Advances in Molecular Marker Technology and their Significance in Plant Improvement Strategies

Abstract

Molecular markers are powerful tools that have revolutionized plant improvement strategies by allowing breeders to select plants with desirable traits at an early stage. These markers are specific DNA sequences that can be used to identify genes responsible for important plant traits such as disease resistance, drought tolerance, and yield potential. Advances in molecular marker technology have greatly improved their efficiency and accuracy, making them an essential tool in plant breeding programs. One such advance is the development of high-throughput sequencing technologies, which allow for the rapid and cost-effective identification of large numbers of molecular markers. Additionally, new marker systems such as SNPs have been developed, which offer a high level of accuracy and reproducibility. The use of molecular markers in plant breeding has several advantages over traditional breeding methods. For instance, markers can be used to identify desirable traits that are not easily observable, or to select plants with multiple desirable traits at once. This has led to the development of new and improved crop varieties that are more resistant to diseases, better adapted to changing environmental conditions, and have higher yields. In conclusion, the continued development of molecular marker technology is crucial for the advancement of plant improvement strategies.

1. Introduction

In the realm of modern agriculture and plant breeding, the integration of advanced biotechnological tools has revolutionized the traditional methods of crop improvement. Among these tools, molecular marker technology stands out as a pivotal innovation that has significantly accelerated the progress of plant breeding [1, 2, 3]. This technology allows breeders to gain insights into the genetic makeup of plants with unprecedented precision, facilitating the selection of desired traits in a more efficient and targeted manner.

Molecular markers are specific DNA sequences that can be easily identified and linked to particular traits or genes of interest. These markers serve as genetic landmarks on chromosomes, aiding in the identification of specific regions associated with advantageous characteristics such as disease resistance, yield potential, nutritional content, and environmental adaptability. By enabling researchers to pinpoint the genetic basis of these traits, molecular markers enhance the accuracy and speed of breeding processes [4, 5, 6, 7, 8].

Traditional plant breeding methods involve the crossbreeding of plants with desirable traits and the subsequent selection of offspring displaying those traits. However, this process is time-consuming and often requires multiple generations to achieve the desired results [6, 7, 8]. Molecular marker technology addresses these challenges by allowing breeders to identify and select plants possessing the desired traits at the molecular level, even before visible traits are expressed. This “marker-assisted selection” greatly expedites the breeding cycle, enabling the development of improved plant varieties within a shorter timeframe [9, 10, 11].

There are various types of molecular markers, including Restriction Fragment Length Polymorphisms (RFLPs), Simple Sequence Repeats (SSRs), Single Nucleotide Polymorphisms (SNPs), and Insertion/Deletion Polymorphisms (InDels), among others. Each type has its unique advantages and applications, making them valuable tools for different stages of plant breeding. With the advent of high-throughput sequencing technologies, genotyping a large number of markers has become increasingly feasible and cost-effective [12, 13].

Furthermore, molecular markers have not only expedited the breeding process but have also paved the way for more precise genetic manipulation through techniques like marker-assisted backcrossing and genome editing. This precision breeding approach ensures that only the desired genetic traits are introduced or modified, minimizing the unintended alterations associated with traditional breeding methods.

2. Classification of molecular markers

Molecular markers are crucial tools in modern plant breeding programs. They enable plant breeders to identify and select desirable traits with greater precision, efficiency, and speed compared to traditional breeding methods. Molecular markers are genetic variations that can be detected at the DNA or protein level. They are used to track specific genes or regions of the genome that are associated with desired traits. Here is a detailed classification of molecular markers used in plant breeding.

2.1 RFLPs (Restriction Fragment Length Polymorphisms)

RFLPs were among the first molecular markers developed. They are based on variations in DNA sequences recognized by specific restriction enzymes. Differences in the fragment lengths resulting from enzyme digestion can be visualized on an agarose gel. RFLPs are informative but require large amounts of DNA and are labor-intensive [14, 15].

2.2 AFLPs (Amplified Fragment Length Polymorphisms)

AFLPs involve selective amplification of DNA fragments using PCR (polymerase chain reaction). They are based on restriction enzyme digestion and subsequent ligation of adaptors. AFLPs generate numerous fragments that can be separated by gel electrophoresis, allowing for the identification of polymorphic markers [16, 17].

2.3 SSRs (Simple Sequence Repeats or Microsatellites)

SSRs are short DNA sequences consisting of tandem repeats (e.g., ATATATAT) that exhibit length polymorphisms due to variations in the number of repeats. They are highly variable, co-dominant, and have wide applications in plant breeding due to their abundance in the genome [18, 19, 20].

2.4 SNP (Single Nucleotide Polymorphism)

SNPs are single base pair changes in DNA sequences. They are the most abundant type of genetic variation and can be efficiently detected using various methods, such as microarrays or next-generation sequencing. SNPs are used in high-throughput genotyping and genome-wide association studies [21, 22].

2.5 CAPS (Cleaved Amplified Polymorphic Sequences) and dCAPS (Derived Cleaved Amplified Polymorphic Sequences)

CAPS and dCAPS markers are based on SNPs that create or abolish restriction enzyme recognition sites. After PCR amplification, digestion with a specific enzyme allows for the discrimination of different genotypes [23, 24].

2.6 RAPDs (Random Amplified Polymorphic DNA)

RAPDs involve random PCR amplification of DNA segments using short, arbitrary primers. These markers are quick and simple to generate but may lack reproducibility and require optimization [25, 26].

