Development and use of molecular markers in plant breeding | Botany Optional for UPSC PDF Download

Use of Molecular Markers in Breeding Programmes

• Marker-aided selection is a breeding technique that involves using genetic markers closely associated with desired traits or genes to indirectly select for those traits in generations of plants where traits segregate or do not segregate. In its simplest form, it can be used as a substitute for evaluating traits that are challenging or costly to assess directly. When a marker is identified that co-occurs with a major gene responsible for an important trait, it can be more convenient and cost-effective to check for the presence of the marker allele linked to the gene instead of assessing the trait itself. Periodic confirmation of the linkage between the marker and the gene is necessary.
• For more complex traits controlled by multiple genes (polygenic traits), breeders face the challenge of combining as many beneficial alleles as possible for the Quantitative Trait Loci (QTLs) that have been detected. In such cases, breeding material can be screened for markers linked to these QTLs. Using this analysis, specific crosses can be planned to create an optimal genotype by merging QTL alleles from various sources. However, applying marker-assisted selection within the current breeding material does not address the issue of limited genetic diversity often found in breeding stocks.
• A different use of marker-aided selection can contribute to the genetic enrichment of breeding material. This involves using markers to help control the introduction of new genetic material. Wild species often carry desirable traits that may be absent in cultivated varieties. These traits can be transferred to elite cultivated material through repeated backcrossing. However, breeders may be hesitant to employ this method due to the unpredictable transfer of unwanted genes, known as linkage drag, alongside the genes controlling the target trait. Eliminating these unwanted genes and restoring the material to an acceptable agronomic value can be a time-consuming process. Markers can assist in identifying the genetic factors responsible for the desired characteristics in the unadapted material. In a backcrossing program, the presence of desired QTL alleles can be continuously confirmed by observing linked markers.

Marker assisted selection (MAS)

• Marker-assisted selection (MAS) is a highly effective tool primarily suited for traits that are challenging to directly select for, such as disease resistance, salt tolerance, drought tolerance, heat tolerance, and quality traits like the aroma of basmati rice or the flavor of vegetables. 
• The MAS approach involves selecting plants in the early generations that possess a fixed and favorable genetic background at specific genetic locations. This selection process is conducted on a large scale using genetic markers while striving to preserve as much as possible the diversity of alleles within the population. Large populations are screened to achieve the objectives of the breeding program. 
• It's important to note that selection is exclusively focused on the targeted genomic regions to maintain the segregation of alleles among the selected genotypes following Mendelian principles. Following the selection with DNA markers, the genetic diversity at unselected loci may still provide opportunities for breeders to develop new varieties and hybrids through traditional breeding methods in alignment with the goals of the breeding program.

Materials Required for Marker-Assisted Selection (MAS) 

To implement Marker-Assisted Selection (MAS), several essential components are needed: molecular markers, a collection of authentic plant lines carrying the desired trait, and a population for validating the markers, typically the F2 or BCF2 generation for each specific trait or gene.
The fundamental prerequisites for MAS are as follows:

• Identification of molecular markers associated with the target trait.
• Validation of these available markers in both parental plants and the breeding population.
• In cases where markers are not readily available, they need to be designed and validated before use. If mapping populations are not already in hand, this process may take 2-4 years.
• Development of a selection plan and breeding strategy.
• Determination of the minimum population size to be assessed to capture all beneficial alleles.
• Diligent record-keeping throughout the selection process.
• Progeny testing to confirm the fixation of desired traits.

Steps Involved in Marker-Assisted Selection (MAS) 

The process of MAS involves several key steps:

• Marker Validation: Initially, DNA is extracted from test individuals to confirm the one-to-one relationship between the marker and the trait of interest.
• DNA Extraction and MAS Application: DNA is extracted from the individuals in the breeding population at the seedling stage, and MAS is applied. Selection of individuals is based on the presence of the desired molecular markers associated with the target trait. For other traits, selection follows classical breeding methods. The number of individuals to be assessed should align with the defined strategy and statistical considerations.

