INTRODUCTIONThe taxonomic difficulty for several systematics, field ecologists, and evolutionary biologists is correctly determining an organism by a rapid, repeatable, and reliable method [1]. Advances in molecular genetics, sequencing technology, and bioinformatics have explored numerous molecular markers that can be easily sequenced and used for inferring phylogenetic relationships. One of these markers is the internal transcribed spacer (ITS), which is part of the non-coding nuclear ribosomal DNA cistron. It consists of the regions ITS1 and ITS2 that are separated by the gene 5.8S. The ITS region displays high rates of substitution that can manifest species and even subspecies divergences [2,3]. Therefore, the ITS sequence is one of the most frequently used DNA markers in plant phylogenetic and DNA barcoding analyses [4,5]. In addition, some genetic incidents exist that may mislead ITS-based phylogenetic investigations [6][7][8]. These incidents include the following: (a) existence of two alleles that may be heterozygous for each nuclear gene, (b) presence of several 18S-5.8S-26S repeats at one or more chromosomal locations, (c) some of these copies degenerate into pseudogenes, and (d) the near universality of most primers commonly used for the polymerase chain reaction (PCR) amplification of ITS sequences. Therefore, the direct sequencing of PCR-amplified ITS sequences would typically produce a mixture of allelic sequences with polymorphic nucleotide sites, which will appear as overlapping peaks. A nucleotide sequence obtained from such a mixed PCR product is consensus for several targets sharing the same priming sites in a single genome [9]. By contrast, DNA sequence data from an individual allele (called a haplotype) can provide considerable information to address various biological questions and to guide several practical applications [7,10]. Various methods have been investigated to obtain haplotype sequences from different genomes. A traditional method is to construct a random cloning library from the PCR products before sequencing of the target fragment [7]. Furthermore, it has been suggested that reducing the amount of genomic DNA and decreasing the number of PCR cycles [11]. In addition to using different PCR primer sets and reaction conditions result in allele bias [3,12,13]. Maize (Zea mays) is one of the most productive and widely grown crops worldwide. It has remarkable morphological and genetic diversity and is considered a foundational model for studying genetics and genomics. An improved de novo assembly and annotation of a maize reference genome have been recently conducted [14]. To investigate the wide diversity of maize genotypes, few studies on ITS sequencing [15,16] and microsatellites or simple sequence repeats [17,18] have been published. The phylogenetic relationships of Zea species inferred from ITS sequences were demonstrated to be highly concordant with Random Amplified Polymorphic DNA (RAPD) evidence [16,19]. The main objective of this study was to identify a molecular marker or a...