D-600

Seeds were sieved through a set of calibrated graduated sieves (Scientific Laboratory Supplies Ltd, Hessle, UK). All seeds used in the experiment were taken from the fraction collected between 2.8mm and 3.35mm mesh sizes. This size was selected as it represented the average seed size of both parental lines. Seeds were surface sterilized by rinsing in 70% (v/v) ethanol for 30 s, followed by transfer to 5% (v/v) sodium hypochlorite solution for 10min, and finally washing three times with sterile water. Sterilized seeds were placed onto moistened germination paper crease-side down and incubated at 4°C for 5 days to synchronize germination. Following cold treatment, seeds were transferred to a light-impermeable box for 48h to complete germination. The box was placed inside the controlled environment room where subsequent phenotyping was conducted (12h photoperiod: 20°C day, 15°C night, with a light intensity of 400 µmol m^–2 s^–1 PAR). Uniformly germinated seeds with roots ~5mm in length were transferred to individual growth pouches. The growth pouch system was based on the design developed for maize (Hund et al., 2009 ). Each pouch consisted of a sheet of germination paper (24×30cm; Anchor Paper Company, St Paul, MN, USA), covered with a black polythene film of equal area (75 µm thick; Cransford Polythene Ltd, Suffolk, UK). The germination paper and film were fixed to an acrylic rod (316×15×5mm; Acrylic Online, Hull, UK) using two 18mm foldback clips. A matrix barcode label affixed to the rod allowed identification of each seedling. A single seedling was placed in each pouch centred 2cm from the top edge and held in place by the adhesion of the polythene sheet to the wet germination paper. Growth pouches were fitted into four aluminium and polypropylene frame assemblies (Fig. 1A) in the controlled environment chamber. Each assembly consisted of an aluminium profile frame (104×62×102cm; KJN Ltd, Leicester, UK) supporting toothed acrylic holders to suspend each pouch in a set position. Black polypropylene side panels (101×31×0.3cm and 63×31×0.3cm; Cut Plastic Sheeting, Devon, UK) maintain the pouches in darkness.
The base of each frame held a black polypropylene tray (99×61×10cm; Stansa Plastic Fabrication Ltd, Suffolk, UK) containing 18 l modified one-quarter Hoagland’s solution (Hoagland and Arnon, 1950 ) with HEDTA as the iron chelator (Piñeros et al., 2005 (link)). The composition (mg l^–1) of the nutrient solution was: (NH₄)₃PO₄, 29mg; Ca(NO₃)₂, 165mg; MgSO₄, 251.87mg; KNO₃, 151.99mg; H₃BO₃, 28.44mg; Cu₂SO₄, 75mg; MnCl₂(H₂O)₄, 10.1mg; MoO₃, 0.2mg; ZnSO₄, 2.29mg; FeHEDTA, 25.49mg. The solution was adjusted to pH 6 using KOH. The volume of nutrient solution in each tray was maintained automatically via a float valve system and header tank containing deionized water. Each frame assembly consisted of three rows of 30 pouches allowing 90 plants per frame. Pouches were suspended so that the bottom 3cm of the pouch was submerged in the nutrient solution.
A randomized block design was used (each table acting as a block). Eighteen lines were germinated for each experiment, with a minimum target of 15 seedlings per line. Phenotyping of the whole mapping population was split over six experiments. After 9 days (two-leaf stage), individual pouches were transferred to a copy stand (model number SGCS-920; Speed Graphic, Hampshire, UK) for imaging using a Nikon D600 DSLR camera (Fig. 1B) controlled using NKRemote software (Breeze Systems Ltd, Camberley, UK). The copy stand was modified with two draw slides (RS UK, Northants, UK), a Nylatron block (600×260×22mm), and white acrylic sheets (330×290×9mm and 290×290×9mm; Cut Plastic Sheeting, Devon, UK) to form a template to ensure consistent placement of each pouch. The polythene film covering each pouch was carefully peeled back leaving the roots fixed to the blotting paper for imaging. The draw slides then enabled the template block to be repositioned allowing shoots to be imaged without moving the pouch. Images were taken of 8–36 seedlings of 92 lines of the mapping population together with the parental lines, resulting in 1709 RSA images.