Limitations of Marker-Assisted Selection (MAS) 

While MAS offers significant benefits, it also has its limitations:

• Cost Factor: MAS can be expensive due to the need for specialized equipment and resources.
• Technical Skill: Implementation of MAS requires technical expertise in molecular biology and genetics.
• Automation: To maximize the benefits of MAS, automated techniques are often necessary.
• Environmental Effects: DNA markers themselves are not influenced by the environment, but the traits they represent can be affected by environmental factors and exhibit gene-environment interactions (G x E). Therefore, when developing markers, phenotyping should be conducted in multiple environments, and the implications of G x E interactions should be understood and considered when using markers.
• Marker Validation: Each breeding population must validate DNA markers individually. Making assumptions about the markers' validity without proper validation can lead to significant issues.

 Marker assisted backcross breeding

• Marker-assisted backcross breeding is a breeding approach that focuses on transferring specific genes from a "donor" line into the genetic makeup of a "recipient" line. This technique has gained significant attention in recent years.
  Molecular markers play a crucial role in these programs by serving two main purposes:
  • Foreground Selection: Molecular markers are used to confirm the presence of the transferred gene of interest when direct evaluation of the trait is not feasible, is costly, or can only be conducted at a later stage of development.
  • Background Selection: Markers are also employed to expedite the process of restoring the genetic background of the recipient parent at other loci. This ensures that while the desired gene is being introduced, the overall genetic makeup of the recipient parent is maintained or gradually restored.
• The assumption underlying this approach is that the presence of the introgressed gene can be accurately detected, and in the theoretical study discussed, the primary focus was on background selection.
• Experimental evidence has demonstrated the efficiency of using molecular markers for background selection in backcross breeding programs. By recurrently backcrossing and introducing favorable alleles of Quantitative Trait Loci (QTLs), the economic value of a plant line can be significantly improved. However, recent findings indicate that many economically important traits are influenced by QTLs with relatively modest effects. In such cases, the economic benefits derived from introducing the favorable allele of a single QTL may not be competitive when compared to the improvements achieved through traditional breeding methods over a similar timeframe.
• The competitive advantage of marker-assisted introgression of superior QTL alleles can be realized when multiple QTLs can be manipulated simultaneously, making it a viable alternative to conventional phenotypic selection. This approach allows for the simultaneous improvement of multiple traits, making it a powerful tool in plant breeding when dealing with complex traits influenced by multiple genes.

Selection scheme for MAS breeding

• The selection strategy for Marker-Assisted Selection (MAS) breeding involves choosing plants in the early generations with a predetermined favorable genetic makeup at specific genetic locations. Subsequently, a comprehensive marker-assisted selection process is conducted, focusing on specific target loci, while striving to maintain genetic diversity in the rest of the genome.
• This scheme consists of two main phases:
  • Identification of Elite Lines: Initially, it is essential to identify elite lines with high allelic diversity and exceptional traits. This step enables the capture of beneficial alleles from different parental lines.
  • Development of Segregating Populations: Following the identification of promising genomic regions in each selected parental line, these lines are crossed to create segregating populations. From these populations, plants homozygous for favorable alleles at the target loci are carefully selected.
• The primary objective of this scheme is to conduct marker-assisted selection only once, thereby minimizing the need for multiple rounds of selection. To achieve this, a minimal number of plants are selected to maintain sufficient allelic diversity at the unselected loci. Consequently, significant selection pressure is applied to the segregating population, necessitating the screening of large populations to achieve the breeding goals. Importantly, no selection is applied outside of the specified target genomic regions to preserve Mendelian allelic segregation among the selected genotypes as effectively as possible.

