Sorghum bicolor

The OrthoFinder^{97 (link)} clustering method was used to classify complete proteomes of 23 sequenced green plant genomes, including A. filiculoides and S. cucullata (Supplementary Table 5), into orthologous gene lineages (that is, orthogroups). We selected taxa that represented all of the major land plant and green algal lineages, including six core eudicots (A. thaliana, Lotus japonicus, Populus trichocarpa, Solanum lycopersicum, Erythranthe guttata and Vitis vinifera), five monocots (O. sativa, Sorghum bicolor, Musa acuminata, Zostera marina and Spirodella polyrhiza), one basal angiosperm (A. trichopoda), two gymnosperms (Pinus taeda and Picea abies), two ferns (A. filiculoides and S. cucullata), one lycophyte (S. moellendorffii), four bryophytes (Sphagnum fallax, P. patens, Marchantia polymorpha and Jungermannia infusca) and two green algae (Klebsormidium flaccidum and C. reinhardtii). In total, 16,817 orthogroups containing at least two genes were circumscribed, 8,680 of which contain at least one gene from either A. filiculoides or S. cucullata. Of the 20,203 annotated A. filiculoides genes and the 19,780 annotated S. cucullata genes, 17,941 (89%) and 16,807 (84%) were classified into orthogroups, respectively. The details for each orthogroup, including gene counts, secondary clustering of orthogroups (that is, super-orthogroups)^{110 (link)} and functional annotations, are reported in Supplementary Table 5.
We used Wagner parsimony implemented in the program Count^{111 (link)} with a weighted gene gain penalty of 1.2 to reconstruct the ancestral gene content at key nodes in the phylogeny of the 23 land plants and green algae species (Supplementary Table 5). The ancestral gene content dynamics—gains, losses, expansions and contractions—are depicted in Supplementary Fig. 5. Complete details of orthogroup dynamics for the key ancestral nodes that include seed plants, such as Salviniaceae, euphyllophytes and vascular plants, are reported in Supplementary Table 5.

Soro is one of the administrative districts of Hadiyya zone which is located in south central Ethiopia. It is situated approximately 272 km southwest of Addis Ababa and in a close proximity to the Gimicho town. Sibiya Arera is geographically located in 7° 9′ 0″–7° 11′ 0″ N latitude and 37° 52′ 30″–37° 54′ 0″ E longitude (Figure 1).
Rainfall distribution in the study area is bimodal, characterized by heavy rainy season from June to September, and light rainy season from March to May. The annual long term average rainfall is 1,107 mm and peak rainfall in September. The long term average annual temperature is 17.2°C [10 (link)]. The mean monthly temperature ranges from 15.98°C in December to 18.91°C in March (Figure 2). These favorable climatic conditions and high population have made the district to be one of the intensively cultivated areas in the south central highlands of Ethiopia. Rain-fed agriculture is the only source of livelihood for the majority of population. It is characterized by a smallholder mixed crop-livestock production.
Soil is a good indicator of the influence of soil parent material and the spatial variability in the degree of weathering, geological, and other factors are responsible for soil formation and development [11 ]. The dominant soil type of the study area is Nitisols that cover extensive areas of agricultural fields are highly suitable for crop production. The local geology is characterized by volcanic basalt flows and Cenozoic pyroclastic fall deposits [10 (link)].
The major land use/land cover types in the district include cultivated land, grazing land, forest land, and built-up areas. Cultivated land is the dominant land use type with 50,454 hectares (73.3% of the total area). At the present time, the local community has been implementing different practices to protect the adverse effect of erosion on their farmland and to improve soil fertility. Sibiya Arera is one of the areas with better implementation of soil and water conservation practices. Model farmers adopted biological and physical conservation practices, however, there is still land without conservation technologies which owned by reluctant farmers showing no willingness to implement soil and water conservation measures.
The farming system of the study area is mainly subsistence farming based on mixed crop-livestock production. Major crops grown in the area include wheat (Triticum aestivum L.), maize (Zea mays L.), barley (Hordeum vulgare L.), sorghum (Sorghum bicolor (L.) Moench), and teff (Eragrostis tef (Zucc.) Trotter). All farmers of the area have been practicing rain-fed agriculture based on continuous cultivation. Previously, diammonium phosphate (DAP) and urea were the main fertilizer types used by a large number of people. However, currently, farmers in the study area have started to use blended fertilizers such as nitrogen, phosphorus, sulfur (NPS), and nitrogen, phosphorus, sulfur, and boron (NPSB).
Arable lands are composed of the landscape without conservation practice, physical soil and water conservation structures (fanya juu), and physical soil and water conservation structures combined with biological practices (fanya juu stabilized with desho grass). Soil and water conservation practices are mechanisms used to reduce erosion and associated nutrient loss, reducing the risk of production; however, are not constructed in some agricultural lands in the study area. As a result soil erosion is major deterioration processes which lead to soil degradation and declining agricultural productivity in nonconserved agricultural land. Fanya juu structures integrated with biological practices are permanent features made of earth, designed to protect the soil from uncontrolled runoff and erosion and retain water where needed. It seeks to increase the amount of water seeping into the soil, reducing the speed and amount of water running off. Erosion is prevented by keeping enough vegetation cover on embankment to protect the soil surface and binds the soil together and maintains soil structure.

The experiment was conducted at Jiangxiaobai Red Sticky Sorghum Planting Demonstration Base (29°07′N, 106°16′E), located in Chongqing, China. The area has a mean temperature of 17.9°C, and a mean annual rainfall of 1,100 mm during the last 30 years (Chongqing Meteorological Bureau). The soil was a neutral yellow soil with a pH 7.5, alkali-dispelled nitrogen 96.15 mg/kg, available phosphorus (Olsen-P) 17.83 mg/kg, and available potassium 90.15 mg/kg.
The red sticky Sorghum bicolor cv. Chuannuoliang 2 is the principal variety planting in this base for Baijiu (distilled liquor) production. Sorghum-Canola rotation system is used in this base. Generally, the sorghum will be sown in May after the canola is harvested, and be harvested in August, then the canola will be sown in October. From August to October, there is about 2 months’ fallow period, which is suitable for the regrowth of the harvested sorghum, called ratooning sorghum. Currently, the ratooning sorghum is chopped and returned to soils or discarded. In this study, on 2th October, 2020 and 10th October, 2021, the ratooning sorghum at their heading and flowering stage were harvested for whole plant silage (above 5 cm from the ground). These ratooning sorghums have the potential to be used as fodder or silage.
A. niger was bought from Beijing Beina Chuanglian Biotechnology Research Institute. Before used as additives, the A. niger was cultured on Potato Dextrose Agar medium (PDA) at 28°C for 72 h, then, the activated A. niger was inoculated into Potato Dextrose Broph liquid medium (PDB) at 32°C for 3 days at 170 ~ 180 r/min (Wang et al., 2007 (link)). When the concentration of A. niger solution reached 2.56 × 10⁷ CFU/mL, it was evenly sprayed on the silage as additives.