Identification of SNP and InDel variations in the promoter and 5′untranslated regions of γ-tocopherol methyl transferase(ZmVTE4)affecting higher accumulation of α-tocopherol in maize kernel

Abhijit Kumar Das, Rashmi Chhabra, Vignesh Muthusamy, Hema Singh Chauhan,Rajkumar Uttamrao Zunjare, Firoz Hossain*

The Indian Council of Agricultural Research(ICAR)-Indian Agricultural Research Institute(IARI),New Delhi 110012,India

Keywords:Maize Vitamin-E InDel SNP Biofortification

A B S T R A C T Deficiency of vitamin-E or tocopherol causes neurological and cardiovascular disorders in humans. Though maize kernel is rich in total tocopherol, the level of α-tocopherol possessing the highest vitamin-E activity is low. Mutant allele of ZmVTE4 with deletion of 7 bp and 118 bp(0/0:most favorable haplotype)in 5′UTR and promoter region,respectively significantly enhances α-tocopherol in maize kernel than wild type haplotype (7/118). αtocopherol estimation in 15 diverse inbreds revealed that mean α-/γ-tocopherol and α-/total-tocopherol was much higher in genotypes with favorable haplotype (1.51 and 0.41)than unfavorable class(0.19 and 0.13),respectively.However even within favorable class,αtocopherol ranged from 4.76 to 30.07 μg g-1. Sequence analysis of part of 5′UTR and promoter of ZmVTE4 among the genotypes with favorable haplotype revealed 14 SNPs(SNP1 to SNP14)and eight InDels(InDel1 to InDel8).SNP7 at 606 bp(G to A),and InDels viz.,InDel1(27 bp), InDel4 (27 bp) and InDel8 (14 bp) differentiated low and high α-tocopherol accumulating inbreds with favorable haplotype.Hence the newly identified SNP and InDels in addition to the already reported InDels can be useful in selection of favorable genotypes with higher α-tocopherol in maize.

1.Introduction

Micronutrient deficiency severely affects human health leading to mental impairment,lower productivity and even death[1]. In children, effects of micronutrient deficiency are more acute and can lead to severe physical and cognitive consequences[2].Worldwide micronutrient deficiency affects nearly two billion people especially in the under-developed and developing countries. Among micronutrients, vitamin-E plays important role for human growth and metabolism [3].The recommended dietary allowance for adult is 15 mg day-1,whereas in children (0-6 month) the same is 4 mg day-1[4].The deficiency of vitamin-E leads to age-related macular degeneration, neurological disorders, cancer, cataracts,Alzheimer's-, cardiovascular-, and inflammatory-diseases[5]. It is estimated that 20% of the examined peoples both in developed and developing countries possess suboptimal level of vitamin-E [6]. Hence sufficient intake of vitamin-E is essential for efficient metabolism. Although various avenues such as food-fortification, medical-supplementation and dietary-diversification are available, genetic enrichment of food through biofortification is regarded as the most costeffective and sustainable solution to alleviate malnutrition[7].

Maize is a popular choice as food throughout the world[8].Maize kernels are rich in total tocopherol consisting α-,β-,γ-,and δ-isoforms. Though all fractions possess vitamin-E activity, α-tocopherol possesses the highest vitamin-E activity,and is preferentially absorbed by receptor transfer protein in human liver [9]. However, α-tocopherol constitutes only～20% of the total tocopherol [10], while γ-tocopherol having the low vitamin-E activity predominates with ～80% proportion. Thus enhancement of α-tocopherol would help in enhancing the vitamin-E activity in maize.

Several key genes/QTL of vitamin-E biosynthesis have been identified in maize [11-13], however two insertion/deletions(InDel7 and InDel118)within ZmVTE4 gene encoding γ-tocopherol methyl transferase significantly affect the level of αtocopherol [6]. InDel7 and InDel118 are located in 5′UTR and promoter region of ZmVTE4,respectively.Favorable haplotype(0/0: deletion at InDel7 and InDel118) increases α-tocopherol by 3.2 folds over unfavorable haplotype (7/118: insertion at InDel7 and InDel118). Though the favorable haplotype significantly enhances α-tocopherol,wide variation of α-tocopherol is observed among the inbreds possessing 0/0 haplotype [14].So far no attempt has been made to find out possible cause of genetic variation in α-tocopherol among the inbreds with favorable haplotype. The present study was therefore undertaken to (i) characterize the 5′UTR and promoter regions harbouring the 7 bp and 118 bp InDels in the ZmVTE4,and(ii)identify nucleotide polymorphism(s), if any, that can further differentiate the high- and low- α-tocopherol inbreds possessing the favorable haplotype of ZmVTE4.