Application of DNA markers in crop improvement

QTL mapping

• Plant breeding encounters significant challenges when it comes to improving traits that exhibit a continuous range of values. These traits, known as quantitative traits, are influenced by multiple genetic factors, and the genetic elements responsible for part of the observed variation are called quantitative trait loci (QTLs). QTLs represent regions on the genome and can encompass single genes or clusters of linked genes, but they differ from individual genes in that they denote genomic regions containing functional genes. Quantitative traits, such as yield, plant height, and flowering time, are crucial in breeding, but they are challenging to work with because the relationship between the observed trait values (phenotype) and the underlying genetic makeup (genotype) is not straightforward. Typically, quantitative traits are controlled by numerous genes, each contributing a small portion to the overall variation. Additionally, environmental variations further complicate the relationship between phenotype and genotype. To address this complexity, large and replicated trials are often conducted to identify genetic differences through statistical analysis.
• Plant breeders aim to utilize the genetic factors responsible for the observed variability in quantitative traits. QTL mapping is a process used to analyze the association between observed trait values and the presence or absence of alleles of molecular markers that have been mapped onto a linkage map. When a clear and significant correlation is established, it is concluded that a QTL has been detected. The size of the effect of the identified QTL can also be estimated.
  The process of identifying molecular markers associated with QTLs involves several steps:
  • Crossbreeding to generate marker data.
  • Constructing linkage maps of molecular markers.
  • Collecting phenotypic data for the QTL trait across different environments in replicated trials.
  • Mapping the QTL.
• The most common method for determining the association between markers and QTLs is through the analysis of phenotypic trait observations and the scoring of molecular data using one-way analysis of variance and regression analysis. In this approach, each marker defines a marker class based on the presence of a specific DNA fragment. If the variance attributed to a particular marker class is significant, the molecular marker associated with that class is considered to be linked to a QTL. Regression values are calculated for markers associated with the quantitative trait, reflecting the extent of the total genetic variation explained by each specific molecular marker.
• Despite the significance of QTL mapping in plant breeding, there are relatively few examples of QTL mapping in pulse crops. However, in chickpea, lentil, mungbean, and other crops, researchers have successfully identified QTLs associated with traits like yield, plant height, disease resistance, and drought tolerance, using various types of molecular markers. These discoveries contribute to the understanding and improvement of quantitative traits in crop breeding programs.

Tagging of disease resistance genes

• DNA markers have proven to be invaluable tools in expediting plant breeding efforts, particularly in the identification of molecular markers closely associated with resistance genes. This enables the efficient incorporation of key genes into elite crop varieties, ultimately reducing costs and speeding up the breeding process. Once resistance genes are tagged with molecular markers, selecting plants with resistance traits in segregating generations becomes much simpler.
• In one example involving chickpea, a linkage map was created using 354 molecular markers, including various types such as STMSs, DAFs, AFLPs, ISSRs, RAPDs, isozymes, cDNAs, SCARs, and markers linked to fusarium wilt resistance. This map was developed from 130 recombinant inbred lines resulting from a cross between C. arietinum and C. reticulatum. The map not only located genes conferring resistance to fusarium wilt races 4 and 5 but also revealed clustering of several fusarium wilt resistance genes, providing a foundation for Marker-Assisted Selection (MAS) and the search for other valuable genes.
• Similarly, DNA markers associated with resistance to fusarium wilt races 4 and 5 in chickpea were identified from a population of 131 recombinant inbred lines derived from a cross between Cicer arietinum and Cicer reticulatum. Through bulk segregant analysis, researchers discovered nineteen new markers in proximity to the fusarium wilt resistance genes. These findings have practical implications for MAS in chickpea breeding programs.
• In pigeonpea, DNA markers have been employed to identify markers linked to resistance genes for diseases like sterility mosaic disease (PPSMD). By using techniques like bulk segregant analysis, researchers were able to pinpoint RAPD markers associated with PPSMD resistance, simplifying the selection process for resistant plants.
• Similar approaches have been applied in other crops like lentil, pea, and Vigna species to identify markers linked to resistance genes for various diseases. These markers hold promise for accelerating breeding programs by allowing for more efficient selection of plants with desired disease resistance traits.
• In summary, DNA markers closely linked to disease resistance genes have revolutionized plant breeding by expediting the process of incorporating valuable traits into elite crop varieties, making it more cost-effective and efficient. These markers simplify the selection of plants with specific resistance traits in segregating populations, streamlining the breeding process.