The transposase and its upstream regulatory region from pBTK30 [50] (link) was PCR amplified and cloned as a BamHI restriction fragment into pSAM_Bt [6] (link). The erythromycin cassette was then removed from the resultant plasmid, pSAM_DYH, using MfeI and XbaI restriction enzymes, and replaced using a PCR fragment containing the gentamicin resistance cassette from pUC19Gm. The resulting plasmid, pSAM_DGm was verified by PCR and sequencing and subsequently transformed into E. coli Sm10λpir [51] (link) to generate a donor strain for conjugation (Figure S10).
The donor strain, E. coli Sm10λpir pSAM_DGm was grown overnight in LB with 10 µg/ml gentamicin, and the recipient P. aeruginosa PA14 [52] (link) was grown in LB without antibiotics. The cells were centrifuged, washed in LB, centrifuged again and re-suspended to O.D._{600 nm} of 2.0. Equal volumes of donor and recipient were mixed and 100 µL of the suspension was pipetted onto pre-warmed LB agar plates. For the preparation of the library, 100 independent conjugation mixtures were used. The plates were left at room temperature for 10 min to allow the liquid to absorb into the agar and the mating continued for 3 hours at 37°C. Each conjugation reaction was scraped off, suspended in 10 ml LB and 150 µl aliquots were plated on 100 LB plates containing irgasan (25 µg/ml) and gentamicin (75 µg/mL). After approximately 12 hours at 37°C, the plates were flooded with LB and colonies scraped off using a cotton swab and pooled. The yield was approximately 300,000 individual colonies from 100 plates. The pooled library was further incubated for two additional hours in LB broth with gentamicin. Following centrifugation of the culture, the library was re-suspended in LB containing 20% glycerol and aliquots were frozen at −80°C.

CARDIA is an ongoing, prospective study of the determinants and evolution of cardiometabolic risk starting in young adulthood. Participants for the baseline examination were randomly selected and recruited by telephone or door to door from census tracts in the four field centers: Minneapolis, MN and Chicago, IL, by telephone exchanges within the Birmingham, AL city limits, and from lists of the Kaiser-Permanente Health Plan membership in Berkeley, CA. Although the source populations and methods of recruitment varied slightly, all centers adhered to the following basic eligibility criteria: 18–30 years of age, Black or white race, and permanent residential address in the target areas. Individuals living with a long-term illness or disability that would prevent participation and pregnant women and those who were up to 3 months postpartum were excluded from recruitment [38 (link),39 (link)]. A total of 5,115 young adults aged 18–30 years were enrolled at baseline in 1985–1986 (Exam Year 0) with balance according to race (Black and white), sex, education (≤high school and >high school), and age (18–24 and 25–30 years) from the population in each of the four metropolitan areas. Follow-up examinations occurred in 1987–1988 (Exam Year 2), 1990–1991 (Exam Year 5), 1992–1993 (Exam Year 7), 1995–1996 (Exam Year 10), 2000–2001 (Exam Year 15), and 2005–2006 (Exam Year 20) and 2010–2011 (Exam Year 25); retention at each exam year was 91%, 90%, 86%, 81%, 79%, 74%, 72% and 72%, respectively. The CARDIA study methods are described in detail elsewhere [38 (link),39 (link)]. Each study participant provided written informed consent, and data were collected under protocols approved by the Institutional Review Boards at each study center and at the University of North Carolina at Chapel Hill [8 ]. The current study included CARDIA exam years 0, 5, 10, 15, 20 and 25. We excluded participants with only one of the six exams used in this analysis. As has been done in previous studies, to minimize bias resulting from illness that may affect body weight, we excluded participants with hypertension, diabetes or cancer at exam year 0 [32 (link),40 (link)]. We further restricted the analytic sample to those with data on diabetes, hypertension, or self-reported cancer diagnoses at each exam and those with waist circumference and BMI data at exam year 0. For individuals included in the primary analytic sample, observations were excluded at given exam years if participants were pregnant or breastfeeding or had implausible energy intakes (<600 kcal/d or >6000/d kcal for women and <800 kcal/d or >8000 kcal/d for men) at any exam or if they were missing exposure, outcome, or covariate data at a given exam year. We censored observations for participants with diabetes, hypertension, or self-reported cancer during follow-up at the year in which the disease was reported. To facilitate sensitivity analyses, a secondary analytic dataset was created in accordance with the primary analytic dataset exclusion criteria except for censoring on diabetes, hypertension, or self-reported cancer. The secondary dataset is described in the (S1 File) [8 ,32 (link),40 (link)].