2. Materials and methods

2.1. Plant materials

Fifteen inbreds from diverse sources were selected for the experiment (Table 1). Five genotypes (CML496, CML497,CML426, CML162, and HP465-41) from CIMMYT, Mexico;three inbreds (DQL785-1-8, DQL653-2-4, and DML420R) from ICAR-Indian Institute of Maize Research, Ludhiana; two inbreds each from ICAR-Vivekananda Parvatiya Krishi Anusandhan Sansthan, Almora (CM145 and V345PV) and Chaudhary Charan Singh Haryana Agricultural University,Uchani(HKI1344 and HKI1532);and one each from Acharya N.G. Ranga Agricultural University, Guntur (BQPML10-1-1);Punjab Agricultural University, Ludhiana (LM13); and ICARIndian Agricultural Research Institute (IARI), New Delhi(CM140Q) were selected for the present study. These inbreds were grown in rainy season of 2017 at ICAR-IARI, New Delhi,and 2-3 plants in each entry were self pollinated to avoid contamination by foreign pollen.

2.2. Genotyping for InDel7 and InDel118

Genomic DNA from young maize seedling was isolated using standard CTAB protocol [15]. Inbreds were characterized for ZmVTE4 haplotype using primers for InDel7 (F-5′-TGCCGGCACCTCTACTTTAT-3′/R-5′-AGGACTGGGAGCAATGGAG-3′) and InDel118 (F-5′-AAAGCACTTACATCATGGGAAAC-3′/R-5′-TTGG TGTAGCTCCGATTTGG-3′) as per Li et al. [6]. 20 μL reaction mixture comprising 1× of OnePCR ready-to-use PCR mix(GeneDireX), 100 ng of genomic DNA from each genotype and 0.25 μmol L-1each of forward and reverse primer) was used to carry out the PCR.Standard PCR protocol consisting of initial denaturation of 94 °C for 5 min,followed by 35 cycles of denaturation at 94 °C for 45 s,annealing at 60 °C for 45 s,and extension at 72 °C for 45 s and final extension at 72 °C for 10 min.Amplified PCR products for InDel7 and InDel118 were resolved on 4%MetaPhor Agarose(Lonza,Rockland,ME,USA)and 2%agarose gel,respectively.

Table 1-Pedigree details,haplotype and tocopherol of inbreds used in the study.

2.3. Estimation of α-tocopherol

To avoid degradation of tocopherol, seeds from self-fertilized cob of each genotype were harvested and stored at 4 °C till the period of extraction. Kernel tocopherols were extracted following the protocol of Saha et al. [16]; however absolute ethanol was used instead of methanol.Fluorescence detector of dionex ultra-high performance liquid chromatography(UHPLC) system was used with an excitation wavelength of 290 nm and an emission wavelength of 325 nm to detect the peak.Injected sample volume was 20 μL and YMC C30 column(5 μm,4.6 mm× 250 mm;Waters Chromatography)was used.Methanol: TBME (tert-butyl methyl ether) in a ratio of 95:5 (v/v) was used as mobile phase with a flow rate of 1 mL min-1.Concentration of α-tocopherols in each sample was estimated from standard curve prepared using standard of each of the components provided by Sigma-Aldrich Co., St. Louis, MO,USA.