2.7 ISSRs (Inter Simple Sequence Repeats)

ISSRs involve PCR amplification between microsatellite sequences using anchored primers. They combine the advantages of SSRs and RAPDs and have been widely used for genetic diversity assessments [27, 28].

2.8 SNP arrays

SNP arrays are high-density microarrays containing thousands to millions of SNP markers. They provide efficient genotyping of large populations and enable genome-wide association studies and genomic selection [29, 30].

2.9 Genotyping-by-sequencing (GBS)

GBS is a next-generation sequencing-based approach that sequences subsets of a genome. It allows for simultaneous genotyping of many individuals at a reduced cost and is suitable for both well-characterized and non-model species [31, 32].

2.10 Methylation-based markers

These markers detect epigenetic modifications, such as DNA methylation. Methylation status can influence gene expression and phenotype, making these markers relevant for studying complex traits [33, 34].

2.11 Gene-specific markers

Markers can be designed to target specific genes of interest, aiding in tracking and selecting specific traits [35].

2.12 Transcriptome-based markers

Transcriptome sequencing can identify gene expression variations linked to traits. These markers are valuable for understanding gene function and regulation [36].

2.13 Exome capture markers

These markers focus on sequencing only the protein-coding regions (exons) of the genome, providing cost-effective genotyping information [37].

2.14 Copy number variation (CNV) markers

CNV markers detect variations in gene copy numbers among individuals, contributing to genetic diversity and phenotypic differences [38].

2.15 Insertion-deletion polymorphisms (InDels)

InDels are variations in the number of nucleotides within a specific genomic region. They are useful for distinguishing closely related individuals.

2.16 Sequence-tagged sites (STS)

STS markers are derived from specific DNA sequences associated with genes of interest. They can be used to directly amplify and analyze specific DNA fragments [39].

The choice of molecular marker depends on the specific breeding objectives, available resources, and the species under consideration. Advances in genomics and sequencing technologies continue to expand the range of molecular markers available to plant breeders, enabling them to accelerate the development of improved crop varieties.

3. Principles and techniques of molecular marker analysis

Molecular marker analysis has revolutionized plant breeding by providing tools to study and manipulate the genetic makeup of plants more effectively [40]. These markers serve as indicators of specific genes or genomic regions linked to desirable traits. Here’s a detailed overview of the principles and techniques of molecular marker analysis in plant breeding.

3.1 Principles of molecular marker analysis

1. Genetic linkage: molecular markers are used to identify regions of the genome that are closely linked to target traits of interest. These markers are inherited along with the target genes, enabling breeders to select plants with desired traits through marker-assisted selection (MAS) [5, 41].

2. Quantitative trait loci (QTL) mapping: by analyzing the co-segregation of molecular markers and phenotypic traits in mapping populations, QTLs—genomic regions influencing quantitative traits—can be identified. This helps in understanding the genetic basis of complex traits [42, 43].

3. Genetic diversity assessment: molecular markers aid in evaluating the genetic diversity within a breeding population, ensuring that genetic resources are effectively utilized to develop improved varieties [20, 44].

4. Marker-assisted breeding (MAB): breeders can use molecular markers to expedite the traditional breeding process by identifying plants carrying desirable genes without having to wait for phenotypic expression [13, 45].

3.2 Techniques for molecular marker analysis

1. DNA extraction: isolation of high-quality DNA is the initial step. Various protocols are used depending on the plant species and tissue type [46].

2. PCR-based techniques: polymerase chain reaction is used to amplify specific DNA fragments using primers designed based on marker sequences.

3. Gel electrophoresis: amplified DNA fragments are separated by size using gel electrophoresis to visualize differences in marker patterns.

4. Hybridization: techniques like Southern blotting are used to identify RFLPs and other DNA fragments based on their hybridization patterns with labeled probes.

5. High-resolution melting (HRM): this technique identifies sequence variations by monitoring the melting behavior of PCR-amplified fragments [47].

6. DNA sequencing: sanger sequencing or NGS can provide the exact DNA sequence information for the marker region [48].

7. Genotyping platforms: SNP arrays and NGS platforms allow for high-throughput genotyping of thousands to millions of markers across the genome [49].

8. Data analysis: various software tools and statistical methods are used to analyze marker data, perform QTL mapping, assess genetic diversity, and make breeding decisions [50, 51].

9. Marker-assisted selection (MAS): breeders use marker information to select plants with desirable traits during the breeding process, improving efficiency and accuracy

10. Genome-wide association studies (GWAS): this technique involves analyzing marker data from diverse germplasm to identify associations between markers and phenotypic traits [52].

4. Applications of molecular marker technology in plant improvement

Molecular marker technology has revolutionized the field of plant improvement by enabling precise and efficient selection of desired traits in crops. These markers are specific DNA sequences associated with particular traits, and they provide a valuable tool for plant breeders to make informed decisions during the breeding process. Here, I’ll cover various applications of molecular marker technology in plant improvement, along with examples of their use:

4.1 Marker-assisted selection (MAS)

This is one of the most common applications of molecular markers in plant improvement. MAS involves identifying markers linked to desirable traits and using them to select plants with those traits more accurately and efficiently than traditional methods [53]. For instance:

1. Disease resistance: breeding for disease-resistant plants by identifying markers linked to resistance genes. Example: development of rice varieties resistant to bacterial blight using Xa21 gene marker [54].

