ton haminus1) measured in both unhusked andhusked conditions Ear numbers were recorded separatelyfor 1st 2nd and 3rd ear emerged per plant and wereconverted to ears per hectare ampe total ear yield wasderived by adding the ear yield of each emerged ear (1st2nd and 3rd) Yield components were measured on huskedear diameter (cm) by HACHI digital caliper 0ndash150mmand husked ear length (cm) by Electronic Compact DigitalKitchen SF-400 A based on ten representative ears perplot







2500 times W (1)














r


re

VP VG + VError

h2bs

VG

VP

(3)


















EN




















EN (ears haminus1)












143a

076b

Rainy Dry

Lute

in (micro

gmiddotgndash1

FW)

00

20

40

60

(a)

Zeax

anth

in (micro

gmiddotgndash1

FW)

224a

132b

Rainy Dry00

20

40

60

(b)

059a 035b

Rainy Dry

Beta

-car

oten

e (microg

middotgndash1

FW)

00

20

40

60

(c)

Tota

l car

oten

oids

(microgmiddot

gndash1 FW

)

570a

386b

Rainy Dry00

20

40

60

(d)






A

B

C

D

L Z BC TC

L

00565



Check 2


Check 3


Check 4


Check 1

02662

04758

06854

0895

11046

15996

20946

25895

30845

35794

00358

03903

07448

10993

14538

18084

24607

3113

37653

44176

50699

Z

B03638

03849

04059

0427

04481

04691

05385

0608

06774

07468

08162

08935

16703

24471

32239

40007

47774

62312

7685

91388

10593

12046

TC









(a) (b) (c) (d) (e)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References



EN2 052lowastlowast













Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1





















































2500 times W (1)














r


re

VP VG + VError

h2bs

VG

VP

(3)


















EN




















EN (ears haminus1)












143a

076b

Rainy Dry

Lute

in (micro

gmiddotgndash1

FW)

00

20

40

60

(a)

Zeax

anth

in (micro

gmiddotgndash1

FW)

224a

132b

Rainy Dry00

20

40

60

(b)

059a 035b

Rainy Dry

Beta

-car

oten

e (microg

middotgndash1

FW)

00

20

40

60

(c)

Tota

l car

oten

oids

(microgmiddot

gndash1 FW

)

570a

386b

Rainy Dry00

20

40

60

(d)






A

B

C

D

L Z BC TC

L

00565



Check 2


Check 3


Check 4


Check 1

02662

04758

06854

0895

11046

15996

20946

25895

30845

35794

00358

03903

07448

10993

14538

18084

24607

3113

37653

44176

50699

Z

B03638

03849

04059

0427

04481

04691

05385

0608

06774

07468

08162

08935

16703

24471

32239

40007

47774

62312

7685

91388

10593

12046

TC









(a) (b) (c) (d) (e)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References



EN2 052lowastlowast













Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1
























































r


re

VP VG + VError

h2bs

VG

VP

(3)


















EN




















EN (ears haminus1)












143a

076b

Rainy Dry

Lute

in (micro

gmiddotgndash1

FW)

00

20

40

60

(a)

Zeax

anth

in (micro

gmiddotgndash1

FW)

224a

132b

Rainy Dry00

20

40

60

(b)

059a 035b

Rainy Dry

Beta

-car

oten

e (microg

middotgndash1

FW)

00

20

40

60

(c)

Tota

l car

oten

oids

(microgmiddot

gndash1 FW

)

570a

386b

Rainy Dry00

20

40

60

(d)






A

B

C

D

L Z BC TC

L

00565



Check 2


Check 3


Check 4


Check 1

02662

04758

06854

0895

11046

15996

20946

25895

30845

35794

00358

03903

07448

10993

14538

18084

24607

3113

37653

44176

50699

Z

B03638

03849

04059

0427

04481

04691

05385

0608

06774

07468

08162

08935

16703

24471

32239

40007

47774

62312

7685

91388

10593

12046

TC









(a) (b) (c) (d) (e)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References



EN2 052lowastlowast













Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1
























































EN




















EN (ears haminus1)












143a

076b

Rainy Dry

Lute

in (micro

gmiddotgndash1

FW)

00

20

40

60

(a)