2.4. Phylogenetic analysis and discovery of polymorphism

Position of InDel7 is within the 5′ untranslated region (UTR),while InDel118 is located within the promoter region, 9 bp upstream of the putative transcriptional start site of ZmVTE4.InDel7 has two frequent alleles (0 bp and 7 bp insertion) and one rare allele with 4 bp insertion, whereas InDel118 possesses two alleles with 0 bp and 118 bp insertion [6]. DNA segment between 5′UTR and promoter region was amplified using forward primer of ZmInDel118(InDel118-F)and reverse primer of InDel7(InDel7-R)which has amplified about 706 bp and 831 bp DNA in 0/0 and 7/118 class, respectively. PCR amplified fragments across inbreds were used for sequencing in four replications at Macrogen Inc.,Seoul,Republic of Korea.Poor quality sequences at the end were trimmed and sequences with high quality score were aligned using ClustalW in BioEdit tool for construction of phylogenetic tree using the UPGMA method[17].Distance matrix was computed using the p-distance method with units of the number of base differences per site[18].The proportion of sites where at least one unambiguous base is present in at least one sequence for each descendent clade is shown next to each internal node in the tree.All positions containing gaps and missing data were eliminated. Multiple sequence alignment files were used to identify any new variants viz., SNPs and/or InDel to specific genotype affecting exceptional accumulation of α-tocopherol.

2.5. Functional characterization of newly identified variants

In-silico analysis of the sequences was performed to identify any new motif or SNP/InDel and their possible role in kernel α-tocopherol accumulation using bioinformatics tools viz.,MEME Suite, GOMo, RegRNA 2.0, SOFTBERRY (NSITE),Neural Network Promoter Prediction program by BDGP and GPMiner.

3. Results

3.1. Variation in kernel tocopherols

Fig.1-Mean concentration of different tocopherols in most and least favorable haplotypes. T,tocopherol.

Fig.2-Proportion of α/γ-tocopherol and α-tocopherol in total tocopherol in inbred kernel.T,tocopherol;TT,total tocopherol.

Diverse inbreds were screened for InDel7 and InDel118, of which eleven genotypes had the most favorable haplotype(0/0), while four genotypes possessed the least favorable haplotype (7/118). Mean α-tocopherol of the genotypes carrying favorable haplotype was 17.40 μg g-1, whereas the same for unfavorable group was 5.58 μg g-1(Fig. 1). Hence favorable haplotype recorded 3.12-fold more α-tocopherol over unfavorable haplotype. Inbreds also showed wide variation in α-tocopherol which ranged from 4.71 to 30.07 μg g-1across haplotypes. However, genotypes within favorable group recorded α-tocopherol as high as 30.07 μg g-1(CM145) compared to as low as 4.76 μg g-1(LM13). In favorable class, nine genotypes viz., CM145(30.07 μg g-1),DQL785-1-8(26.10 μg g-1),V345PV(20.24 μg g--1), CM140Q (25.15 μg g-1), HP465-41 (18.38 μg g-1), DQL653-2-4 (17.38 μg g-1), HKI1532 (16.01 μg g-1), BQPML10-1-1(15.60 μg g-1), and HKI1344 (11.05 μg g-1) were considered as high α-tocopherol genotypes. Similarly in unfavorable class, four genotypes viz., DML420-R (5.60 μg g-1), CML497(4.71 μg g-1), CML426 (5.47 μg g-1), and CML162 (6.53 μg g-1)and two genotypes in favorable class CML496 (6.70 μg g-1)and LM13 (4.76 μg g-1) were identified as low α-tocopherol genotypes based on Duncan's multiple comparison test(Table 1). γ-Tocopherol ranged from 3.75 μg g-1(BQPML10-1-1) to 52.57 μg g-1(V345PV) in favorable class and 24.78 μg g-1(DML420R) to 37.32 μg g-1(CML426) in unfavorable class. The range of kernel δ-tocopherol varied from 2.68 μg g-1(HP465-41) to 8.79 μg g-1(V345PV) across 15 genotypes with mean of 5.91 μg g-1. V345PV recorded the highest total tocopherol (81.60 μg g-1) whereas the least was recorded by LM13 (16.15 μg g-1).

Fig.3- Phylogenetic tree using nucleotide sequence.Red and yellow circles represent favorable and unfavorable haplotypes,respectively.Green circle represents reference sequence available in the public domain.

