The work, led by Sunita Munda with contributions from Rahul Chandrakant Kaldate, Priyabrata Sen, Sanjay Kumar Chetia and Jyoti Lekha Borah, targets the bacterial blight resistance genes xa5, xa13 and Xa21, the blast resistance gene Pi2, and the drought tolerance quantitative trait locus qDTY2.1. These loci represent some of the most valuable tools in rice breeding. Xa21, first genetically characterized in the early 1990s, confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae, the bacterium behind bacterial leaf blight, while xa5 and xa13 contribute complementary, recessive and semi-recessive layers of defense. Pi2 belongs to a well-mapped resistance gene family that recognizes the rice blast fungus Magnaporthe oryzae, and qDTY2.1 is one of a set of quantitative trait loci shown in earlier multi-institutional work to maintain yield under reproductive-stage drought.

Gene pyramiding, the practice of combining multiple resistance or tolerance genes in one line, is the standard strategy for building durable, climate-resilient cultivars. The bottleneck is not identifying the genes but tracking them through breeding populations. In conventional marker-assisted selection, each locus is assayed separately by polymerase chain reaction, meaning a single plant carrying six target loci would need six independent reactions, six sets of reagents and six lanes of gel electrophoresis. When thousands of seedlings from a backcross or recombinant population must be genotyped, the costs of enzymes, primers, plastics and technician hours multiply rapidly, and the pace of a breeding cycle is dictated by the slowest screening step.

Multiplex PCR addresses this by amplifying several targets in a single tube, but the technique is notoriously finicky. Primers designed for separate assays can bind each other, compete for the same nucleotides, or produce bands of overlapping sizes that cannot be distinguished on a gel. Annealing temperatures that suit one primer pair may be wrong for another. The Indian team therefore began with six candidate markers, including a functional marker for the blast resistance gene Pi54 alongside those for the five target loci, and evaluated whether they could coexist in one reaction. Parental polymorphism screening, the first gatekeeping step in any marker-assisted program, revealed that the Pi54 marker showed no variation between the two parental lines, making it useless for selection in this particular cross and it was excluded from the final assay.

What emerged from the optimization were two workable multiplex combinations: one detecting xa5, xa13, Xa21 and Pi2 simultaneously, and another detecting Xa21, qDTY2.1 and Pi2 together. The researchers validated the system on the parental lines and on real breeding populations, including a BC1F4 population derived from the cross MSM × DRR Dhan 44 and an F7 population from the cross MSM × No. 29. Across these materials, the multiplex assay produced distinct, reproducible bands for all five loci, with segregation patterns among the progeny that were clear enough to classify individual plants by the alleles they carried. Band intensity and specificity remained high despite the crowded reaction environment, indicating that primer concentrations and cycling conditions had been successfully balanced.

Critical to the credibility of the new assay is its agreement with the gold standard. The genotypic profiles generated by the multiplex PCR matched the results of conventional single-locus, or monoplex, PCR for the selected breeding lines, confirming that compressing the reactions had not introduced genotyping errors. In molecular breeding, where a single misclassification can propagate a wrong allele through generations of crossing, this kind of concordance is the difference between a laboratory curiosity and a tool a breeding program can actually deploy. The authors report that the optimized system exhibited high amplification efficiency and supported the effectiveness of marker-assisted selection for accelerating the development of pyramided lines.

The economics are equally significant. By consolidating what would have been five separate reactions into fewer tubes, the multiplex system reduced reagent consumption and labor by 60 to 75 percent. For publicly funded breeding institutes in rice-growing countries of South and Southeast Asia, where budgets per marker data point are often the binding constraint, savings of this magnitude translate directly into more plants screened per season and faster delivery of improved varieties to farmers. The study builds on earlier demonstrations, including a single-tube functional marker assay for the three bacterial blight genes published in Rice Science in 2016 and a 2024 cost-effective multiplex assay covering bacterial leaf blight, blast and brown planthopper resistance, extending the concept to a combined biotic and abiotic stress panel.

The inclusion of a drought tolerance QTL alongside disease resistance genes is what makes the system genuinely climate-oriented. Drought is quantitatively inherited and environmentally sensitive, which is why major-effect QTL such as qDTY2.1, previously introgressed into varieties like Pusa 44 to produce drought-tolerant near-isogenic lines, are prized by breeders working on rainfed lowlands. Combining such loci with resistance to bacterial blight and blast in a single genotyping pipeline means a breeder can select seedlings that carry all the desired traits before they ever reach the field, collapsing what would otherwise be sequential screening rounds into one laboratory pass. The work was supported by the Department of Biotechnology, Government of India, under the DBT-NECAB Phase-III program, with field facilities provided by the AAU-Assam Rice Research Institute in Titabar.

