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Sub-atomic marker; Genetic diversity of plants; Polymorphism; Fundamental idea; Hereditary transformation

Introduction

The period of sub-atomic marker improvement and applications started during the 1980s. A decade later, the development of PCR-based DNA markers followed this significant milestone in plant genomics research [1]. From that point forward, the utilizations of numerous sub-atomic markers have been accounted for in different parts of plant sub-atomic reproducing and genomics. Using specific or arbitrary oligonucleotide primers, the PCR method allows specific DNA sequences to be practically amplified from genomic DNA sections. Currently available tools for plant improvement research include molecular markers. The majority of these markers are polymorphic nucleic acids that differ between individuals or populations [2]. Hereditarily, genotypes show differentiating pools of pieces because of point changes in oligonucleotide preparing locales. Now and again, the distance between the ends successions changed by inclusion or erasure transformation occasions could prompt polymorphism.

In plant genomics research, well-known molecular marker techniques like Arbitrarily Amplified DNA (AAD) markers like Amplified Fragment Length Polymorphism (AFLP), Inter Simple Sequence Repeat (ISSR), and Random Amplified Polymorphic DNA (RAPD) are more prevalent and utilized extensively. Newly developed molecular marker techniques are less commonly used in plant breeding programs than the well-known AAD markers. However, these brandnew, cutting-edge molecular marker techniques have recently been utilized by a significant number of plant molecular breeding projects to accomplish a variety of research goals. Throughout the long term, atomic marker research has zeroed in generally on the advancement of sub-atomic markers that are more productive for the genomic examination of harvests of monetary interest [2]. On the other hand, very few resources from research have been used to create molecular markers for the genomic study of underutilized crops that do not have a significant impact on the economy. As a result, there is still a grave lack of sequence information or data to assist in primer design in the majority of underutilized crops. Therefore, some DNA marker techniques are still inapplicable to these crops [3]. However, it is anticipated that when the development of molecular markers becomes less expensive and the cost of DNA sequencing significantly decreases, these crops will also be covered. This overview of molecular marker techniques will make it possible to use DNA marker techniques in plant breeding in a more effective and efficient way to promote sustainable agricultural production and use.

The idea of DNA or molecular markers

The difference in DNA nucleotide sequence between distinct species or organisms that is close to or tightly linked to a trait-expressing target gene is known as a molecular or DNA marker. The closely linked molecular marker and the expressed trait, biological function, or target gene are typically inherited together. The molecular marker's precise genomic location within chromosomes, which is referred to as a locus or loci, may or may not be known [4]. It is significant that sub-atomic or DNA markers don't impact qualities related with the articulation or capability of the connected quality or qualities. The tight relationship of sub-atomic markers to a characteristic or quality of a specific natural capability, makes the markers act as viable signs or banners that signal a specific quality locus and help the location or distinguishing proof of the related characteristics whether the qualities included are known or obscure and whether the gene(s) can be identified or not. If there are differences in the marker nucleotide sequences that are referred to as polymorphisms between individuals or species, then DNA markers can be used to identify individual genotypic differences in the same or different species. There are many different kinds of DNA mutations that cause nucleotide sequence differences between organisms or between species, resulting in molecular marker polymorphisms.

Point mutations involving single nucleotide substitutions, rearrangements involving insertions or deletions, DNA section duplication, translocations, and inversions, as well as mistakes in tandemly repeated DNA replication, typically result in marker polymorphisms in organisms [5]. Polymorphic markers are signals from molecular markers that can be used to identify genotypic differences between individuals due to differences in marker sequence. Monomorphic markers, on the other hand, are DNA markers that cannot be used to distinguish between genotypes. A good and very useful DNA marker is easily assayable, inexpensive, multiplexed, and able to be automated, ubiquitous and evenly distributed throughout the genome. To effectively differentiate between homozygotes and heterozygotes, an ideal molecular marker must also be highly polymorphic, co-dominant in expression, highly reproducible, and able to share generated data among laboratories. A very good molecular DNA marker also has the properties of being genome-specific, having multiple functions, and not having a negative effect on phenotype [6].