Mean γ-, δ-, and total tocopherol of favorable and unfavorable groups were also determined to find the effect of haplotypes. The mean γ-tocopherol was significantly higher in unfavorable class (30.11 μg g-1) than favorable class(22.78 μg g-1) (Fig. 1). Hence, mean proportion of α-/γ-tocopherol was much higher in favorable class (1.51) than unfavorable one (0.19) (Fig. 2). However, mean δ-tocopherol was almost comparable in favorable class (5.50 μg g-1) and unfavorable class (7.04 μg g-1). Mean total tocopherol of both the groups was also of similar magnitude (favorable class:45.69 μg g-1; unfavorable class: 42.72 μg g-1). Higher amount of α-tocopherol of favorable class and equitable total tocopherol with unfavorable class resulted into higher proportion of α-/total-tocopherol (41%) compared to (13%) in the unfavorable class(Fig.2).

3.2. Phylogenetic analysis

Phylogenetic tree was constructed to understand whether the haplotypes alone can discriminate these genotypes or any other mutations are also affecting the genetic distance of inbreds(Fig.3).Sequence for ZmVTE4(GRMZM2G035213)from B73 retrieved from maize GDB (www.maizegdb.org) was used as reference. Three distinct clades were clearly visible in the tree. Four genotypes (DML420R, CML497, CML426, and CML162) having lower level of α-tocopherol with least favorable haplotype formed one separate cluster. Similarly nine genotypes (V345PV, HKI1344, DQL785-1-8, DQL653-2-4,CM145, HKI1532, BQPML10-1-1, CM140Q, and HP465-41) from favorable class formed second cluster. However, CML496 and LM13 from favorable class with least α-tocopherol were the most distinct genotypes and formed a separate cluster.Hence,sequence variations in the amplified region might be present in LM13 and CML496, which made them distinct from their own class.

Table 2-SNPs in genotypes with most favorable haplotype.

3.3. Sequence variations among favorable haplotypes

Sequences of favorable and unfavorable haplotypes were aligned separately to identify within group variants.Fourteen SNPs(SNP1 to SNP14)and eight InDels(InDel1 to InDel8)were identified in the amplified fragments of favorable genotypes(Tables 2, 3). Eleven of these SNPs were present in CML496 alone.CM140Q(SNP1)and LM13(SNP9)had one SNP each and SNP7 was common to both CML496 and LM13 (Table 2). Four out of eight InDels were of 1 bp, two with 27 bp (InDel1 and InDel4)and one each 6 bp(InDel2)and 14 bp(InDel8).Majority of the InDels (InDel1, InDel5, and InDel8) were observed in CML496 alone and one each in CM140Q(InDel7),BQPML-10-1-1 (InDel2), and LM13 (InDel4), while InDel6 was common to both BQPML10-1-1 and CML496 (Table 3). InDel3 was present in four genotypes viz.,HKI1344,LM13,DQL785-1-8,and HP465-41. Presence of much of the variations specifically in both these genotypes (CML496 and LM13) indicated the role of these SNPs and InDels in reduction of kernel α-tocopherol despite of presence of favorable haplotype at ZmVTE4. SNP7 at 606 bp (transition: G to A) could clearly differentiate CML496 and LM13 from rest of the genotypes suggesting its effect on kernel α-tocopherol concentration. Further, InDel1(at position 1)in CML496,overlapping InDel4(at position 608)in LM13 and InDel8 (at position 621) in CML496 differentiated the high and low α-tocopherol inbreds with favorable haplotype (Fig. 4). Among the inbreds, frequency of transversion mutation (86%) was higher than transition mutation (14%),and SNPs and InDels were observed after every 45 bp and 79 bp, respectively.

3.4. Sequence variation among unfavorable haplotypes

In unfavorable haplotype possessing insertion of both 7 bp and 118 bp four SNPs viz., SNP15 and SNP16 in CML162,SNP17 in both CML497 and DML420R and SNP18 in CML497 were found in the sequenced amplicons. Two InDels(InDel9 and InDel11) in DML420R and one (InDel10) in CML426 of 1 bp were also observed. Across the four inbreds, transversion was in higher frequency (75%) compared to transition (25%). SNPs and InDels were observed after every 190 bp and 253 bp, respectively in the unfavorable haplotype.