2. Abiotic stress tolerance: identifying markers associated with drought, salinity, and cold tolerance to develop crops better suited for challenging environments [55, 56].

3. Genetic diversity and germplasm characterization: molecular markers help assess the genetic diversity of plant populations and germplasm collections, aiding in the conservation and utilization of genetic resources [57].

4. Fingerprinting: establishing genetic profiles of varieties to prevent misidentification and support intellectual property rights [58].

4.2 Phylogenetic studies

The use of molecular markers in phylogenetic studies has revolutionized the field by providing a more accurate and detailed understanding of evolutionary relationships among species [59, 60]. These molecular markers, such as RFLPs, SSRs, SNPs, and InDels, allow researchers to examine variations in DNA sequences [61, 62]. Identify unique genetic markers that can be used to determine relatedness and trace ancestral lineages. By analyzing these markers, researchers can create phylogenetic trees and reconstruct evolutionary histories. Furthermore, molecular markers have significantly improved our knowledge about past events that have shaped the genetic diversity within species [63].

4.3 Quantitative trait loci (QTL) mapping

The use of molecular markers in plant QTL mapping has revolutionized the field of plant breeding and genetics. By using molecular markers, researchers can identify and track specific regions of the genome that are associated with desirable traits in plants [61, 64, 65]. This helps to determine the criteria of selection and also combines molecular markers with conventional breeding methods to obtain the best results.

The progress in quantitative trait loci mapping in plants depends on the availability of DNA markers and efficient biometric tools, which lead to the accurate detection of QTLs [66, 67].

4.4 Marker-assisted backcrossing (MABC)

Marker-assisted backcrossing has emerged as a revolutionary approach in plant breeding, seamlessly integrating modern molecular techniques with traditional breeding methods. This strategy involves the use of molecular markers to identify and track specific desirable traits within the genome of a plant, facilitating the efficient transfer of these traits into elite breeding lines through a series of backcrosses. One of the most successful examples of this technique is the development of disease-resistant rice varieties. By identifying and incorporating markers linked to genes conferring resistance to devastating pathogens like bacterial blight and blast, breeders have rapidly produced high-yielding rice cultivars with enhanced disease resistance. Another notable achievement is the creation of drought-tolerant maize varieties. Through marker-assisted backcrossing, scientists have successfully transferred drought-responsive genes into commercially valuable maize lines, mitigating the impacts of water scarcity on crop productivity. These examples underscore the transformative potential of marker-assisted backcross breeding in addressing pressing challenges in agriculture and securing global food production.

4.5 Marker-assisted introgression of alien genes

Transferring beneficial genes from wild or related species into cultivated varieties to enhance traits like disease resistance, quality, or yield. Wheat rust resistance: incorporation of rust resistance genes from wild wheat relatives into cultivated wheat to combat fungal diseases [68, 69].

4.6 Genomic selection

Using genome-wide marker information to predict the breeding value of plants without having to observe the trait directly. This accelerates the breeding process by selecting individuals with the highest genetic potential. Animal forage quality: improving the quality of forage crops for livestock feed based on predicted genetic values [70, 71, 72].

4.7 Genetic mapping and genome assembly

Molecular markers aid in constructing genetic maps and assembling plant genomes, facilitating further research on gene function and evolution. Human consumption quality: enhancing the taste, texture, and nutritional content of fruits and vegetables for improved consumer satisfaction [73, 74].

4.8 Detection of genetic mutations

Identifying and characterizing mutations responsible for specific traits, diseases, or disorders. Seedless fruit development: understanding and manipulating the genetic mutations that lead to seedlessness in fruits like grapes and watermelons [75, 76].

4.9 Marker-based cloning of genes

Molecular markers help in identifying and isolating genes responsible for specific traits, enabling a deeper understanding of plant biology. Flower color modification: cloning genes associated with flower color to create ornamental plants with novel hues [77, 78].

4.10 Transgene detection and purity testing

Molecular markers can be used to detect the presence of transgenes in genetically modified (GM) plants and assess the purity of seed stocks. Bt cotton: identifying the presence of Bt toxin genes in cotton plants for insect resistance [79].

4.11 Marker development

Continual advances in molecular marker technologies, like Single Nucleotide Polymorphisms (SNPs) and high-throughput genotyping, allow the development of more efficient and cost-effective markers for specific traits [80].

In summary, molecular marker technology has profoundly impacted plant improvement by expediting the breeding process, increasing precision, and enhancing the ability to select and manipulate desired traits in crops. Its diverse applications have contributed to the development of improved crop varieties that address challenges ranging from disease resistance to environmental adaptation and nutritional content.

5. Marker Assisted Selection (MAS) in plant breeding

Marker-assisted selection (MAS) is a powerful technique in plant breeding that allows breeders to select plants with desired traits more efficiently and accurately by utilizing genetic markers associated with those traits. This approach has revolutionized the field of plant breeding by significantly speeding up the process of developing new crop varieties with improved characteristics. Here’s a detailed explanation of marker-assisted selection, along with some examples of its applications.

5.1 What is marker-assisted selection (MAS)?

Marker-assisted selection involves the use of molecular markers, which are specific DNA sequences that can be easily detected and analyzed in a laboratory setting [81]. These markers are linked to particular traits of interest, such as disease resistance, yield potential, nutrient content, and other agronomic characteristics. By identifying the presence or absence of these markers, breeders can predict the phenotype of a plant and make informed decisions about whether to continue breeding or discard certain plants.