Technically, the study illustrates the practical logic of marker system design. Sequence-tagged site markers and simple sequence repeat markers were chosen for their ability to discriminate between the parents, and the final panel was tuned so that amplicon sizes could be resolved on standard agarose gels, avoiding the need for expensive capillary instrumentation. The exclusion of the monomorphic Pi54 marker is a useful cautionary tale: a marker that performs brilliantly in one genetic background can be blind in another, and parental polymorphism screening remains an indispensable first step before any multiplex panel is locked in. The authors note that no datasets beyond those described in the study were generated or analysed, and they declare no competing interests.

For a world in which rice blast and bacterial blight continue to cause yield losses measured in millions of tonnes annually, and in which drought increasingly shapes planting decisions across Asia and Africa, tools that compress the breeding cycle carry outsized importance. This multiplex marker system does not discover new genes; its contribution is infrastructural, turning a laborious six-assay workflow into a fast, economical routine that a modestly equipped laboratory can run at scale. As gene pyramiding becomes the default strategy for climate-resilient rice, assays of this kind may well become the quiet workhorses of the breeding station, screening thousands of seedlings a season and quietly assembling the genetic armor that tomorrow’s varieties will carry into the field.

Subject of Research: Development of a multiplex PCR marker system for pyramiding disease resistance and drought tolerance loci in rice breeding

Article Title: A Multiplex Marker System for Simultaneous Pyramiding of Bacterial Blight Resistance, Blast Resistance and Drought Tolerance Loci in Rice Breeding Populations

Article References: Munda, S., Kaldate, R. C., Sen, P., Chetia, S. K., & Borah, J. L. (2026). A Multiplex Marker System for Simultaneous Pyramiding of Bacterial Blight Resistance, Blast Resistance and Drought Tolerance Loci in Rice Breeding Populations. Indian Journal of Genetics and Plant Breeding. https://doi.org/10.1007/s44489-026-00050-z

Image Credits: AI Generated

DOI: 10.1007/s44489-026-00050-z

Keywords: rice breeding, multiplex PCR, marker-assisted selection, bacterial blight, blast resistance, drought tolerance, gene pyramiding, xa5, Xa21, Pi2, qDTY2.1, quantitative trait loci

Samba Mahsuri occupies a special place in Indian agriculture. The variety, developed decades ago, is prized for its excellent cooking and eating quality, with slender, fine grains that command strong consumer preference across southern and eastern India. Its popularity, however, has always been tempered by two weaknesses. It matures late, extending the crop’s exposure to end-of-season water shortages, and it is highly sensitive to both drought and flash flooding. In the rainfed lowland agro-ecosystem, where fields depend on erratic monsoon rainfall rather than assured irrigation, these weaknesses translate into devastating yield losses in bad years. Farmers who grow Samba Mahsuri do so for its market value, but they accept a substantial risk every time they sow it.

The strategy the researchers employed is known as QTL pyramiding. Quantitative trait loci, or QTLs, are stretches of the genome that harbor genes influencing complex, quantitatively inherited traits such as yield under stress. Unlike single-gene resistances, drought tolerance is controlled by many loci of small to moderate effect, and breeding programs have identified several major-effect QTLs that reliably improve grain yield when water is scarce. Two of the most consistent are qDTY2.1 on chromosome 2 and qDTY3.2 on chromosome 3, both of which have been shown in previous studies to enhance yield under reproductive-stage drought across multiple genetic backgrounds. By combining these with the Sub1 locus, which confers remarkable tolerance to complete submergence for up to two weeks, the team aimed to build a single variety capable of withstanding the two most damaging water-related stresses of the rainfed lowlands.

The breeding material itself was elegant in design. The researchers crossed two advanced lines, each of which already carried the Sub1 submergence-tolerance locus in the Samba Mahsuri background: SM-Sub1-DTY2.1, which carries qDTY2.1 and is known as DRR Dhan 50, and SM-Sub1-DTY3.2, which carries qDTY3.2. From this cross, they generated a population of 990 F2 plants. Each plant was genotyped using simple sequence repeat markers linked to the two drought-tolerance QTLs, allowing the team to identify individuals that had inherited both genomic regions in homozygous form. Of the 990 plants screened, 62 such QTL-pyramided lines were identified and advanced to the F3 generation, each one now carrying the drought-tolerance loci from both parents alongside the Sub1 submergence-tolerance gene.

