Take to source, DNA extraction, and you may genome sequencing

Here by the whole genome sequencing of 55 honey bees by developing a leading quality recombination map in the honey bee, i learned that crossovers are of this GC content, nucleotide variety, and you can gene occurrence. We in addition to verified the previous suggestion one to family genes expressed when you look at the staff member brains possess unusually high CO pricing. Our very own analysis support the evaluate one to diversity regarding employee choices, not protected form, was a driver of your own large crossing-more than speed inside bees. We find no research that crossing-more speed try with a leading NCO rate.

Procedures and you will product

Five territories regarding honeybees (Apis mellifera ligustica Twist) had been obtained off an excellent bee ranch inside the Zhenjiang, Asia. Per nest contained you to king, all those drones, and you will a huge selection of gurus. Bees away from about three territories were chose to own entire genome sequencing.

The DNA each and every private is extracted having fun with phenol/chloroform/isoamyl alcohol method. To minimize the risk of bacterial contaminants, the stomachs out-of bees had been removed in advance of DNA removal. From the step 3 ?g of DNA out-of per shot were utilized to own whole genome resequencing because the left DNA are remaining to own PCR and you may Sanger sequencing. Design of the DNA libraries and you can Illumina sequencing were performed on BGI-Shenzhen. During the temporary, paired-stop sequencing libraries which have enter measurements of five-hundred bp have been created for each sample with respect to the manufacturer’s recommendations. After that dos ? one hundred bp matched-end reads had been generated to your IlluminaHiSEq 2000. New queens was in fact sequenced from the whenever 67? publicity typically, drones during the everything thirty-five? visibility, and you can workers during the just as much as 29? publicity (Dining table S1 within the More document dos). This new sequences was transferred from the GenBank databases (accession no. SRP043350).

SNP getting in touch with and you can marker character

Honeybee site genome was installed out of NCBI . The newest sequencing checks out was in fact basic mapped onto source genome having bwa then realigned with stampy . Then regional realignment as much as indels is actually performed from the Genome Studies Toolkit (GATK) , and you may variants was indeed named because of the GATK UnifiedGenotyper.

Because of the lower reliability off getting in touch with indel variants, only understood SNPs are utilized because the markers. Very first, 920,528 to help you 960,246 hetSNPs was titled into the each king (Table S2 when you look at the Most document 2). Up coming, whenever twenty two% of those had been removed due to the fact that the web sites are hetSNPs in one haploid drone (this might reflect non-allelic succession alignments considering CNVs, sequencing mistake, otherwise lowest sequencing top quality). Comparable proportions of brand new hetSNPs also was present in human jizz sequencing . Finally, 671,690 so you’re able to 740,763 reliable hetSNPs inside for each nest were used as markers to detect recombination situations (Dining table S2 during the More document 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 https://datingranking.net/flirt-review/ ‘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.