5.2 Steps in marker-assisted selection

1. Marker discovery: in this step, researchers identify and develop molecular markers that are associated with the desired traits. These markers can be Single Nucleotide Polymorphisms (SNPs), microsatellites (SSRs), or other types of genetic variations.

2. Marker validation: once potential markers are identified, they are validated across a diverse range of plant genotypes to ensure their consistency and reliability in predicting the desired trait.

3. Marker-assisted selection: after validation, the markers are used to screen and select plants during breeding. The presence of specific markers is used as an indicator of the presence of the desired trait.

4. Traditional breeding: the selected plants are then further evaluated through traditional breeding methods to confirm their performance and combine multiple traits.

5. Marker-assisted backcrossing: if needed, selected plants can undergo backcrossing, where they are crossed with a recurrent parent to recover their genetic background while retaining the desired trait.

5.3 Examples of marker-assisted selection

1. Disease resistance: one of the earliest successful applications of MAS was in developing disease-resistant crops. For instance, in rice breeding, markers associated with resistance to diseases like bacterial blight and blast have been used to identify and select plants with enhanced resistance, leading to improved crop yields [82, 83].

2. Quality traits: MAS has been employed to enhance the quality of crops, such as improving protein content in wheat and oil quality in canola. Genetic markers linked to these quality traits are used to identify and select plants with superior nutritional attributes [84].

3. Drought tolerance: with the increasing challenges of climate change, breeding for drought-tolerant crops is crucial. MAS allows breeders to identify plants with genetic markers associated with drought tolerance, enabling the development of varieties that can thrive in water-limited conditions [85, 86].

4. Yield enhancement: genetic markers associated with high yield potential have been utilized to accelerate the breeding of crops with improved productivity, helping to address global food security concerns.

5. Nutrient efficiency: breeders have used MAS to develop plants with improved nutrient uptake and utilization efficiency, resulting in crops with enhanced nutritional value and reduced fertilizer requirements [87].

6. Fruit traits: in fruit crops, MAS has been employed to select traits such as fruit size, color, texture, and taste, leading to the development of more appealing and marketable varieties [88].

7. Herbicide resistance: in crops like soybeans and corn, MAS has aided in the development of herbicide-resistant varieties by identifying genetic markers linked to resistance traits, allowing farmers to control weeds more effectively [89].

6. Genomic selection in plant improvement

Genomic selection (GS) is a cutting-edge plant breeding technique that leverages genetic information from an organism’s entire genome to predict its potential performance and select the best individuals for breeding. This approach has revolutionized traditional plant breeding by allowing breeders to make more informed decisions and significantly accelerate the improvement of crop plants [90].

Genomic selection is based on the concept that the genetic information contained within an organism’s genome can be used to predict its phenotypic traits, such as yield, disease resistance, and quality. This is achieved by analyzing a large number of genetic markers distributed across the genome, often in the form of Single Nucleotide Polymorphisms (SNPs). These markers are associated with specific traits, allowing breeders to create predictive models to estimate the potential performance of individual plants or animals.

6.1 Steps in genomic selection

1. Genotyping: the first step involves genotyping a large number of plants with molecular markers, typically SNPs, distributed throughout the genome. This creates a genomic profile for each individual.

2. Phenotyping: phenotypic data, such as yield, disease resistance, and other relevant traits, are collected from each plant in the breeding population.

3. Model training: statistical methods are then used to associate the genetic markers with the phenotypic data. This step involves building predictive models, such as genomic prediction models or genome-wide association studies (GWAS), which highlight the genetic markers associated with specific traits.

4. Model validation: the accuracy of the models is assessed using independent data sets. This helps ensure that the models are reliable and can predict the traits of interest accurately.

5. Selection: once validated, the predictive models are used to select plants or individuals with the desired traits for further breeding. This can greatly expedite the breeding process as only the most promising individuals are chosen for advancement.

6.2 Examples of genomic selection in plant improvement

1. Maize (corn): researchers have successfully applied genomic selection in maize breeding. By using a high-density SNP array, they created predictive models for traits like yield, drought tolerance, and disease resistance. This has led to the development of maize varieties with improved performance under challenging environmental conditions [91].

2. Wheat: genomic selection has been used to improve wheat yield and quality. Researchers have identified genomic regions associated with traits like disease resistance, grain size, and nutritional content. By selecting wheat plants based on their genomic profiles, breeders have produced varieties with enhanced yield potential and nutritional value [92].

3. Rice: in rice breeding, genomic selection has enabled the development of varieties with increased resistance to pests and diseases. By identifying genetic markers linked to resistance traits, breeders can efficiently select plants that are less susceptible to yield-reducing factors [93].

4. Soybean: genomic selection has played a crucial role in soybean breeding. Researchers have employed this technique to enhance traits such as oil content, protein content, and resistance to pathogens. This has resulted in the creation of soybean varieties that meet the demands of both food and industrial applications [94].

5. Apple: in fruit tree breeding, such as apple breeding, genomic selection has been used to improve traits like fruit quality, disease resistance, and tree architecture. By selecting apple trees based on their genomic profiles, breeders can develop varieties that exhibit improved fruit characteristics and resilience [95].