Tagging of male sterility genes

• The development of a cytoplasmic male sterile (CMS) system is highly advantageous for hybrid seed production because it eliminates the labor-intensive process of manual emasculation. CMS is a trait that is inherited maternally and is characterized by the inability to produce viable pollen while leaving female fertility unaffected. It is often associated with rearrangements, mutations, and editing of mitochondrial DNA. To better understand and utilize the CMS system in various crops, several restorer loci have been identified using techniques like Random Amplified Polymorphic DNA (RAPD) and Sequence-Tagged Sites (STS). DNA markers linked to these loci facilitate the molecular study of CMS.
• These co-dominant markers play a crucial role in identifying homozygous restorer genotypes following the backcrossing process, which is essential for producing restorer lines. This molecular approach allows for the expedited development of restorer lines compared to traditional breeding methods. In one study conducted by Souframanien et al. in 2003, a RAPD marker linked with the male sterility gene was identified. Specifically, the primer OPC-11 produced a unique DNA fragment of 600 base pairs in male sterile (A) lines, such as 288A (derived from C. scarabaeoides) and 67A (derived from C. sericeus). This amplicon was absent in their respective maintainer and putative restorer (R) lines, represented as TRR 5 and TRR 6. Furthermore, when assessing the genetic distance based on a similarity index, substantial genetic variation was observed between male sterile lines, the two putative restorer lines, and the donors of male sterility genes. This finding underscores the potential of DNA markers in elucidating the genetics of male sterility and facilitating the efficient production of restorer lines.

Diversity Evaluation

• The assessment of diversity and the preservation of the stability and identity of crop varieties have become increasingly important, particularly in the context of plant breeder's rights and farmers' rights. Traditionally, the evaluation and conservation of biodiversity and genetic variability have relied on methods such as comparative anatomy, morphology, embryology, and physiology. While these methods provide valuable information, they offer relatively low genetic resolution.
• In recent years, molecular biology advancements have provided powerful genetic tools capable of delivering rapid and detailed genetic insights. Molecular marker-based genotyping involves the development of a unique marker profile for an individual, creating an unambiguous pattern for crop varieties known as "DNA fingerprinting." This technique was first developed by Alec Jeffery in 1985 for humans and was subsequently applied to crops, with rice being one of the first examples in 1988 for cultivar identification.
• The choice of molecular markers for DNA fingerprinting depends on factors such as technical expertise, available funds, and specific experimental requirements. However, two critical considerations in the selection of molecular techniques are their discrimination power and reproducibility. Among the commonly used markers, Random Amplified Polymorphic DNA (RAPD) markers are known to have lower discrimination power compared to Simple Sequence Repeat (SSR) and Amplified Fragment Length Polymorphism (AFLP) markers. Microsatellites, or SSRs, have become the preferred method for varietal identification due to their abundance, high polymorphism, and simplicity of use.
• Odeny et al. (2007) demonstrated DNA polymorphism in pigeonpea using 113 primers designed from genomic SSR markers and an additional 220 soybean primers.
• Sivaramakrishnan et al. (2002) showed that Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (mtDNA) can be employed to analyze the diversity of pigeonpea. They used restriction enzyme-digested fragments from 28 accessions representing 12 species of the genus Cajanus, including cultivated and wild species. Intra-specific diversity was observed among wild species and between wild and cultivated species of pigeonpea.
• Saxena et al. (2010b) designed 23 primer pairs from a genomic library enriched with SSRs for pigeonpea. These primers produced expected amplification fragments, and 13 of them were found to be polymorphic among 32 cultivars and 8 wild accessions from six species.
• Ratnaparkhe et al. (1995) developed DNA fingerprints for cultivated and wild pigeonpea accessions using RAPD markers. They found low polymorphism among cultivated species but high polymorphism among wild species, demonstrating the utility of RAPD in genetic fingerprinting of pigeonpea.
• Ganapathy et al. (2011) generated 561 AFLP loci to cluster cultivated and wild pigeonpea accessions. Their analysis indicated greater diversity within wild species, which clustered into several groups, while most cultivated accessions were grouped into a major cluster.
• Hamwieh et al. (2009) developed a new set of microsatellite markers in lentil to assess molecular diversity, and Souframanien and Gopalkrishna (2004) utilized RAPD and IISR markers to deduce genetic diversity among 18 blackgram cultivars.

