Large-scale phenotyping and multi-trait selection identify low-methane Napier grass (Cenchrus purpureus) accessions under contrasting environmental conditions

Abstract Napier grass (Cenchrus purpureus, NG) is an important forage in tropical and subtropical livestock systems, but the extent of naturally occurring variation in its methane (CH4) production and its potential for selecting low-CH4 emitting genotypes remains poorly characterised. This study evaluated variation in CH4 yield among NG accessions, examined the effects of season and water stress, validated contrasting CH4 phenotypes across donor cows, and identified elite accessions combining low CH4 yield with favourable digestibility. A total of 750 NG samples representing 84 accessions grown under dry and wet seasons and moderate and severe water-stress were evaluated using a high-throughput in-vitro fermentation system. To further identify forages combining low-CH4 production with acceptable digestibility, a multi-trait selection approach was applied using biologically defined thresholds for CH4 yield and in vitro organic matter digestibility (IVOMD), followed by a weighted index assigning 60% weight to CH4 yield and 40% to IVOMD. Methane yield varied widely among accessions, ranging from 0.02 to 5.07 mL/g DM and was affected by growing conditions. Methane production was generally lower during the wet season, while water stress was associated with changes in forage quality and fermentation characteristics. Low-CH4 producing accessions generally had lower IVOMD, crude protein and metabolizable energy and relatively higher fibre than high-CH4 producing accessions. Accessions initially classified as low and high-CH4 emitters based solely on CH4 yield were subsequently validated using rumen inoculum from three individual donor cows. Although the magnitude of CH4 production varied among donors, the contrast between low- and- high-CH4 accessions was generally retained, supporting the robustness of accession differences in CH4 production. Several accessions also maintained relatively low CH4 production across contrasting season and water-stress conditions, indicating that favourable CH4 phenotypes were not entirely environment dependent. A multi-trait selection identified ten elite accessions that produced 0.019-0.810 mL CH4/g DM and had 55.2-69.0% IVOMD, whereas ten poor-performing accessions produced 2.80-3.89 mL CH4/g DM and had 50.1-54.7% IVOMD. Differences in hexose fermentation and CH4 produced per unit of fermented substrate further indicated contrasting fermentation efficiency between the groups. These results demonstrate substantial variation in NG and identify accessions with low-CH4 production, favourable digestibility and relatively stable performance across growing conditions. In conclusion, incorporating CH4 yield and digestibility into forage selection could support the development of lower-emission forage cultivars for sustainable climate-smart livestock production.