Zeax

anth

in (micro

gmiddotgndash1

FW)

224a

132b

Rainy Dry00

20

40

60

(b)

059a 035b

Rainy Dry

Beta

-car

oten

e (microg

middotgndash1

FW)

00

20

40

60

(c)

Tota

l car

oten

oids

(microgmiddot

gndash1 FW

)

570a

386b

Rainy Dry00

20

40

60

(d)






A

B

C

D

L Z BC TC

L

00565



Check 2


Check 3


Check 4


Check 1

02662

04758

06854

0895

11046

15996

20946

25895

30845

35794

00358

03903

07448

10993

14538

18084

24607

3113

37653

44176

50699

Z

B03638

03849

04059

0427

04481

04691

05385

0608

06774

07468

08162

08935

16703

24471

32239

40007

47774

62312

7685

91388

10593

12046

TC









(a) (b) (c) (d) (e)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References



EN2 052lowastlowast













Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1





























































EN (ears haminus1)












143a

076b

Rainy Dry

Lute

in (micro

gmiddotgndash1

FW)

00

20

40

60

(a)

Zeax

anth

in (micro

gmiddotgndash1

FW)

224a

132b

Rainy Dry00

20

40

60

(b)

059a 035b

Rainy Dry

Beta

-car

oten

e (microg

middotgndash1

FW)

00

20

40

60

(c)

Tota

l car

oten

oids

(microgmiddot

gndash1 FW

)

570a

386b

Rainy Dry00

20

40

60

(d)






A

B

C

D

L Z BC TC

L

00565



Check 2


Check 3


Check 4


Check 1

02662

04758

06854

0895

11046

15996

20946

25895

30845

35794

00358

03903

07448

10993

14538

18084

24607

3113

37653

44176

50699

Z

B03638

03849

04059

0427

04481

04691

05385

0608

06774

07468

08162

08935

16703

24471

32239

40007

47774

62312

7685

91388

10593

12046

TC









(a) (b) (c) (d) (e)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References



EN2 052lowastlowast













Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1





















































143a

076b

Rainy Dry

Lute

in (micro

gmiddotgndash1

FW)

00

20

40

60

(a)

Zeax

anth

in (micro

gmiddotgndash1

FW)

224a

132b

Rainy Dry00

20

40

60

(b)

059a 035b

Rainy Dry

Beta

-car

oten

e (microg

middotgndash1

FW)

00

20

40

60

(c)

Tota

l car

oten

oids

(microgmiddot

gndash1 FW

)

570a

386b

Rainy Dry00

20

40

60

(d)






A

B

C

D

L Z BC TC

L

00565



Check 2


Check 3


Check 4


Check 1

02662

04758

06854

0895

11046

15996

20946

25895

30845

35794

00358

03903

07448

10993

14538

18084

24607

3113

37653

44176

50699

Z

B03638

03849

04059

0427

04481

04691

05385

0608

06774

07468

08162

08935

16703

24471

32239

40007

47774

62312

7685

91388

10593

12046

TC









(a) (b) (c) (d) (e)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References



EN2 052lowastlowast













Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1



















































A

B

C

D

L Z BC TC

L

00565



Check 2


Check 3


Check 4


Check 1

02662

04758

06854

0895

11046

15996

20946

25895

30845

35794

00358

03903

07448

10993

14538

18084

24607

3113

37653

44176

50699

Z

B03638

03849

04059

0427

04481

04691

05385

0608

06774

07468

08162

08935

16703

24471

32239

40007

47774

62312

7685

91388

10593

12046

TC









(a) (b) (c) (d) (e)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References



EN2 052lowastlowast













Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1






















































(a) (b) (c) (d) (e)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References



EN2 052lowastlowast













Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1
















































4 Conclusions


Data Availability


Disclosure




Acknowledgments


References



EN2 052lowastlowast













Mag

nitu

de o

f pea

rson

rsquos co

rrel

atio

n

09

05

0

ndash05

ndash09

EN1

EN2

EN2 M

TEN

UY1

TUY

HY1

THY

AD

EH

EL1

EL2

ED1
















































variabilityinprolificacy,totalcarotenoids,lutein,and … · 2020. 11. 19. · while additive...

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