Heterosis Breeding

• DNA markers have also been utilized in the prediction of heterosis in hybrid plants. Heterosis, also known as hybrid vigor, refers to the phenomenon where hybrid offspring exhibit superior traits, such as higher yield or improved disease resistance, compared to their parent plants. Assessing hybrids for heterosis or combining ability through field trials can be costly and time-consuming. Molecular markers have been employed to establish a connection between genetic diversity and heterosis in various cereal crops, including rice, oats, and wheat.
• Studies have indicated that measures of genetic similarity based on Restriction Fragment Length Polymorphism (RFLP) markers and pedigree information can be employed to forecast superior hybrid combinations. However, the associations between heterosis and DNA-based genetic distance have shown variability, with some investigations revealing weak correlations while others demonstrating stronger correlations.
• As an example, in soybean, a study conducted by Cern et al. (1997) examined the relationship between heterosis and molecular diversity (isozyme and RFLP) among parents in three maturity groups. The results revealed that parental RFLP diversity did not exhibit a significant correlation with mid-parent and better-parent heterosis concerning yield. This suggests that, in the context of soybean, the manifestation of heterosis in yield may not be strongly linked to genetic diversity at the molecular level, as determined by RFLP markers. Nevertheless, isozyme diversity among parents did exhibit an association with yield heterosis, albeit with limited significance due to the reduced number of assessable isozyme loci in soybean.
• In summary, DNA markers have been explored as a tool to predict heterosis in hybrid plants. However, the relationship between molecular-level genetic diversity and heterosis can fluctuate depending on the crop species and the specific markers employed. While some correlations have been observed, it is crucial to consider other influencing factors in breeding programs.

Hybrid seed purity testing

To assess the quality of hybrid seeds, it is essential to confirm that the intended crossbreeding has taken place, that self-pollination between the female parents is within acceptable purity limits, and that the resulting seeds meet the required quality standards. Traditionally, the primary method for verifying hybrid seed purity has involved growing out the seeds and visually inspecting the plants. However, more contemporary approaches employ DNA markers such as RAPD and RFLP to assess the purity of F1 hybrid seeds. For instance, Kumar et al. (2011) utilized male-specific markers like SSR 218, SSR 306, and the Ty2 gene CAPs gene marker to evaluate the F₁ purity of tomato hybrids resulting from crosses like Pbc × EC 538408, Pbc × EC 520061, and H 86 × EC 520061. 

Gene pyramiding

• Gene pyramiding is a method used to incorporate multiple genes into a plant variety, either to provide resistance against different pests or to enhance resistance against a single pest through distinct host pathways. One approach to increase the long-term effectiveness of resistance is to integrate several resistance genes into a single crop variety. When these pyramided genes have not been individually deployed, it is believed that this resistance could remain effective for up to 50 years. However, it can be challenging to confirm the exact number of successfully pyramided resistance genes during the cultivar development process. Interestingly, plants with just one resistance gene can exhibit similar levels of resistance as those with three resistance genes, although the latter might offer more enduring resistance.
• For example, Li et al. (2010) employed gene pyramiding to combine phytophthora tolerance quantitative trait loci (QTLs) in soybean. They created a population of recombinant inbred lines by crossing two soybean cultivars, Conrad and Hefeng 25, known for their phytophthora tolerance. Through the use of 161 SSR markers, they identified seven environmentally stable QTLs (QPRR-1 and QRR2 from Conrad, and QRR3 to QRR7 from Hefeng 25). Interestingly, the higher the number of QTLs integrated, the greater the level of tolerance observed.

