Dataset: Diazo Distribution
Deployment: KM0703

Refer to individual platform deployments

Materials and Methods
Water samples were collected from the R/V Kilo Moana (cruise KM0703) in 18 March - 14 April, 2007.

Quantification of diazotrophs. Diazotroph abundances were determined using quantitative PCR (qPCR) targeting the nifH gene. Samples for DNA were collected with Niskin bottles from 8 depths at each station. Four to five liters of water was filtered first through 10 um (Osmonics) and next through 0.2 um Supor (Pall-Gelman) filters using a peristaltic pump. Filters were placed in bead beater tubes with 0.1 g sterile glass beads and immediately frozen in liquid N. Nucleic acid samples were kept in liquid N or liquid N fumes during the cruise and transportation to the University of California Santa Cruz where they were stored at –80ºC. DNA was extracted using a modified Qiagen Plant Minikit protocol (S1).

A new qPCR primer-probe set (pps) was designed, tested and applied targeting Crocosphaera watsonii. The pps was designed using Primer Express software (Applied Biosystems). The pps has 100% nucleotide identity with C. watsonii WH8501 draft genome and an environmental clone DQ118216 (clone HT3312A10) from station ALOHA in the North Pacific Ocean. Specificity of the new primer probe set was tested against plasmid dilution series of 1-10⁷nifH copies from other diazotroph groups including UCYN-A, Trichodesmium, symbiotic heterocystous cyanobacteria Het-1, Het-2, and Het-3. None of these diazotrophs were detected by the Crocosphaera primer probe set and all no template controls (blanks) were negative. QPCR TaqMan primer-probe sets from previous studies were used to target UCYN-A, the filamentous cyanobacterium Trichodesmium spp., heterocystous filamentous cyanobacterium Richelia associated with the diatom Rhizosolenia (Het-1) (S2-3), Richelia symbiont associated with the diatom Hemiaulus (Het-2) (S4-5), and a γ-Proteobacterial diazotroph for clone 24774A11 (GenBank accession number EU052413) (probe γ-Prot 24774A11) (S1). The probes were 5’FAM and 3’TAMRA labeled (Sigma Genosys). For qPCR template, DNA from 0.2-10 um size fraction was either diluted 1:10 (vol:vol) or used undiluted, and DNA extracted from the >10 um size fraction was used undiluted as the template. The 0-10 um size fraction samples were processed for UCYN-A, Crocosphaera, and y-Prot 24774A11, and all other targets except UCYN-A and γ-Prot 24774A11 were detected in >10 um size fraction samples. Gene copy abundances of Crocosphaera in the two size fractions were pooled to obtain total abundance. Three surface samples (5, 15, and 30 m) from station 25 were omitted as outliers in the Crocosphaera dataset (total n=98); low density surface water lens influenced vertical distributions at this station. Two uL of template DNA was added into the reaction mix at a 25 uL final volume that consisted of 12.5 uL ABI TaqMan Gene Expression mix, forward and reverse primers at 0.4 uM final concentration, and 0.2 uM final concentration of the TaqMan probe, and the volume was adjusted to 25 uL with water. Standards were included in duplicate with each set of samples run on the qPCR instrument, and composed of a dilution series with a range of 1 to 10⁷nifH gene copies of linearized plasmid (pGEM-T, Promega) with the target insert. Four no template controls were run with each set of samples. Inhibition tests were carried out for each DNA sample by adding to the reaction mix two ul sample and two μl of standard containing 10⁴ or 10⁵ gene copies. QPCR was carried out using an ABI 7500 Real time PCR instrument (Applied Biosystems). The qPCR run conditions were 2 min at 50°C, then 45 cycles of 15 s at 95°C and 1 min at 60°C. The number of gene copies per sample was calculated as described in Short and Zehr (S6). One gene copy per reaction was determined as the limit of detection and 8 gene copies per reaction was determined as a limit of quantification (DNQ, "detected but not quantifiable"). In data analysis, DNQ was replaced with 1 gene copy L^-1. In regression analyses, values from the upper mixed layer (UML) were included. Determination of the bottom of the UML is approximate, therefore values below UML were also included if UCYN-A or Crocosphaera abundance was in the same order of magnitude or higher than that in the UML.