These are just a few examples of how genomic selection has been applied to plant improvement across various crops. The technique’s ability to rapidly and accurately predict the performance of plants based on their genetic makeup has significantly advanced the field of plant breeding, enabling the development of new varieties that are better adapted to changing environments, more resilient to diseases, and capable of meeting the needs of a growing global population.

7. Marker Assisted Backcross Breeding (MABB) in plant breeding

Marker-Assisted Backcrossing (MABC) is a powerful technique employed in plant breeding to accelerate the transfer of desirable traits from one plant variety (donor parent) to another (recipient parent) while retaining the genetic background of the recipient parent. This technique combines traditional backcrossing methods with modern molecular marker technology, enabling breeders to select plants carrying the desired traits more efficiently and accurately [96].

7.1 Steps in MABB

1. Trait identification: breeders identify a desirable trait, such as disease resistance, drought tolerance, or enhanced yield, that they wish to introduce into a particular plant variety.

2. Marker selection: molecular markers, specific DNA sequences associated with the desired trait, are chosen. These markers are polymorphic, meaning they exhibit variations between different plants or varieties. Common types of markers include SSRs (Simple Sequence Repeats), SNPs (Single Nucleotide Polymorphisms), and AFLPs (Amplified Fragment Length Polymorphisms).

3. Donor parent selection: a donor parent possessing the desired trait is chosen. This parent serves as the source of the target trait and provides the genetic material needed to introduce the trait into the recipient parent.

4. Recipient parent selection: the recipient parent, usually a commercially valuable or locally adapted variety, is selected. This parent provides the genetic background that will be maintained throughout the breeding process.

5. Backcrossing: the donor parent is crossed with the recipient parent, resulting in the first generation of hybrids. The progeny from this cross is then backcrossed to the recipient parent for several generations. During each backcross, molecular markers linked to the target trait are used to select offspring that carry the desired trait.

6. Marker-assisted selection (MAS): molecular markers associated with the target trait are used to screen the progeny at each backcross generation. This allows breeders to select plants that carry the trait of interest while minimizing the incorporation of unwanted genetic material from the donor parent.

7. Selfing and selection: after each backcross generation, selected plants are self-pollinated to produce homozygous lines with a high proportion of the recipient parent’s genome. These lines are then subjected to further rounds of selection using the molecular markers.

8. Genotype analysis: throughout the process, genotyping techniques are used to identify and confirm the presence of the target trait and monitor the proportion of recipient parent genetic material in the selected lines. Final Evaluation and Release: Once the desired trait has been successfully introgressed into the recipient parent’s genetic background, the selected lines are subjected to extensive field trials and evaluations for various agronomic and quality traits. Once the lines meet the desired standards, they can be released as new improved varieties.

8. Marker Assisted Trait Mapping

Marker-Assisted Trait Mapping (MATM) is a powerful technique in plant breeding that involves the identification of genetic markers associated with specific traits of interest. These markers are then used to accelerate the process of selecting and breeding plants with desirable traits [97]. This technique has revolutionized plant breeding by enabling breeders to make informed decisions based on molecular data, leading to more efficient and targeted breeding programs.

8.1 Process of marker-assisted trait mapping

1. Trait selection: the first step in MATM is to select the trait of interest for improvement. This could be anything from disease resistance and yield to nutritional content and drought tolerance.

2. Marker identification: once the trait is chosen, researchers identify genetic markers that are linked to the trait. These markers can be Single Nucleotide Polymorphisms (SNPs), microsatellites, or other types of DNA variations. The idea is to find markers that are physically close to the gene responsible for the trait.

3. Population development: a diverse population of plants is developed for analysis. This population typically consists of individuals with varying degrees of the trait under investigation.

4. Genotyping: the DNA of each individual in the population is genotyped using techniques like Polymerase Chain Reaction (PCR) or high-throughput sequencing. This results in a dataset of genetic markers for each individual.

5. Phenotyping: the individuals in the population are evaluated for the trait of interest. This could involve measuring yield, disease resistance, or any other relevant phenotype.

6. Statistical analysis: the genetic marker data is statistically analyzed to identify associations between specific markers and the trait. This is usually done using methods like genome-wide association studies (GWAS) or linkage analysis.

7. Validation: the identified marker-trait associations are validated in different environments and populations to ensure their reliability.

8. Marker-assisted selection (MAS): once validated, these markers serve as tools for breeders. They can now use these markers to indirectly select plants with the desired trait in early generations, without waiting for the phenotype to become apparent. This significantly speeds up the breeding process.

9. High-throughput genotyping platforms in plant breeding

Plant breeding plays a crucial role in developing new crop varieties with improved traits, such as yield, disease resistance, and nutritional content. Genotyping, the process of identifying an individual’s genetic makeup, is a vital component of modern plant breeding efforts. High-throughput genotyping platforms have revolutionized the field by allowing researchers to rapidly analyze thousands of genetic markers across plant genomes. These platforms enable the identification of genes responsible for desired traits and the development of markers for selection, accelerating the breeding process. This article provides an in-depth overview of high-throughput genotyping platforms used in plant breeding, highlighting key contributions from various researchers.

9.1 Polymerase chain reaction (PCR)-based techniques

Random Amplified Polymorphic DNA (RAPD): RAPD is a simple PCR-based technique that uses random primers to amplify DNA segments. Although less commonly used today, it paved the way for more sophisticated genotyping approaches. Amplified Fragment Length Polymorphism (AFLP): AFLP combines PCR with restriction enzyme digestion to generate DNA fragments for analysis. It was widely used in the late 1990s and early 2000s for plant breeding studies [98].

