Try supply, DNA extraction, and genome sequencing

Here of the entire genome sequencing away from 55 honey bees and also by creating a high resolution recombination chart during the honey bee, we discovered that crossovers try from the GC articles, nucleotide variety, and you may gene thickness. I also verified the previous idea you to definitely genes expressed when you look at the staff member thoughts have strangely higher CO cost. The research contain the see you to definitely variation out of worker conclusion, not immune mode, is actually a driver of your own highest crossing-more than price when you look at the bees. We find zero research your crossing-more speed are accompanied by a premier NCO rates.

Strategies and you can material

Five territories from honeybees (Apis mellifera ligustica Twist) had been amassed away from a bee ranch in Zhenjiang, Asia. For each nest consisted of that king, all those drones, and you can numerous workers. Bees from about three territories have been picked for entire genome sequencing.

This new DNA of any individual was removed using phenol/chloroform/isoamyl alcohol means. To reduce the risk of bacterial toxic contamination, the latest stomachs away from bees was in fact removed prior to DNA removal. From the step three ?g off DNA away from for every test were utilized to have entire genome resequencing once the left DNA try leftover having PCR and you can Sanger sequencing. Design of DNA libraries and you may Illumina sequencing was in fact performed from the BGI-Shenzhen. During the temporary, paired-avoid sequencing libraries having enter size of 500 bp was in fact constructed for each and every shot with regards to the manufacturer’s information. After that dos ? one hundred bp matched-avoid reads was in fact produced towards the IlluminaHiSEq 2000. The newest queens have been sequenced at up to 67? publicity typically, drones from the as much as 35? publicity, and you will workers in the as much as 29? exposure (Table S1 during the Additional document dos). The latest sequences were deposited on GenBank database (accession zero. SRP043350).

SNP getting in touch with and you will marker identity

Honeybee site genome try downloaded of NCBI . The fresh sequencing reads have been first mapped to resource genome with bwa after which realigned that have stampy . Next local realignment up to indels are did of the Genome Research Toolkit (GATK) , and you will alternatives have been entitled from the GATK UnifiedGenotyper.

Considering the lower precision of contacting indel alternatives, only identified SNPs are utilized as indicators. Basic, 920,528 in order to 960,246 hetSNPs was in fact titled in for each queen (Table S2 inside Additional file dos). Following, just as much as twenty-two% ones was in fact eliminated because the web sites also are hetSNPs for the one haploid drone (this could reflect low-allelic sequence alignments for the reason that CNVs, sequencing error, otherwise reduced sequencing quality). Similar proportions of the fresh hetSNPs as well as were found in individual sperm sequencing . Finally, 671,690 to help you 740,763 credible hetSNPs inside the for each and every colony were used just like the indicators to help you locate recombination occurrences (Table S2 from inside the Most file 2).

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.