Sorghum

Studies were carried out in Walukuba subcounty, Jinja District (00° 26′ 33.2″ N, 33°13′ 32.3″ E); Kihihi subcounty, Kanungu District (00°45′ 03.1″ S, 29°42′ 03.6″ E); and Nagongera subcounty, Tororo District (00°46′ 10.6″, N 34°01′ 34.1″ E) (Figure 1). Jinja is in the southeast region at an elevation of 1,215 m above sea level and the study site is peri-urban, close to a swampy area near Lake Victoria. The major malaria vector species here was Anopheles gambiae s.s. ten years ago [16 (link)], but it is now Anopheles arabiensis[17 (link)]. Kanungu is a rural area of rolling hills in western Uganda located at an elevation of 1,310 m above sea level, where farmers grow bananas, millet, rice, cassava, potatoes, sweet potatoes, tomatoes, maize, groundnuts, and beans. The main vector here is An. gambiae s.s.. Tororo is located in the eastern region at an elevation of 1,185 m above sea level in an area of savannah grassland interrupted by bare rocky outcrops and low-lying wetlands, where maize, rice, cassava, sweet potatoes, sorghum, groundnuts, soya beans, beans, and millet are cultivated. The major malaria vector species reported for the region are Anopheles gambiae s.s. and Anopheles funestus with small numbers of An. arabiensis[16 (link),18 (link)]. There are typically two rainy seasons in Uganda (March to May and August to October) with annual rainfall of 1,000-1,500 mm.

Based on the physiological results, samples from the 0 and 10 μM CdCl₂ treatments were selected for transcription and metabolic analysis. The roots of the treated and control sorghum seedlings were washed with sterilized distilled water, and were immediately frozen in liquid nitrogen. Total RNA was extracted using the NEBNext^®UltraTM RNA Library Prep Kit for Illumina^®(NEB, USA) following manufacturer’s recommendations. The purity, concentration and integrity of RNA were assessed using a NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA), Qubit^® RNA Assay Kit in Qubit^®2.0 Flurometer (Life Technologies, CA, USA) and RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively (Ren et al., 2022 (link)). One μg high quality RNA per sample was then used to construct the cDNA library, and was sequenced using an Illumina HiSeq sequencing platform by Metware Biotechnology Co., Ltd. (Wuhan, China). The software fastp v 0.19.3 was used to clean and trim the original data to obtain high-quality clean reads (reads with adapters were filtered out, as were paired reads were the N content exceeded 10% of the base number of the reads, or when the number of low-quality (Q ≤ 20) bases contained in the read exceeds 50% of the bases). Clean data were then mapped to the sorghum reference genome (Sbicolor_454_v3.0.1) using the HISAT v2.1.0 software. The expression abundance of reads was quantified using the fragments per kilobase of transcript per million base pairs (FPKM) value. The differentially expressed genes (DEGs) in the control and treatment groups were screened using DESeq2 v1.22.1 (Ross Ihaka, University of Auckland, New Zealand) with a threshold of |log₂ Fold Change | ≥1 and False Discovery Rate (FDR)< 0.05.

The plant height, root length and the number of lateral roots were immediately measured after 3 days of growth in a Cd stress environment (Jia et al., 2017 (link)). The shoots and roots were detached and used for fresh and dry weight measurements. The shoots and roots immediately weighed to obtain the fresh weight and then dried to constant weight at 80°C to obtain the dry weight.
The Cd content in the sorghum seedlings was assayed according to a method described previously with slight modifications (Xiao et al., 2021 (link)). Briefly, roots and aboveground parts of the sorghum plants were sampled and rinsed with distilled water. Samples were then dried in an oven at 80°C until constant weight and then pulverized to a fine powder by passing through a mesh (100 mesh fractions). Afterwards, the obtained ash residues were digested using the nitric-perchloric acid (9:1, v/v) wet digestion method. The amount of Cd was measured using an atomic absorption spectrophotometer (GGX-810, Haiguang Instrument Co., Ltd, Beijing, China).