3.5. Sequence variation between favorable and unfavorable haplotypes

Eleven haplotypes of favorable class, four from unfavorable class along with reference gene (ZmVTE4) of B73 were aligned together to identify any new mutation which can differentiate these two groups along with already reported InDel7 and InDel118 (Fig. 5). ZmVTE4 of B73 possessed both 7 bp and 118 bp InDels and thus was designated as unfavorable haplotype. Four SNPs (SNP19 to SNP22) could clearly differentiate the favorable and unfavorable groups(Table 4).

Table 3-InDels in genotypes with most favorable haplotype.

4. Discussion

4.1. Variability of α-tocopherol

Considering the significance of ZmVTE4 gene in affecting kernel α-tocopherol in maize and its possible role in mitigation of vitamin-E deficiency, the present study was conducted to find new nucleotide polymorphisms in ZmVTE4 gene across 15 diverse maize inbreds differing in kernel α-tocopherol content.The inbreds possessed wide genetic variation in α-tocopherol.Similar range of variation (3.4 to 34.3 μg g-1and 0.70 to 31.35 μg g-1)for kernel α-tocopherol in maize has been reported by Muzhingi et al.[3]and Lipka et al.[19],respectively.However,much higher range of variation for α-tocopherol (0.40 to 61.08 μg g-1) was reported by Li et al. [6]. The fold change in αtocopherol among favorable haplotype over unfavorable haplotype was of similar degree as reported earlier by Li et al.[6].The favorable allele of ZmVTE4 efficiently converts more γ- to αtocopherol, therefore possess higher α-/γ-tocopherol and α-/total-tocopherol ratio compared to unfavorable haplotype[14].

Tocopherol biosynthesis pathway in maize is well characterized [20]. Precursor for vitamin-E is contributed by two separate pathways. Shikkimate pathway contributes the‘head' group common to all tocochromanols and the ‘tail'group consisting of phytyldiphosphate (PDP) and geranylgeranyl pyrophosphate (GGPP) is produced from plastidic isoprenoid pathway. In the first committed step, phydroxyphenylpyruvate (HPA) is converted to homogentistic acid (HGA) through cytosolic enzyme phydroxyphenylpyruvic acid dioxygenase (HPPD). Thereafter,HGA is converted to 2-methyl-6-phytyl-1,4-benzoquinol(MPBQ) in the plastid through condensation reaction by help of PDP using enzyme homogentisate phytyl transferase(VTE2). MPBQ is converted to γ-tocopherol by the action of methyl transferase(VTE3)and tocopherol cyclase(VTE1)or δtocopherol by solely tocopherol cyclase (VTE1). Finally, γtocopherol methyl transferase (VTE4) converts the δ- and γtocopherols to β-and α-tocopherols,respectively[20].Among various genes, mutant version of VTE4 with two insertion/deletions(InDel7 and InDel118)in 5′UTR and promoter region is significantly associated with higher accumulation of αtocopherol in maize kernel [6]; 118 bp InDel affects the transcript level, while 7 bp InDel regulates the translational efficiency of the ZmVTE4 thereby affecting the accumulation of α-tocopherol.

4.2. Newly identified nucleotide polymorphisms

The study revealed presence of several new SNPs and InDels in the promoter and 5′UTR other than 7 bp and 118 bp InDels.Ching et al. [21] also reported similar frequency of SNP (1 per 31 bp)and InDel(1 per 85 bp)in maize.Bhattramakki et al.[22]also observed that one bp InDels are the major fraction. In maize, favorable alleles of crtRB1 and lcyE caused due to deletion of transposable element accumulate more provitamin A (proA) in kernels [23,24]. Vignesh et al. [25] analyzed a set of maize inbreds possessing favorable allele of crtRB1 gene,and identified two SNPs and two InDels in the 3′UTR that clearly differentiated the high and the low β-carotene accumulating genotypes. Similarly, Zunjare et al. [26] identified four SNPs at positions 446,458,459,and 483 in 5′UTR that discriminated the low- and high- provitamin A lines having favorable allele of lcyE gene.

