Millets’ Chloroplast Genomes Reveal Codon Preferences Shaped by Natural Selection

Millets have quietly fed humanity for thousands of years, thriving on dry, marginal land where rice and wheat would wither. Now a team of researchers in India has taken a close look at the smallest but arguably most important archive of information these hardy grasses carry: the chloroplast genome. In a study published in the Indian Journal of Genetics and Plant Breeding, J. Bevin Nishanth, Raju Balaji, Ganesan Govindhan, Suji Somasundaram and colleagues dissected the codon usage patterns of eight millet species, and their findings point to a genome shaped less by random mutation than by the steady hand of natural selection.

Codons are the three-letter DNA words that specify amino acids during protein synthesis. Because the genetic code is degenerate, most amino acids can be encoded by several synonymous codons, yet organisms rarely use these synonyms equally. This unevenness, known as codon usage bias, is far from trivial. It influences how efficiently genes are translated into proteins, how quickly chloroplasts can respond to environmental stress, and even how successfully a foreign gene can be expressed if it is engineered into a plant. Understanding which codons a species prefers, and why, therefore has both evolutionary and very practical implications.

The eight species examined span the diversity of cultivated and related millets: barnyard millet (Echinochloa esculenta), finger millet (Eleusine coracana), proso millet (Panicum miliaceum), little millet (Panicum sumatrense), kodo millet (Paspalum scrobiculatum), pearl millet (Pennisetum glaucum), foxtail millet (Setaria italica) and sorghum (Sorghum bicolor). All belong to the grass family Poaceae, and all are prized for their nutritional value and resilience to drought, making them increasingly important crops in a warming world. Yet, as the authors note, comprehensive analyses of their chloroplast genomic architecture, and of codon usage in particular, had been lacking.

To fill that gap, the team deployed a battery of standard but powerful bioinformatic measures. The relative synonymous codon usage (RSCU) quantifies how often each codon appears compared with what would be expected if all synonyms were used equally. The effective number of codons (ENC) summarizes how many distinct codons a gene actually draws upon, ranging from 20, meaning extreme bias with only one codon per amino acid, to 61, meaning no bias at all. GC content and GC3, the fraction of guanine and cytosine at the third, wobble position of codons, reveal compositional tendencies. Neutrality plots and parity rule 2 (PR2) analysis then help distinguish between two competing forces: mutational pressure, which pushes base composition in a direction dictated by the chemistry of DNA repair and replication, and natural selection, which favors codons that are translated more efficiently or accurately.

The results were strikingly consistent. Across all eight species, the chloroplast genomes showed a strong preference for codons ending in adenine or thymine. GC3 values hovered around 29 to 30 percent, a clear signature of compositional bias toward AT-richness. Thirty high-frequency codons, defined as those with an RSCU greater than 1, were identified in each genome, and every one of them ended in A or T. Some of these favored codons, such as TGT, CCT and TTA, were shared across all eight species, hinting at a common ancestral or functional logic. But many optimal codons differed from species to species, suggesting that each lineage has fine-tuned its translational machinery in its own way.

The neutrality plot analysis delivered the study’s central evolutionary verdict. If mutation were the dominant force, GC3 would track the overall GC content of genes closely, and the neutrality plot would show a strong diagonal relationship. Instead, the ENC values and neutrality results indicated that natural selection has played the decisive role in shaping codon usage bias in these chloroplast genomes, outweighing mutational pressure. This makes biological sense for an organelle whose genes encode the photosynthetic machinery on which the entire plant depends. Efficient, accurate translation of photosystem proteins, rubisco subunits and ATP synthase components confers a direct fitness advantage, particularly in the harsh, water-limited environments where millets excel.

The PR2, or parity rule 2, analysis added another layer of nuance. Under purely neutral conditions, adenine should equal thymine and guanine should equal cytosine within a DNA strand. Deviations from this parity reveal directional forces at work. In the millet chloroplast genomes, the PR2 plots showed a preference for thymine over adenine and for guanine over cytosine at the third codon position, a pattern consistent with selection acting on translational efficiency rather than with random drift. Correspondence analysis, a multivariate technique that clusters genes according to their codon usage signatures, revealed that most genes share a similar codon usage profile, but a subset of genes deviated noticeably, possibly reflecting differences in gene function, expression level or adaptation to environmental conditions.

Why does this matter beyond evolutionary theory? Chloroplasts have become a favored target for plant genetic engineering. They are maternally inherited in many crops, which reduces gene flow through pollen, they support very high levels of foreign protein expression, and they offer a natural containment strategy. But chloroplast expression depends on the translational preferences of the plastid itself. Codon optimization, rewriting a transgene to match the host’s preferred codons, has been shown in prior work to dramatically enhance expression levels and yield insights into chloroplast translation. The codon preference data assembled for these eight millets provide exactly the reference table that synthetic biologists would need when designing transgenes for millet improvement, whether the goal is stress tolerance, enhanced photosynthesis or nutritional fortification.

The study also contributes to phylogenetics. Codon usage patterns can carry historical signal, and comparative chloroplast genome analyses across the Poaceae have already been used to resolve relationships among grass lineages. By documenting shared and species-specific optimal codons, the researchers have generated a dataset that can inform both evolutionary reconstructions and the identification of conserved regulatory features in millet chloroplasts. Given that millets are increasingly promoted as climate-resilient staples for future food security, any tool that accelerates their improvement is welcome.

For a crop group often described as orphan grains, overlooked by mainstream breeding programs despite their nutritional virtues, this work represents a meaningful step into the genomic era. It confirms that the chloroplast genomes of millets are not passive repositories of sequence but actively curated libraries, their codon choices sculpted by selection to keep the photosynthetic engine running smoothly under stress. As gene editing and chloroplast transformation mature, the humble codon table may turn out to be one of the most valuable things these ancient grains can teach us.

Subject of Research: Codon usage bias in the chloroplast genomes of eight millet species

Article Title: Comprehensive Analysis of Codon Usage Bias Pattern in Chloroplast Genomes of Eight Millet Species

Article References: Nishanth, J. B., Balaji, R., Govindhan, G., & Somasundaram, S. (2026). Comprehensive Analysis of Codon Usage Bias Pattern in Chloroplast Genomes of Eight Millet Species. Indian Journal of Genetics and Plant Breeding, 86(2), 177-189. https://doi.org/10.1007/s44489-026-00018-z

Image Credits: AI Generated

DOI: 10.1007/s44489-026-00018-z

Keywords: codon usage bias, chloroplast genome, millets, RSCU, effective number of codons, GC3 content, neutrality plot, natural selection, translational selection, codon optimization, Poaceae, plant biotechnology