Take to resource, DNA extraction, and genome sequencing

Here by the whole genome sequencing out-of 55 honey bees by creating a high resolution recombination map inside the honey bee, we unearthed that crossovers is in the GC posts, nucleotide range, and you will gene thickness. We together with confirmed the former suggestion one to family genes expressed from inside the personnel minds provides strangely high CO prices. The analysis keep the examine you to definitely diversity out of worker choices, although not immune setting, was a drivers of your highest crossing-more than price inside the bees. We discover no evidence that the crossing-more rates are followed by a high NCO price.

Steps and you can material

Five territories off honeybees (Apis mellifera ligustica Spin) was collected out of a good bee farm inside the Zhenjiang, Asia. For every single colony contained one queen, dozens of drones, and a huge selection of pros. Bees out-of around three territories were selected for whole genome sequencing.

The fresh new DNA of each and every individual are extracted playing with phenol/chloroform/isoamyl alcoholic drinks method. To minimize the risk of microbial contaminants, the fresh new abdomens from bees were removed before DNA extraction. From the step three ?g out-of DNA out-of per decide to try were used to possess whole genome resequencing once the left DNA is actually kept to have PCR and Sanger sequencing. Framework of your own DNA libraries and Illumina sequencing was did during the BGI-Shenzhen. For the short term, paired-avoid sequencing libraries having enter size of five hundred bp was indeed developed for every shot according to manufacturer’s information. After that 2 ? 100 bp matched-stop checks out was in fact produced to your IlluminaHiSEq 2000. The new queens was indeed sequenced within just as much as 67? coverage on average, drones during the up to thirty-five? visibility, and gurus at around 31? coverage (Desk S1 inside A lot more document 2). The fresh new sequences have been placed in the GenBank databases (accession no. SRP043350).

SNP getting in touch with and marker identification

Honeybee site genome are installed away from NCBI . This new sequencing reads was earliest mapped onto source genome that have bwa right after which realigned having stampy . After that regional realignment up to indels is did from the Genome Study Toolkit (GATK) , and you will versions was named from the GATK UnifiedGenotyper.

Considering the lower reliability from calling indel variants, just recognized SNPs are utilized since the markers. Basic, 920,528 to 960,246 hetSNPs was in fact named within the each king (Desk S2 in Even more file dos). Up coming, as much as 22% of those was indeed eliminated due to the fact that those sites are also hetSNPs from inside the one haploid drone (this might echo non-allelic sequence alignments considering CNVs, sequencing mistake, otherwise reduced sequencing top quality). Similar dimensions of the brand new hetSNPs also was observed in person jizz sequencing . In the long run, 671,690 in order to 740,763 reputable hetSNPs into the for each colony were used as the indicators so you can find recombination events hookup bars San Jose (Table S2 inside Extra file dos).

Haploid phasing

For each colony, the identified markers were used for haploid phasing. The linkage of every two adjacent markers was inferred to determine the two chromosome haplotypes of the queen by comparing the SNP linkage information across all drones from the same colony. Detailed methods were described in Lu’s study . In brief, for each pair of adjacent hetSNPs, for example A/G and C/T, there could be two types of link in the queen ‘A-C, G-T’ or ‘A-T, G-C’. Assuming recombination events are low probability, if more ‘A-C, G-T’ drones are found than ‘A-T, G-C’ drones, then ‘A-C, G-T’ is assumed to be the correct link in the queen and vice versa. The two haplotypes can be clearly discriminated between >99% of ple). For linkage of the <1% markers, as shown in Additional file 1: Figure S2B, between markers at ‘LG1:20555174' and ‘LG1:20555456' , there are 14 ‘A-A or G-G' type drones against 1 ‘A-G or G-A' type drone, so ‘A-A, G-G' is assumed to be the correct link in queen and a recombination event is identified at this site in sample I-9.