4.3. Functional analysis of sequence variations in favorable haplotype

Alteration of gene function and phenotypes in crop plants due to presence of SNPs and InDels are well known phenomena[27,28]. SNPs in the coding region of a gene can lead to functional change by altering the amino acids sequence of protein or by changing the folding pattern of the proteins[29].SNPs and InDels in the 5′UTR of genes have been reported to regulate gene expression by various means.Jackson et al.[30]reported that control elements on 5′UTR of genes like 7mG cap,secondary structure and nucleotide sequence nearby AUG start codon could affect the translational efficiency of the gene.Most of the SNPs and InDels in 5′-and 3′-UTRs regulate the translational efficiency of genes by interacting with RNAbinding protein[31].Presence of specific sequence motifs and alteration in upstream open reading frames(ORF)in the 5′UTR can also modify the translational efficiency[32].

Fig.4- Multiple sequence alignment of inbreds having favorable haplotype of ZmVTE4.

Fig.5-Sequence alignment of favorable and unfavorable haplotypes.

SNP1 was observed only between CM140Q and other favorable genotypes(“C”in CM140Q and“A”in other favorable haplotypes).As the position of the SNPs(SNP2 to SNP14)were very close to each other,functional analysis using GOMO was performed by motif search of DNA sequences carrying all the SNPs in three consecutive searches which include SNP2 to SNP5 (Search-1), SNP6 to SNP11 (Search-2), and SNP12 to SNP14 (Search-3) and individual SNP within the search fragment was replaced each time to find out the effect of that SNP.Wild type motif carrying SNP2 to SNP5 was found to control translation process; however none of these SNPs had any effect on the motif function. Search-2 revealed that wild type motif was probably involved in ATP-dependent helicase activity and in the process of translation with specificity of 52% and 20%, respectively. While mutated motif at SNP7,SNP8, SNP10 and SNP11 have abolished the helicase activity and both the activities were disrupted in mutated SNP6.SNP1 and SNP9 did not affect the function of the motif. Fritsche et al. [33] reported SNPs and InDels within genes involved in tocopherol biosynthesis in Brassica napus.They reported seven polymorphisms within PDS1 gene responsible for an enzyme that catalyzes the production of homogentisate, an essential substrate for the formation of aromatic head group of the tocopherol forms. Three SNPs at positions 35, 174, and 207 were significantly linked to total tocopherol. SNP at position 543 was associated with α-tocopherol and total tocopherol.Further, SNP285 in VTE3 gene was found to be significantly associated with ratio of α- and γ-tocopherol, while SNP342 was linked to γ-tocopherol [33]. Shaw et al. [34] also reported five SNPs in VTE4 gene of soybean,out of which first SNP was involved in alanine to threonine change in peptide sequence and second SNP resulted in methionine to valine change.These sites were predicted to be associated with phosphorylation changes and hence may be responsible for posttranslational modifications thereby regulating the tocopherol biosynthesis pathway[34].

Table 4-Sequence polymorphisms differentiating favorable and unfavorable haplotypes.

In the present study, among 14 SNPs present in favorable class, SNP7 could differentiate the low α-tocopherol accumulating inbreds (CML496 and LM13) from other high α-tocopherol based inbreds carrying favorable haplotype. G to A transition in the low α-tocopherol accumulating inbreds leads to disruption of helicase activity and thereby affecting the process of transcription.Functional analysis of InDel of favorable class revealed the involvement of few deletions viz., InDel1, InDel4, and InDel8 through loss of function of different motifs in CML496 and LM13.Motif carrying InDel1 was involved in interaction with TBF(TATA box binding factor), whereas InDel4 and InDel8 acted as TF-cis acting element while InDel4 also possessed TATA-box.Mutations due to disruption of TATA box have been reported by various researchers. Variation in expression of ADH1 gene in different maize tissues due to variation in TATA box sequence has been proposed [35]. Grace et al. [36] also reported that the sequence and spacing of TATA box elements are important for precise initiation from β-phaseolin promoter. Mutation in the TATA box and its flanking nucleotides was reported to influence promoter function in transient transformation of tobacco[35-37].Transcriptional factors are proteins which bind to DNA through sequence specificity and modify the transcription process either directly by binding an activator to a promoter region of DNA and RNA Polymerase II for transcription initiation or indirectly through cofactors. These DNA motifs are frequently ranged from 5 to 15 bp in length. Sequence variations of nucleotides within this motif have the capacity to alter the degree of binding and functional implications. However, the nature and extent of transcriptional modification differ between genes, type of transcription factors, and cell types [38]. In eukaryotes, TBF is used by RNA polymerase to recognize the TATA box. For RNA polymerase II, TBF is found with other protein in a complex TFIID. Cis-regulatory elements (CREs) are regions of non-coding DNA in the vicinity of a gene and regulate transcription of the genes by binding to transcription factors. SNP and InDels identified here thus possibly regulate biosynthesis of αtocopherol through differential expression of ZmVTE4.However,it needs further validation through an elaborate experiment involving expression analyses among diverse set of maize inbreds varying for α-tocopherol.