9.2 Microarray-based platforms

1. Genotyping by microarray: microarrays allow the simultaneous detection of thousands of DNA markers. Researchers design microarrays with probes that hybridize to specific target sequences. This technique has been used for genotyping crops like rice and maize [99, 100].

2. Diversity Arrays Technology (DArT): DArT involves probing genomic representations with a diverse set of DNA fragments. It is a cost-effective method for generating large amounts of genotyping data [101].

9.3 Next-generation sequencing (NGS)-based platforms

1. Restriction-Site Associated DNA Sequencing (RAD-Seq): RAD-Seq uses restriction enzymes to selectively sequence DNA fragments adjacent to recognition sites. It’s particularly useful for detecting Single Nucleotide Polymorphisms (SNPs) [102].

2. Genotyping by Sequencing (GBS): GBS involves digesting genomic DNA with restriction enzymes and sequencing the resulting fragments. It has been used to discover SNPs and other genetic variants in various crop species [103].

3. Specific-Locus Amplified Fragment Sequencing (SLAF-Seq): SLAF-Seq combines reduced representation libraries with NGS to identify and genotype SNPs [104].

9.4 Single Nucleotide Polymorphism (SNP)-based platforms

1. Illumina Infinium BeadChip Arrays: these arrays contain thousands to millions of immobilized SNP probes, enabling high-throughput SNP genotyping. They have been extensively used in various crop species [105].

2. Kompetitive Allele Specific PCR (KASP): KASP is a flexible SNP genotyping platform that uses allele-specific primers to target specific SNP loci. It’s known for its scalability and cost-effectiveness [106].

9.5 CRISPR-Cas and genotyping

The CRISPR-Cas system has revolutionized genome editing. Researchers have used this technology to develop genotyping methods that exploit its precision and efficiency [107].

10. Genotyping by Sequencing (GBS) and Next Generation Sequencing (NGS) in plant breeding

10.1 Genotyping-by-Sequencing (GBS) in plant breeding

Genotyping-by-Sequencing (GBS) is a high-throughput genotyping method that has gained popularity in plant breeding and genetics. GBS involves the selective sequencing of a subset of the genome, focusing on specific genetic markers, to provide information about genetic variations within a population. This method allows researchers to genotype a large number of samples simultaneously, making it well-suited for plant breeding applications [108].

Here’s how GBS works:

1. Library preparation: DNA is extracted from the plant samples of interest. The DNA is then digested using restriction enzymes to create DNA fragments. Unique DNA barcodes (indexes) are added to each sample to allow for multiplexing, where multiple samples are sequenced in the same run.

2. Fragment selection: after digestion, the DNA fragments are size-selected to target specific regions of the genome. This selection can focus on specific genetic markers, such as Single Nucleotide Polymorphisms (SNPs), that are known to be associated with traits of interest.

3. Amplification and sequencing: the selected DNA fragments are amplified using PCR (polymerase chain reaction), and then the amplified fragments are sequenced using next-generation sequencing (NGS) technologies.

4. Data analysis: the sequencing data is processed to identify the genetic variations in the selected regions. By comparing the sequences to a reference genome, researchers can determine the presence of specific alleles and variants associated with traits.

10.2 Next-Generation Sequencing (NGS) technologies in plant breeding

Next-Generation Sequencing (NGS), also known as high-throughput sequencing, refers to a set of modern sequencing technologies that allow rapid and cost-effective sequencing of DNA or RNA molecules. NGS has revolutionized the field of plant breeding by enabling comprehensive analysis of genetic diversity, marker discovery, and trait association studies [109]. Here are some NGS technologies commonly used in plant breeding:

1. Illumina sequencing: illumina platforms, such as HiSeq and NovaSeq, use reversible dye-terminator chemistry to sequence DNA. These platforms offer high-throughput, accurate, and cost-effective sequencing, making them widely used in plant genomics. They have been used for GBS as well as whole-genome sequencing in various plant species.

2. Ion torrent sequencing: ion torrent sequencing is based on the detection of hydrogen ions released during DNA synthesis. This technology is known for its simplicity and speed, making it suitable for targeted sequencing and genotyping applications.

3. Pacific Biosciences (PacBio): PacBio platforms use single-molecule real-time (SMRT) sequencing to generate longer reads compared to Illumina. This longer read length can be valuable for resolving complex regions of the genome, such as repeat-rich areas.

4. Oxford Nanopore Technologies (ONT): ONT’s nanopore sequencing technology passes DNA strands through nanopores, and the changes in electrical current as DNA passes through are used to identify the sequence. ONT offers long read lengths and has been used for the de novo assembly of plant genomes.

10.3 Examples of applications

1. Trait mapping: researchers have used GBS to identify genetic markers associated with desirable traits in crops. For example, GBS has been used to map genes responsible for disease resistance, yield, and nutritional content in various plant species.

2. Diversity analysis: NGS technologies have enabled a comprehensive analysis of genetic diversity within crop populations. This information is crucial for selecting diverse parents for breeding programs, conserving germplasm, and understanding the genetic basis of adaptability.

3. Marker-assisted selection (MAS): GBS-derived markers have been integrated into breeding programs for the efficient selection of individuals carrying desired traits. This accelerates the breeding process by allowing early identification of superior genotypes.