Pyramiding of Bt genes

• The use of insecticidal genes known as cry genes, derived from Bacillus thuringiensis (Bt), has been employed for insect control in both biopesticides and genetically modified (transgenic) plants. The discovery of new insecticidal genes is crucial to delay the development of resistance in targeted insects. The wide range of Bt strains makes it possible to isolate new varieties of cry and vip (vegetative insecticidal protein) genes. Polymerase Chain Reaction (PCR) is a valuable tool for rapidly screening Bt strains, allowing for their classification and prediction of insecticidal properties. PCR, along with other analytical methods such as Restriction Fragment Length Polymorphism (RFLP), gene sequencing, electrophoresis, immunological assays, and chromatographic analysis of Cry proteins, as well as insect bioassays to assess toxicity, has been utilized to identify novel insecticidal proteins. 
• Additionally, innovative approaches have been developed. Numerous Bt strains containing unique insecticidal genes have been identified. To create an enhanced biopesticide, a desired combination of Cry proteins can be constructed using site-specific recombination vectors and introduced into a recipient Bt strain. In the context of pest management in transgenic crops, it is often necessary to stack multiple insecticidal genes. Another promising approach involves modifying Cry proteins through protein engineering to increase their toxicity and broaden their spectrum, although this requires a deep understanding of the structure and function of these proteins and the analysis of toxin-receptor interactions. 
• The effective implementation of resistance management practices is essential to maintain the efficacy of Bt transgenic crops, ensuring maximum economic and environmental benefits. For instance, cry1Ac and cry1C Bt genes were introduced into a broccoli line, resulting in hybrid plants that produced stable Cry1Ac and Cry1C proteins at levels comparable to those found in parental plants carrying individual genes. These plants with pyramided cry genes showed no leaf damage and effectively controlled Diamondback Moth (DBM) larvae, even those resistant to Cry1A or Cry1C.

Map based cloning of genes

• Map-based or positional cloning is a method used to identify and isolate specific genes of interest. The first step in this process involves identifying a molecular marker that is located close to the target gene. Initially, a gene search is conducted using a small mapping population. Subsequently, it is essential to create a highly saturated genetic map in order to successfully clone the gene.
• Once an initial marker is identified, the region around this marker is saturated with markers that have a high density. A large number of individuals are then screened to find a marker that rarely recombines with the gene of interest. The next step involves screening a large-insert genomic library, such as a BAC (Bacterial Artificial Chromosome) or YAC (Yeast Artificial Chromosome) library, with the closely linked molecular markers that were identified earlier. The aim is to isolate clones from the library that hybridize with the marker.
• When two flanking markers that are linked to the target gene are identified, researchers use a technique known as chromosomal walking to locate the target gene. The objective is to find clones that contain a set of flanking markers that co-segregate with the gene of interest. These potential clones are then introduced into individuals that lack the target gene. If a transgenic organism is able to rescue the mutant phenotype, it indicates that the newly cloned gene is likely the target gene. Subsequent detailed molecular and biochemical analyses are conducted to characterize this gene.
• As an example, map-based cloning was used to clone the Pto gene (which confers resistance to bacterial speck disease) in tomatoes. Initially, a genetic population of 251 F2 plants was screened using DNA probes, and the TG 538 locus was found to cosegregate with the Pto gene. A YAC library was screened with the TG 538 probe, leading to the identification of the clone PTY538-1. When susceptible plants were transformed with PTY 538, they exhibited a recovered resistant phenotype.