Cabbage

Four hosts from the Williams’ classification system, including “Badger Shipper,” “Jersey Queen,” “Laurentian,” and “Wilhelmsburger” were used in this study. The above four differential hosts and eleven CR inbred lines of Chinese cabbage were infected with 37 isolates for the selection of possible hosts to develop a new clubroot differential system, named the Sinitic clubroot differential (SCD) set (Table 1). An inbred line of Chinese cabbage, “BJN3-1,” was used as a clubroot-susceptible control. Isolate Collection and Plant Inoculation
In total, 132 field isolates collected from the infected roots of Chinese cabbage, canola, broccoli, or wild mustard from China and Korea were used in this study (Table S1). Of these, 37 (numbers 1–37) were used to inoculate to 4 hosts from Williams’ set (1966 ) and 12 Chinese cabbage inbred lines (Table 1), for the development of the clubroot differential set. The remaining 95 isolates were used to inoculate eight selected CR hosts (Table 2, H01–H08) to validate and evaluate the practicability, stability and extensibility of the developed SCD system. To accurately evaluate the pathotypes of P. brassicae, the resistance tests lasted from 2015 to 2019. All these materials were planted in 72-well multi-pots with 3 replications and maintained in a greenhouse at 20°C to 25°C under a 16-h photoperiod until inoculation with P. brassicae. Each replication contained 24 plants.
All field isolates were propagated with the “BJN3-1” line under controlled environments, and fresh galls were stored at −20°C for further use. Preparation of the resting spores of P. brassicae was according to Williams (1966) with minor modifications. Briefly, after the galls were ground in sterile distilled water with a homogenizer, the mixtures were filtered through 8 layers of cheesecloth. The resting spores were collected by centrifugation at 2,500g and quantified with a hemocytometer (Neubauer improved, Marienfeld, Germany). The concentration of resting spores was adjusted to 1 × 10⁷/ml, and 1 ml of the suspension was inoculated to the 5-day-old seedlings of each host. The potting mixture (Fanyu, Shenyang, China) was kept moist until 6 weeks after P. brassicae inoculation.

Quantitative real-time PCR (qPCR) analyses were performed as detailed previously (Jiang et al., 2018 (link)), using primers compiled in Supplementary Table S1. Sampling of plants at the seedling and flowering stages was performed as published previously (Jiang et al., 2018 (link)), while sampling at the rosette and heading stages was performed at the 6^th and 8^th weeks, respectively. When rosette stage sampling was performed, three leaves were collected from each of nine individual ‘Chiifu’ plants (three biological replicates, three plants per replicate). With the oldest leaf numbered as leaf one, samples were collected from different positions (top, middle, bottom) on three different leaves (outer leaf, first leaf; middle leaf, 10^th leaf; inner leaf, 20^th leaf), designated as the RL1, RL2, and RL3 from the outer to the inner leaves. In the heading stage, three leaves (outer leaf, first leaf; middle leaf, 20^th leaf; inner leaf, 40^th leaf) were similarly collected from each of nine ‘Chiifu’ plants, with these samples being respectively designated as HL1, HL2, and HL3. For BR1 expression in bolting resistant or bolting non-resistant Chinese cabbage, ten inbred lines from heading stage were selected from our laboratory, the top point of short stem (GP) and the top point of inner leaf (TP) were sampled, TP was used as control. Three biological replicates with three plants of different lines per replicate were sampled. After collection, samples were snap-frozen with liquid nitrogen and stored at −80°C for subsequent RNA isolation. qPCR primers for the expression analyses of representative mark genes were listed in Supplementary Table S1.

B. rapa ‘Chiifu’ cultivar, Chinese cabbage inbred lines, A. thaliana ecotype ‘Columbia-0’ (Col-0), and the transgenic A. thaliana lines were cultivated as in our prior studies (Jiang et al., 2018 (link); Jiang et al., 2020b (link)). Nicotiana benthamiana was cultivated as in prior study (Zhan et al., 2022 (link)).

A semi-quantitative FFQ to estimate dietary Zn intake in the Indonesian population was developed, focusing particularly on pregnancy and the period of infancy. In an initial phase, participants filled out an online questionnaire to report their recollection of all foods consumed in the previous 24 h (Q-24 h) to gather information on foods commonly consumed in Indonesia. The food items gathered from the Q-24 h were used in the development of a FFQ to be used to estimate habitual Zn intake over a longer period (LFFQ). As this study focused on Zn intake, food items not captured through the Q-24 h but known to be good sources of Zn were added. The LFFQ comprised 82 food items.
A series of food photographs were produced to enable participants to estimate their usual food portion and were based on the recommendations of a previous study (22 (link)). Each food was presented as four portion sizes comprising 25%, 50%, 100% and 125% of a portion commonly consumed or portion on the package label of commercial products. Portions were measured out using an electrical scale (TANITA digital food scale). The amount of Zn in each food was obtained from USDA Nutrient Database for Standard Reference (United States Department of Agriculture 2013; https://data.nal.usda.gov/dataset/usda-national-nutrient-database-standard-reference-legacy-release), the Indonesian food database Nutrisurvey 2007 (http://www.nutrisurvey.de), or from previous studies (23 (link), 24 (link)). A plate or bowl containing the food was arranged together with a spoon and fork on each side. Food was photographed on a white background using a digital camera with a macro lens (Nikon 3100D) and photographs were printed at a size of 4 cm × 8 cm. In parallel, a shorter version of the FFQ (S-FFQ), which comprised fewer food items (28 items), was developed with the aim of reducing the required time for completion and thus pressure on the interviewer and participant during the clinic visit. To develop the S-FFQ, the number of food items was reduced by focusing on Zn-rich foods, such as red meat, offal, avocado, broccoli, spinach, grouping vegetables with lower Zn content, such as cabbage, carrot and lettuce, into a category of “other vegetables” and excluding items that were found to be rarely or never consumed by this population, such as brown rice, veal and pork. The L-FFQ and S-FFQ were compared with one and other and with a 3-day food record (Q3-d).
Both the L-FFQ and the S-FFQ consisted of five sections, which were: (1) personal information about child and parents, which included name, date of birth, birth weight, parents' educational background and occupation; (2) prenatal and birth history; (3) post-natal history, including feeding in the first six months, weaning age and foods, and consumption of food supplements; (4) retrospective record of foods consumed during pregnancy; (5) retrospective record of foods consumed by the child during infancy (from weaning up to age one year old); and (6) record of foods consumed by the child at the point of sampling. The S-FFQ is included as supplementary information.