4.4.Functional analysis of sequence variations in unfavorable haplotype

SNPs in least favorable genotypes were distant from each other and were studied separately.Four SNPs,viz.,SNP15(G to T in CML162), SNP16 (A to T in CML162), SNP17 (A to C in CML497 and DML420R), and SNP18 (C to T in CML497) were found. However, SNPs and InDels of least favorable haplotypes could not be assigned with any specific function.

A total of four SNPs (named as SNP19 to SNP22) differentiated the favorable and unfavorable haplotypes. The regions possessing these SNPs were scanned for functional motifs and promoter regions.SNP22 is a part of GCC box and there was an ERF7 binding factor site. ERF7 contains a repression domain(FDLNFPP)[39].Some of the Arabidopsis ERFs were reported to have an active repression mechanism because they suppress the trans-activation of other transcription factors without competing for the same DNA binding site[40].

4.5. Utilization of sequence variations in future breeding programme

Estimation of tocopherol through UHPLC requires high cost(US$30-35 per sample),besides requiring infrastructure and technical expertise. PCR-based co-dominant markers are easy to use and much cheaper(US$0.5 per sample)than UHPLC estimation,and can be effectively used to identify desired genotype by differentiating heterozygotes and homozygotes in segregating populations [41]. Li et al. [6] has reported PCR based co-dominant markers for 7 bp and 118 bp InDels to identify favorable haplotypes of ZmVTE4. However, the present study suggested that genotypes may accumulate lower α-tocopherol even in presence of these two InDels,and that additional SNP and InDels further regulate the accumulation of α-tocopherol. Similar findings has been reported for provitamin A in crtRB1 [25] and lcyE[26]genes.Hence,this information on newly identified SNP and InDels can be utilized to develop co-dominant markers like cleaved amplified polymorphic sequence (CAPS) for identification of most favorable allele of ZmVTE4. CAPS marker was developed from SNP and used in marker-assisted selection(MAS)to improve provitamin A in Cassava [42]. In Brassica juncea, two CAPS markers were developed from SNPs that could differentiate low and high erucic acid genotypes[43].Thus such SNPs are can be effectively utilized in MAS [44,45]. Hence the present study identified new SNP and InDels in ZmVTE4 that cause further variation in accumulation of α-tocopherol in maize kernels.

5. Conclusions

We demonstrated that presence of already known InDel7 and InDel118 in the promoter and 5′UTR of ZmVTE4 cannot always identify the high α-tocopherol genotypes. SNP and InDels identified in the present study further affect the accumulation of α-tocopherol among the inbreds possessing the favorable haplotype of ZmVTE4. Present investigation identified new SNP and InDels in the promoter and 5′UTR of ZmVTE4.Allelespecific co-dominant markers can be developed from these SNP and InDels, and can be employed in molecular breeding for enhancement of vitamin-E in maize.

Conflict of interest

Authors declare that there are no conflicts of interest exists.

Acknowledgments

Financial support from ICAR-Indian Agricultural Research Institute, New Delhi, India in conducting the study is duly acknowledged. First author is thankful to Indian Council of Agricultural Research, New Delhi, India and ICAR-Indian Institute of Maize Research, Ludhiana, India for providing the study leave for the doctoral programme. We thank CIMMYT-Mexico, HarvestPlus, and centres of All India Coordinated Research Project (AICRP)-Maize for sharing their inbred lines. The help of Mr. Manish Kapasia, technical assistant for management of field activities and pollination programme is thankfully acknowledged.