Molecular markers and marker-assisted selection
DNA markers (RFLP, RAPD, AFLP, SSR, SNP) compared, how MAS and marker-assisted backcrossing work, gene pyramiding, and Indian rice successes.
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Conventional breeders select plants by what they can see or measure, such as height, disease symptoms or yield. Many important traits are hard to see. Some only show up under a particular disease race, after a drought, at maturity, or in the grain chemistry. A molecular marker is a DNA sequence variant whose position in the genome is known. If a marker lies very close to a gene for a desirable trait, the two are inherited together, so checking the marker in a leaf sample tells the breeder whether the gene is present. This is marker-assisted selection (MAS).
MAS lets breeders select at the seedling stage, in any season, without disease screening. It can distinguish homozygous from heterozygous plants and stack several genes that would be impossible to tell apart by eye.
Types of markers
Markers can be morphological (visible traits such as flower colour), biochemical (isozymes and seed storage proteins) or DNA-based. DNA markers are the most useful because there are almost unlimited numbers of them and the environment does not affect them.
| Marker | Basis | Dominance | Notes |
|---|---|---|---|
| RFLP | Restriction digestion and probe hybridisation | Co-dominant | First DNA marker (1980s); slow, needs much DNA |
| RAPD | PCR with short random primers | Dominant | Quick and cheap but poorly reproducible |
| AFLP | Selective PCR of restriction fragments | Dominant | Many bands per reaction; used for fingerprinting |
| SSR (microsatellite) | PCR across short tandem repeats such as (CA)n | Co-dominant | Multi-allelic, reproducible; long the workhorse of MAS |
| SNP | Single-base differences | Co-dominant | Most abundant; suited to high-throughput chips and KASP assays |
A co-dominant marker shows both alleles, so heterozygotes can be identified. A dominant marker shows only whether a band is present or absent. A good MAS marker is polymorphic between the parents, co-dominant, cheap, reproducible and tightly linked to the target gene, ideally less than 1–2 cM away. The best case is a gene-based or functional marker, which lies within the gene itself.
From gene to marker
Finding a usable marker involves several steps:
- Mapping the trait in a segregating population, such as an F2, recombinant inbred lines or doubled haploids. A single major gene is mapped like any Mendelian locus. Traits controlled by several genes are mapped as QTLs (quantitative trait loci) using statistical interval mapping.
- Validating the marker in other genetic backgrounds, to confirm it still predicts the trait.
- Converting the marker into a cheap, high-throughput assay.
Linkage is never perfect. If a marker is r cM from a gene, a recombination between them occurs with frequency of about r ⁄ 100 per meiosis, and selection can go wrong. Using flanking markers, one on each side of the gene, reduces errors, because a double recombination is very rare.
How reliable is a linked marker?
A marker is 5 cM from a resistance gene (r = 0.05). Selecting F2 plants homozygous for the marker allele, the chance that a plant is also homozygous for the gene is about (1 − 0.05)2 ≈ 0.90, so about one in ten selected plants could be wrong. With flanking markers 5 cM on each side, a plant is misclassified only if both intervals recombine, about 0.05 × 0.05 = 0.0025 per gamete. That is why breeders prefer flanking or gene-based markers.
Uses of MAS
Marker-assisted backcrossing (MABC)
MABC is the most successful use of markers. It uses three levels of selection:
- Foreground selection checks for the target gene.
- Recombinant selection looks for crossovers close to the gene on either side, which reduces linkage drag: unwanted donor DNA carried along with the gene.
- Background selection uses markers spread across the genome to pick plants with the most recurrent-parent DNA.
With background selection, the recurrent parent genome can be recovered in two or three backcrosses rather than six.
Gene pyramiding
Several genes for resistance to the same disease can be combined in one variety. This is hard by phenotype, because once one gene gives resistance the others cannot be seen. Pyramided resistance is more durable.
Indian examples
- Improved Samba Mahsuri (ICAR–IIRR and CCMB, released 2008) carries three bacterial blight resistance genes (Xa21, xa13 and xa5) in a popular fine-grain rice.
- Improved Pusa Basmati 1 (Pusa 1460, released 2007) carries xa13 and Xa21.
- Swarna-Sub1 has the Sub1 gene for submergence tolerance and can survive about two weeks under water.
- Other examples include drought-tolerant rice with yield QTLs, salt-tolerant varieties with Saltol, and pearl millet hybrid HHB 67 Improved, with downy mildew resistance added by MABC.
Limitations
- Markers must be developed and validated for each trait and population.
- MAS works best for genes with large effects. For yield and other traits controlled by many small-effect genes, QTL-based MAS has had limited success. This is the reason for genomic selection, which uses thousands of markers at once.
- Laboratory costs and skills can limit adoption in smaller programmes, although SNP genotyping services have made it much cheaper.
Key points
- A molecular marker is a mapped DNA variant; MAS selects plants through markers tightly linked to target genes.
- RFLP and SSR markers are co-dominant; RAPD and AFLP are dominant; SNPs are the most abundant and best suited to automation.
- An ideal marker is polymorphic, co-dominant, reproducible and tightly linked, preferably within the gene.
- MABC uses foreground, recombinant and background selection to transfer a gene with minimal linkage drag.
- Gene pyramiding stacks resistance genes, as in Improved Samba Mahsuri (Xa21, xa13, xa5).
- MAS works for major genes; genomic selection targets complex traits.
What the exams ask
- Classify molecular markers and compare dominant and co-dominant markers.
- Explain foreground and background selection in MABC.
- Define QTL, linkage drag and gene pyramiding.
- Name Indian varieties developed through MAS and the genes they carry.
- List the properties of an ideal marker.
Described in general terms: the kind of examination, not a past paper. Laisha never reproduces exam questions.
Questions people ask
Are marker-assisted varieties genetically modified?
No. MAS only uses DNA tests to choose which plants to keep. The genes come from normal crosses within the species, and no foreign DNA is inserted, so MAS varieties are regulated like conventional varieties.
What is a cM?
A centimorgan is the distance between two loci at which 1% of gametes are recombinant, per meiosis. Over short distances, cM and percentage recombination are roughly equal. The physical length of DNA per cM varies widely between species and chromosome regions.
Why use SSRs if SNPs are better?
SSRs need only simple PCR and a gel, so any laboratory can run them, and each SSR often has several alleles. SNPs require more specialised equipment, but they become cheaper and faster than SSRs once thousands of samples or markers are involved. Many programmes have now switched to SNP assays such as KASP.
Sources
- Collard, B. C. Y. and Mackill, D. J. "Marker-assisted selection: an approach for precision plant breeding in the twenty-first century." Philosophical Transactions of the Royal Society B 363, 557–572 (2008).
- Sundaram, R. M. et al. "Marker assisted introgression of bacterial blight resistance in Samba Mahsuri, an elite indica rice variety." Euphytica 160, 411–422 (2008).
- Singh, B. D. and Singh, A. K. Marker-Assisted Plant Breeding: Principles and Practices. Springer, 2015.
- Hospital, F. "Selection in backcross programmes." Philosophical Transactions of the Royal Society B 360, 1503–1511 (2005).
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