For all intents and purposes, a sub-atomic marker isn't simply the related polymorphism yet the entirety of the point by point conventions or systems for its location or ID. A molecular marker is frequently viewed solely in terms of individual-to-individual variations in DNA sequence or polymorphism. However, it is instructive to note that a molecular marker may in some instances merely consist of a primer or set of primers, restriction enzyme(s), or other relevant components, as well as the procedures for running the marker. The implication is that a complete package of primers, restriction enzymes, or other relevant components as well as the established detailed method for the detection of that particular molecular marker must be known or available for a DNA section to be considered a molecular marker. A sequence polymorphism cannot be useful as a molecular marker in the absence of such a comprehensive collection of information specific to the marker. For sure, this total assortment of data for all intents and purposes characterizes a sub-atomic marker totally.

Random fragment length polymorphism (RFLP)

DNA probe hybridization in Southern blotting and restriction enzyme cleavage of genomic DNA at random but specific recognition sites generate RFLP, a polymorphism that is dependent on DNA sequence length variation. By determining whether or not specific endonucleases used in the restriction of DNA samples produce fragments of varying base pair sizes or lengths, RFLP reveals variation in DNA sequences [7]. Restriction endonucleases are bacterially isolated enzymes. These enzymes break down DNA into smaller pieces at specific recognition sites. Scientists typically employ industrially isolated restriction enzymes to digest double-stranded DNA at specific locations using specific enzyme recognition DNA sequences. Each restriction endonuclease cuts DNA at distinct but distinct recognition sequences because it recognizes distinct four- to six-base-pair restriction sites. By implication, after restriction digestion, varying numbers and fragment lengths are produced by variations in the number of restriction site repeats and the random distribution of particular restriction enzyme recognition sites. Individuals' restriction recognition sequences differ in length due to the random distribution of cut sites for restriction enzyme recognition [8]. As a result, organisms differ in the length of the random DNA restriction fragments produced by enzyme digestion. Typically, RFLP bands are associated with DNA fragments between 2 and 10 kb in size.

Majority of RFLP markers are co-dominant, allowing them to identify both alleles in heterozygous samples. RFLP markers are tolerably polymorphic, exceptionally locus-explicit, and profoundly reproducible. Additionally, RFLP markers are randomly distributed and abundant throughout the genome. Nonetheless, RFLP examination is basically a difficult, tedious, and in fact requesting strategy. For each DNA digestion, a significant amount of purified, high-molecularweight DNA is required. Furthermore, RFLP marker analysis cannot be automated. In addition, the cost of designing probes may be prohibitive for plant species without suitable probes. The development of less complicated and less expensive DNA marker profiling technologies based on polymerase chain reaction (PCR) has rendered RFLP obsolete [9]. In addition to gene mapping studies, RFLPs have been extensively utilized to reveal individual genetic variation and phylogenetic associations.

Randomly amplified polymorphic DNA (RAPD)

PCR is used to randomly amplify large genomic DNA sections using arbitrary oligonucleotide short primers, typically 8 to 15 nucleotides in length, as part of the RAPD marker application procedure. Due to the use of arbitrary primers, prior DNA sequence information is not necessary for RAPD analysis. A primer sequence chosen for RAPD must contain at least 40% GC (guanine and cytosine), though 50%–80% is typically preferred. The primer will be able to function effectively at the annealing temperature that enables DNA polymerase to influence DNA elongation at this GC content [10]. Additionally, there cannot have been a palindromic sequence in the primer. By looking at previous works that have been described by various researchers, primer sequences are chosen based on their high polymorphism. The chosen RAPD groundworks can then be bought from various organizations, either as preliminary sets or as individual groundworks, utilizing the chosen preliminary groupings got from a writing survey of prior examinations. However, due to the fact that RAPD primer polymorphism can vary even within the same cultivar, a substantial set of approximately 50–100 primers is typically tested first with the cultivar of interest. A smaller number—roughly 10 to 20—of these primers are chosen for the main analysis because they are the most instructive or highly polymorphic.