A crucial complication soon emerged. Because both donor parents had been selected for earlier maturity than the original Samba Mahsuri, the pyramided lines segregated for time to flowering and maturity. Maturity is not a trivial trait: farmers time their sowing, harvesting, and water management around it, and any new version of a mega-variety must match the photoperiod and duration expectations of the regions where it will be grown. The team therefore carefully selected only those lines showing uniform maturity, advancing them through the F4 to F6 generations until lines true-breeding for both the stacked QTLs and an acceptable maturity class were obtained. This step illustrates a lesson that runs through modern marker-assisted breeding: stacking useful genes is only half the job, and the other half is restoring the agronomic package that made the original variety successful.

Four of the resulting QTL-pyramided lines were then evaluated in detail for agronomic and physiological performance under both control and drought-stress conditions. The researchers measured a battery of traits, including grain yield, plant height, tiller number, leaf rolling, chlorophyll content, and relative water content, comparing the pyramided lines against the recurrent parent Samba Mahsuri and the single-QTL parental lines. The results were unambiguous. The pyramided lines lost significantly less grain yield under drought stress than either Samba Mahsuri or the lines carrying only one drought QTL, confirming that the two loci act additively or even synergistically when combined in the same genetic background. Physiological measurements supported the yield data, indicating that the pyramided lines maintained better plant water status and photosynthetic capacity as soil moisture declined.

The standout performer was a line designated QPL 62. This line showed the least reduction in grain yield under drought of any material tested, and it also matured 16 days earlier than Samba Mahsuri, an advantage that reduces its exposure to terminal water stress and allows earlier harvesting. Just as importantly, molecular profiling showed that QPL 62 retained more than 95 percent similarity to the Samba Mahsuri genetic background, and its grain and cooking quality characteristics remained essentially indistinguishable from those of the original variety. In other words, the breeders had inserted the drought and flood insurance policies without disrupting the qualities that farmers and consumers actually care about. This recovery of the recurrent parent genome is the central promise of marker-assisted backcrossing and pyramiding approaches, and the present study shows it can be achieved even when combining multiple stress-tolerance loci simultaneously.

The implications extend well beyond a single variety. Rainfed lowlands account for a substantial share of rice production in South and Southeast Asia, and climate projections suggest that both the frequency of mid-season droughts and the intensity of flood events will increase in the coming decades. Varieties that combine tolerance to multiple abiotic stresses are therefore a central goal of national and international breeding programs. The Sub1 locus has already been successfully introgressed into numerous popular varieties, including Swarna and Samba Mahsuri itself, producing flood-tolerant versions now grown on millions of hectares. Adding yield-enhancing drought QTLs on top of Sub1, as this study demonstrates in Samba Mahsuri, represents the logical next generation of climate-resilient rice, and the pyramiding framework can in principle be replicated in any elite cultivar for which Sub1 versions and drought-QTL donors are available.

The authors note that QPL 62 is now ready to be taken forward into multi-location QTL near-isogenic line trials, the standard pathway toward commercial release in India. If those trials confirm its performance across diverse environments, the line could eventually be notified as a climate-resilient version of Samba Mahsuri, giving farmers the same prized grain quality with dramatically reduced risk from drought and flooding. The work was supported by the Indian Council of Agricultural Research Network Project on Translational Genomics in Crop Plants and by an Anusandhan National Research Foundation JC Bose National Fellowship. As extreme weather increasingly tests the world’s rice fields, studies like this one show that the tools of genomics can convert a beloved but vulnerable variety into one that can weather the storm, and the flood, on its own genetic merits.

Subject of Research: Marker-assisted pyramiding of drought and submergence tolerance QTLs in rice variety Samba Mahsuri to develop a climate-resilient cultivar.

Article Title: Pyramiding of Multiple QTL for Drought and Submergence Tolerance for Infusing Climate-Resilience in Rice Variety Samba Mahsuri

Article References: Jain, P., Kapoor, R. T., Kumar, M., Yadav, N., Rani, M., Joshi, R., Rai, V., Bisht, D. S., Gaikwad, K., & Singh, N. K. (2026). Pyramiding of Multiple QTL for Drought and Submergence Tolerance for Infusing Climate-Resilience in Rice Variety Samba Mahsuri. Indian Journal of Genetics and Plant Breeding, 86(3), 279-291. https://doi.org/10.1007/s44489-026-00028-x

Image Credits: AI Generated

DOI: 10.1007/s44489-026-00028-x

Keywords: rice, Samba Mahsuri, QTL pyramiding, drought tolerance, submergence tolerance, qDTY2.1, qDTY3.2, Sub1, marker-assisted selection, climate resilience, molecular breeding, rainfed lowland