Juglans

A total of 55,159 single nucleotide polymorphisms (SNPs) were genotyped in the 298 accessions, with 52,041 analyzed using an Illumina Infinium assay [45 (link)] and 3,072 SNPs analyzed using the Illumina GoldenGate assay following the protocol described by Fan et al. [49 (link)] and Hyten et al. [50 (link)]. In addition, there were 46 SNP markers genotyped by direct sequencing and a single-base-extension method using the Luminex flow cytometer as described by Choi et al. [51 (link)]. A total of 1,363 SNPs were analyzed using both Illumina GoldenGate and Illumina Infinium assays. After eliminating redundant SNPs, non-polymorphic SNPs and SNPs with >25% missing data, a total of 42,368 SNPs (Additional file 5) remained. The physical positions of these markers in the soybean genome were determined using the whole genome assembly of Williams 82 soybean (Glyma 1.01) at the U.S. Department of Energy, Joint Genome Institute, Walnut Creek, CA (http://www.phytozome.net/soybean).

We used direct, PCR-based sequencing of genomic DNA, with primers designed from the A. thaliana reference sequence to cover the genome relatively uniformly. To achieve uniform density of our fragments, the reference genome (releases January 7, 2002, and April 17, 2003) was first divided into equally spaced regions. The last 10 kb of each region then served as an input record to Primer3 (v. 0.6). The designed primer pairs returned from Primer3 for each region were then screened for uniqueness and quality. To screen for uniqueness, all primer pairs were BLASTed (BLAST v. 2.2.3) against both the reference genome as well as BAC datasets downloaded from the Arabidopsis Information Resource (http://www.arabidopsis.org/). Any primer pair that produced a hit in the same region (≤2,300 bps) was removed. Self-amplifying primers were also removed based on this same criterion. Additionally, primers with more than five BLAST hits against the reference were also discarded. To improve the quality of each fragment, any primer pair that amplified a target sequence that contained a homonucleotide run of nine bases or more was removed.
All sequencing was done using ABI 3700 automated sequencers (Applied Biosystems, Foster City, California, United States). All fragments were sequenced in both directions.
Chromatograms were initially base-called with Phred (v. 0.020425.c) and trimmed based on quality value. The start and end of each read was trimmed until the average quality value was 25 in a window of ten bases, and internal bases were converted to missing data when their quality value was below ten. Accessions missing one read of data were trimmed more severely (different setting were used). A combination of Phrap (v. 0.020425.c) and ClustalW (v. 1.82) was used for producing alignments using a modified weight matrix that allowed us to incorporate quality values into the ClustalW algorithm. Alignments were then visually inspected and adjusted as necessary using Consed (v. 13.0). Polyphred (v. 4.20) was used to flag potential heterozygotes, which were confirmed by visual inspection of chromatograms.
Additional trimming was performed as necessary for accessions with multiple false polymorphisms and low-quality sequence after a visual inspection of chromatograms. Whenever two reads from the same accession disagreed, the final call was made by visually inspecting chromatograms unless the difference in quality value made the final call obvious.
Potential polymorphisms in each alignment were then verified by a second person. All alleles found only once or twice in the sample were verified by visually inspecting the chromatograms. When this inspection did not reveal a chromatogram peak clearly different from the other accessions, the base was changed to missing data. This would, if anything, produce a slight underestimate in alleles of frequency one and two. Higher-frequency polymorphisms with generally low-quality values (20 or lower) were also verified by checking the chromatograms.
A total of 876 high-quality fragment alignments were obtained from 979 PCR primers and used for the analyses in this paper. Of the remaining PCR primers, some failed at the stage of PCR amplification and sequencing, while some produced sequencing output that could not be base-called with certainty when the sequence quality was particularly low or when there was evidence that the primer pairs amplified two or more different products in some of the accessions.
To calculate genetic distances, we used a set of markers that have been genetically mapped to the Lister and Dean recombinant inbred lines and that also can be mapped to the AGI reference genome. Some markers were removed so that both physical position and genetic position were monotonically increasing functions.
All data are publicly available through our Web site (http://walnut.usc.edu/2010, and also as Dataset S1.

A clonal line of Aiptasia pallida (clone CC7, available through the Pringle lab) hosting Symbiodinium of clade A was established from a single tiny propagule in a population obtained from Carolina Biological Supply (Burlington, NC) and grown into an abundant stock. Given the Symbiodinium clade harbored by this population, it is likely that the Aiptasia individual originated from the Florida Keys lineage. Approximately 500 anemones of various sizes were harvested from this stock under normal growth conditions (~26°C; salinity, ~33 ppt; light, ~40 μmol m^-2 s^-1 photosynthetic photon flux; 12-h light-dark cycle), blotted to remove excess water, and immediately frozen in liquid nitrogen. The anemones were then ground to a fine powder under liquid nitrogen using a ceramic mortar and pestle. The powder was weighed (~4 g) while still frozen and mixed with a proportional volume (50 ml) of TRIzol Reagent (Invitrogen, Carlsbad, CA); extraction was then performed in accordance with the manufacturer's instructions yielding ~5 mg of total RNA. This RNA was sent to Open Biosystems (Huntsville, AL), where it was tested for quality; mRNA was then isolated using oligo(dT)-coated magnetic particles (Seradyn, Indianapolis, IN), and cDNA was synthesized. Double-stranded cDNA was size fractionated to enrich for long reads, cloned into the vector pExpress1 (Express Genomics, Frederick, MD), and electroporated into E. coli strain DH10B. The resulting library was determined to contain ~96% recombinants with an average insert size of 1.95 kb. Sequencing was performed on 96-well capillary sequencing platforms (ABI 3700) at the DOE Joint Genome Institute (JGI, Walnut Creek, CA) and at the Genome Core Facility at the University of California, Merced, USA, CA.