In numerous other marker frameworks, two groundworks containing forward and turn around short DNA groupings, are expected in a similar PCR response. However, in RAPD PCR, only one oligonucleotide primer is used in each reaction; one copy is oriented in the forward direction, and another copy is oriented in the reverse direction. Typically, RAPD primers can simultaneously produce PCR products from one to ten genomic DNA sites. The typical length of a RAPD PCR fragment is between 0.5 and 5 kb. It is commendable noticing that preliminaries may effectively enhance a part of DNA or neglect to intensify PCR sections. The presence, distribution, and location of primer complementary sequences on the template genomic DNA determine whether or not the RAPD PCR is successful [11]. No PCR fragment is amplified if the 3′ ends of the two primer copies on the template are not oriented correctly or the two primer copies anneal at a distance that is too great from one another. Additionally, in situations where change modifies a segment that was already correlative to a groundwork, the tempering of that preliminary at the adjusted site will be upset, and subsequently, no PCR item will be created. In an electrophoretic gel, individuals with these mutation effects will exhibit distinct DNA banding patterns. A 200 bps fragment was produced when primers 1 and 2 were bound to the depicted DNA section in Accession I. Similar to this, another 375 bps-sized fragment was amplified by the binding of primers 3 and 4 to the Accession I DNA section. As a result, Accession I produced two distinct RAPD fragment sizes. In Accession II, a mutation marked by a red asterisk is found at the binding site of primer 2 [12]. As a consequence of this lack of the primer 2 binding site, only one RAPD fragment (350 bps in size) is amplified in Accession II. The 375 bp section is monomorphic in light of the fact that it couldn't separate between the promotions. However, the 200 bps fragment is polymorphic because it differentiated between accessions.

When two annealing template DNA locations have similar sequences, are appropriately separated by a small amount, and anneal to the template with their orientations facing each other, PCR amplification takes place. Therefore, the nucleotide composition of the template DNA sequence, the species' genome size or complexity, and the primer(s)' sequence and length all play a significant role in the success of fragment PCR amplification. RAPD parts are effortlessly isolated in agarose gels by electrophoresis. UV light is used to visualize the gel, which is stained with ethidium bromide or any other suitable stain. Random annealing of arbitrary RAPD primers to various parts of the target organism's genome results in the production of intricate patterns of PCR products. The presence or absence of various types of mutations that determine the binding or non-binding of the respective primers to various sections of each accession's DNA is the cause. DNA bands indicating RAPD polymorphisms are typically found due to differences in sequence size between individuals' primer binding sites and the target DNA [13]. On a fundamental level, RAPD polymorphisms emerge essentially from varieties in the preliminary hybridization or toughening positions in the objective genome. However, it's important to keep in mind that not all primer annealing sites in the target genome will always result in amplified PCR fragments. Subsequently, essentially, RAPD polymorphisms are section-length varieties in really created PCR items in the middle between groundwork toughening destinations in the objective genome.

Retrotransposon-based markers

Long terminal repeats (LTRs) are extremely conserved sequences at the ends of retrotransposons. Eukaryotic retrotransposons are the most common mobile genetic molecules. These mobile genetic molecules or elements frequently reside close to known genes in genomic regions [14]. Several retrotransposon sequences have been generated. There is a high degree of heterogeneity and insertion polymorphism in many retrotransposons, both within and between plant species. Furthermore, it is known that plant genomes contain a large number of retrotransposons that are dispersed. The overflow and inescapable irregular circulation of retrotransposon atoms all through different plant genomes have been investigated for DNA marker studies. The LTRs groupings are utilized to direct the groundwork plan for retrotransposon-based marker investigation. Typically, retrotransposon inclusions are irreversible, and along these lines, proper especially for phylogenetic investigations. There are a number of retrotransposonbased markers that can be altered and altered in other ways. The primary retrotransposon-based markers discussed in this review are as follows: IRAP, REMAP, RBIP, and IPBS [15]. The correlations of a few significant properties of these markers have been framed. There are also other useful molecular markers that have been used recently in a variety of plant species.

Conclusion

The most widely used and widely used molecular markers for crop breeding and improvement have been explained in detail. One of the most comprehensive and extensive overviews of molecular markers in a single presentation, thirty-four markers have been presented. The well-established Arbitrarily Amplified DNA (AAD) marker techniques, microsatellite-based marker techniques, and retrotransposon-based molecular marker approaches make up the majority of the discussed molecular marker techniques. Indeed, molecular genomic research can take advantage of numerous opportunities provided by DNA or molecular marker techniques. However, these markers should not be viewed as an alternative to the other agro-morphological or biochemical markers; rather, molecular markers should be used in conjunction with genomics and plant breeding techniques to provide a more comprehensive understanding of the variety of germplasm that is available and the ways in which this diversity can be used to improve agricultural production and ensure a sustainable supply of food and nutrition.

Acknowledgement

None

Conflict of Interest

None

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