4. Genome-wide association studies (GWAS): NGS data, including GBS, have been used in GWAS to identify genomic regions associated with complex traits. This helps breeders understand the genetic basis of trait variation and potentially predict performance.

5. Genome sequencing and annotation: NGS technologies have facilitated the sequencing and annotation of complete plant genomes. This knowledge enhances our understanding of gene function, evolution, and diversity.

6. Molecular breeding: Both GBS and NGS have enabled the development of molecular breeding strategies that exploit genetic information to improve crop varieties for specific traits, leading to faster and more targeted breeding processes.

These examples showcase the power of GBS and NGS technologies in advancing plant breeding research and providing valuable insights into the genetic basis of plant traits and diversity. As technology continues to evolve, these methods will likely play an even more significant role in shaping the future of plant breeding.

11. Integration of molecular marker technology with conventional breeding methods

The integration of molecular marker technology with conventional breeding methods has revolutionized the field of plant breeding, leading to more efficient and targeted breeding strategies. This approach is commonly referred to as “marker-assisted selection” (MAS) or “marker-assisted breeding” (MAB). It involves the use of molecular markers, which are specific DNA sequences associated with particular traits, to aid in the selection of desired traits in plants.

Here’s how the integration works and the benefits it brings to plant breeding:

1. Marker identification: first, researchers identify molecular markers that are closely linked to the target traits of interest, such as disease resistance, yield potential, quality characteristics, and more. These markers are typically identified through techniques like genetic mapping and sequencing.

2. Marker selection: once the markers associated with the target traits are identified, breeders can use these markers to select plants that carry the desired traits. This eliminates the need to wait for the trait to be visually expressed in the plant’s phenotype, which can take several generations through traditional breeding methods.

3. Early screening: molecular markers allow for early screening of plants in their juvenile stages or even at the seedling level. This rapid screening enables breeders to identify and discard plants that do not possess the desired traits, saving time and resources.

4. Trait introgression: molecular markers can aid in transferring specific traits from one plant variety to another. For example, if a wild plant possesses a desired trait (e.g., disease resistance) but lacks other favorable characteristics (e.g., yield potential), molecular markers can help breeders introgress the desired trait into a high-yielding commercial variety while retaining its positive attributes.

5. Pyramiding traits: breeders can use molecular markers to combine multiple desirable traits into a single plant variety through a process called trait pyramiding. This results in plants with enhanced performance and adaptability.

6. Reduced generation time: by using markers to assist in selecting plants with desired traits, breeders can reduce the number of generations required to develop a new variety. This accelerates the breeding process and allows for faster release of improved varieties to farmers.

7. Precision and accuracy: molecular markers provide a more precise and accurate way to select specific traits compared to relying solely on phenotypic observations, which can be influenced by environmental factors. This precision leads to higher success rates in achieving the desired trait combinations.

8. Preservation of genetic diversity: molecular markers help breeders maintain genetic diversity by allowing them to select specific traits while preserving the overall genetic background of the plant variety.

9. Expanding breeding possibilities: molecular marker technology enables the utilization of traits that may be difficult to select through traditional breeding due to their complex inheritance patterns or low heritability.

In summary, the integration of molecular marker technology with conventional breeding methods offers a powerful toolset for plant breeders to develop new and improved crop varieties more efficiently. This approach has contributed to the development of crop varieties that are better suited to challenges such as pests, diseases, changing climates, and consumer preferences.

12. Challenges and limitations of molecular marker technology

Molecular marker technology has revolutionized plant breeding by allowing for a more precise and efficient selection of desirable traits in plants. However, like any technology, it comes with its own set of challenges and limitations. Here are some of the major challenges and limitations of molecular marker technology in plant breeding:

1. Cost: developing and implementing molecular markers can be expensive, particularly for small-scale breeding programs or in developing countries. The initial investment required for equipment, reagents, and personnel training can be a significant barrier.

2. Marker-trait associations: the accuracy of marker-trait associations is crucial for successful marker-assisted selection (MAS). However, not all markers are perfectly linked to the target trait due to factors like genetic recombination and environmental influences. This can result in false positives or negatives, reducing the efficiency of selection.

3. Marker density and coverage: using a limited number of markers may not capture the entire genetic variation associated with a trait, leading to incomplete information and potential bias in selection. High-density marker panels can mitigate this, but they come with higher costs and computational challenges.

4. Trait complexity: many traits of interest in plant breeding are controlled by multiple genes and their interactions. Molecular markers often identify individual genes or loci, making them less effective for complex traits influenced by multiple genetic factors.

5. Linkage disequilibrium: over time, the linkage between molecular markers and target genes can break down due to genetic recombination, reducing the accuracy of MAS. This is particularly relevant when markers are developed in one population and applied to another.

6. Genetic diversity and transferability: molecular markers developed in one plant population may not be directly transferable to other populations due to differences in genetic background and marker polymorphism. This can limit the broad applicability of certain markers.

7. Ethical and regulatory concerns: the use of genetically modified organisms (GMOs) or markers associated with patented genes can raise ethical and regulatory challenges. This can complicate the adoption of marker-assisted breeding strategies.

8. Limited trait information: for many traits, the underlying genetic mechanisms are not fully understood. In such cases, even if markers are available, their effective use in breeding may be limited by incomplete knowledge of the trait’s genetic basis.

9. Data analysis and interpretation: managing and analyzing large datasets generated by high-throughput genotyping technologies can be computationally demanding and require specialized expertise. Misinterpretation of results can lead to erroneous breeding decisions.

