Authors: Deepak V Pawar1, Mahesh Mahajan1, Rakesh Kumar Prajapat1, Kishor U Tribhuvan1
1ICAR-NRCPB, I.A.R.I, New Delhi-12
Development of new crop varieties can take almost 25 years. However, biotechnology has considerably shortened the time by 7-10 years for new varieties to be brought to market. One of the tools that make it easier and faster to select plant traits is marker-assisted selection (MAS).
Molecular shortcut
The differences that distinguish one plant from another are encoded in the plant’s genetic material, the DNA. These are packaged in chromosome pairs, one coming from each parent. The genes, which control characteristics, are located on specific segments of each chromosome. Together, all of the genes make up the genome. Some traits may be controlled by only one gene. Others, however, like crop yield or starch content, may be influenced by several genes. Traditionally, plant breeders select plants based on visible or measurable traits, called the phenotype. However, this can be difficult, slow, and costly. Plant breeders now use marker-assisted selection (MAS). To identify specific genes, scientists use molecular or genetic markers. Markers are sequences of nucleic acid which makes up a segment of DNA. Markers are located near the DNA sequence of the desired gene and are passed from generation to generation together with the desired gene. This is called genetic linkage. The presence of the marker also indicates the presence of the desired gene. As scientists learn the locations of markers on a chromosome and their distance to genes, they create a genetic linkage map. This would show locations of markers and genes, and their distance from other genes.
Using detailed genetic maps, researchers are able to determine if a plant has the desired gene using just a piece of plant tissue from seedlings. If a plant doesn’t have the desired gene, they are discarded until they only have plants with the gene. However, molecular breeding through MAS is limited in scope compared to genetic engineering or modification because:
1. it works only for traits already present in a crop
2. it cannot be used effectively to breed crops with long life cycles and
3. it cannot be used effectively with crops that are propagated through cloning.
Molecular markers
Several marker systems have been developed and are applied to various crop species. These are the Restriction Fragment Length Polymorphisms (RFLPs), Random Amplification of Polymorphic DNAs (RAPDs), Sequence Tagged Sites (STS), Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs) or microsatellites, and Single Nucleotide Polymorphism (SNPs). The advantages and disadvantages of these marker systems are shown in Table 1. These techniques have been used to check differences in DNA sequences in and among species. They also allow the creation of new sources of variation by introducing desirable traits from wild varieties. While RFLP markers have been the basis for most genetic work in crops, AFLPs and SSRs are currently the most popular techniques used due to ease in detection and automation. The adoption of the new marker system, SNPs, is now highly preferred, with the increasing amount of sequence information, and the determination of gene function due to genomic research.
| Feature | RFLPs | RAPDs | AFLPs | SSRs | SNPs |
| DNA required (ug) | 10 | 0.02 | 0.5-1.0 | 0.05 | 0.05 |
| DNA quality | High | High | Moderate | Moderate | High |
| PCR-based | No | Yes | Yes | Yes | Yes |
| Number of polymorphic loci analyzed | 1.0-3. | 0 1.5-50 | 20-100 | 1.0-3.0 | 1.0 |
| Ease of use | Not easy | Easy | Easy | Easy | Easy |
| Amenable to Automation | Low | Moderate | Moderate | High | High |
| Reproducibility | High | Low | High | High | High |
| Development cost | Low | Low | Moderate | High | High |
| Cost per analysis | High | Low | Moderate | Low | Low |
Table 1. Comparison of most commonly used marker systems
Applications of molecular markers for crop genetic studies
The main uses of these molecular markers in crop genetic studies are as follows:
- Assessment of genetic variability and characterization of germplasm
- Identification and fingerprinting of genotypes
- Estimation of genetic distances between population, inbreeds, and breeding materials
- Detection of monogenic and quantitative trait loci (QTL)
- Marker-assisted selection
- Identification of sequences of useful candidate genes
The decisions in conventional plant breeding are based on phenotypic evaluation for the target traits. The value of a quantitative trait phenotype for selection depends on the heritability of the trait. Therefore, quantitative traits have to be evaluated in replicated trials preferably conducted under different environments. This increases the evaluation costs and limits the trials to such locations and seasons that allow meaningful expression of the concerned traits. Therefore, off-season nursery and greenhouse facilities cannot be used for selection for traits like yield. Further, traits like fruit/seed characteristics and yield can be evaluated only at maturity. As a result, the selected plants cannot be used for hybridization in the same generation/season. The phenotypic evaluation for many traits may require specific environments, including inoculation with a specific race of the concerned pathogen. The creation of some environments may be difficult or demanding. In addition, phenotypic evaluation for some traits may take time, may be tedious or may be expensive. In some cases, the results from phenotypic evaluation may not be reliable due to the environmental effects.
One of the chief limitations of phenotype based breeding is the non-availability of an effective selection scheme during the early segregating (F2–F4) generations from crosses. Since individual plants are selected in these generations, selection is effective only for highly heritable traits. Another major limitation relates to the selection of parents for hybridization for the improvement of quantitative traits. A variety of approaches based on performance of the parents themselves or of the progeny (F1 or a later generation) from their crosses have been proposed, but none of them is effective in all the cases.
MAS for pathogen resistance in tomato
One of the major problems in tomato cultivation are severe harvest losses caused by several pathogens, including viruses, bacteria, fungi, and nematodes. Although conventional breeding has had a significant impact on improving tomato resistance, the long duration of breeding makes it difficult to cope with new virulent pathogens. Molecular markers are now being used for breeding tomato. More than 40 genes that confer resistance to tomato pathogens have been mapped, cloned, and/or sequenced. These maps have allowed for “pyramiding” resistance genes in tomato through MAS, where several resistance genes can be engineered into one genotype. Currently, tomato breeding through MAS has resulted in varieties with resistance or tolerance to one or more specific pathogens.
About Author / Additional Info:
I am PhD research scholar, pursuing PhD at IARI, New Delhi in the discipline of Molecular Biology and Biotechnology. I am working on blast disease resistance in O. sativa