10. Public acceptance: some consumers and stakeholders may have concerns about the use of molecular marker technology in breeding due to perceptions of genetic modification or other safety concerns. Public acceptance can influence the adoption of marker-assisted breeding practices.

11. Breeding timeframes: while molecular markers can expedite the breeding process, developing and validating markers, as well as conducting MAS, still require time. The overall breeding process can be constrained by other factors, such as crop growth cycles.

Despite these challenges and limitations, molecular marker technology continues to play a pivotal role in modern plant breeding, enhancing the efficiency and precision of trait selection. Ongoing advancements in genotyping techniques, bioinformatics, and our understanding of plant genetics will likely help address some of these limitations over time.

13. Future perspectives and emerging trends in molecular marker technology

1. High-throughput sequencing and genotyping: advances in high-throughput sequencing technologies have allowed for cost-effective and rapid sequencing of entire genomes. This has led to the development of genotyping-by-sequencing (GBS) approaches, enabling the identification of a large number of molecular markers across the genome. This trend is likely to continue with improvements in sequencing platforms and data analysis techniques.

2. Precision breeding and genome editing: molecular markers play a critical role in precision breeding, where specific genetic traits can be targeted for modification using genome editing techniques like CRISPR-Cas9. Molecular markers are used to track the edited genes and to ensure the absence of unintended modifications. This technology holds promise for creating new crop varieties with improved traits.

3. Phenomics and genomics integration: integrating genomic data with high-throughput phenotyping data is becoming essential in modern plant breeding. This enables breeders to associate specific genetic markers with desirable phenotypic traits, leading to more informed selection and breeding decisions.

4. Multi-omics approaches: the integration of multiple omics datasets, including genomics, transcriptomics, proteomics, and metabolomics, allows for a more comprehensive understanding of the genetic and molecular mechanisms underlying complex traits. This approach can provide deeper insights into plant biology and aid in identifying key regulatory pathways for targeted improvement.

5. Big data and machine learning: the growing volume of genomic and phenotypic data requires sophisticated computational tools for analysis. Machine learning algorithms can help identify patterns, predict trait outcomes, and assist breeders in selecting the best candidates for breeding programs.

6. Marker-assisted selection (MAS) and genomic selection (GS): these approaches continue to gain traction. MAS involves using specific markers linked to known traits of interest, while GS uses a dense set of markers across the genome to predict an individual’s breeding value. These techniques enhance the efficiency of selection by reducing the need for time-consuming phenotypic evaluations.

7. Functional genomics and trait discovery: molecular marker technology is being used in conjunction with functional genomics approaches to decipher the roles of specific genes in trait expression. This can accelerate the discovery of genes responsible for important agronomic traits.

8. Disease resistance and stress tolerance: the identification of markers associated with disease resistance and stress tolerance is of significant interest in plant breeding. Developing crops that are resilient to biotic and abiotic stresses is crucial for ensuring food security.

9. Epigenetics and epigenomics: epigenetic modifications can play a significant role in gene expression and phenotypic variation. Integrating epigenetic data into breeding programs can provide insights into transgenerational effects and potentially improve trait stability.

10. Public-private partnerships and open data sharing: collaboration between public institutions, private companies, and researchers is important for advancing molecular marker technology. Open data sharing fosters innovation and accelerates progress in crop improvement.

14. Conclusions

Molecular marker analysis has significantly advanced plant breeding by enabling breeders to efficiently select and manipulate desired traits. The choice of marker type and analysis technique depends on factors like the target species, available resources, and research objectives. Advances in genomics and technology continue to shape the field, with newer marker types and genotyping platforms being developed over time. Marker-assisted backcrossing is a valuable tool in modern plant breeding that facilitates the efficient transfer of specific traits from donor parents to recipient parents. It combines traditional breeding methods with molecular marker technology, allowing breeders to make precise selections and shorten the time required to develop improved plant varieties with desired traits. Marker-assisted selection has transformed the field of plant breeding by enabling breeders to make more informed and targeted selections in developing improved crop varieties. Its applications are vast and continue to expand, ranging from disease resistance to quality improvement, drought tolerance to yield enhancement, and much more. This technology has played a significant role in accelerating the development of crops that can address the challenges of a changing world.

High-throughput genotyping platforms have significantly advanced plant breeding efforts by providing researchers with powerful tools to dissect the genetic basis of important traits. Over the years, various researchers have contributed to the development and refinement of these platforms, enabling the acceleration of crop improvement through targeted breeding approaches. As technology continues to evolve, genotyping platforms are likely to become even more sophisticated and accessible, facilitating more efficient and precise plant breeding strategies. The power of GBS and NGS technologies in advancing plant breeding research and providing valuable insights into the genetic basis of plant traits and diversity. As technology continues to evolve, these methods will likely play an even more significant role in shaping the future of plant breeding.

In conclusion, molecular marker technology represents a paradigm shift in the field of plant breeding, offering a powerful set of tools that enhance the efficiency, precision, and effectiveness of crop improvement efforts. By enabling breeders to identify and manipulate desired traits at the molecular level, this technology has ushered in a new era of agriculture, where the challenges of global food security and sustainability can be tackled with greater agility and innovation. As our understanding of plant genomics deepens and molecular techniques continue to evolve, the potential for developing novel and improved plant varieties becomes increasingly promising.

Conflict of interest

The authors declare no conflict of interest.