Try provider, DNA removal, and genome sequencing

Here of the whole genome sequencing out of 55 honey bees and by developing a top solution recombination chart inside honey-bee, i learned that crossovers is of this GC blogs, nucleotide assortment, and gene density. I along with affirmed the previous idea that family genes conveyed for the staff brains have oddly high CO cost. Our very own analysis hold the have a look at you to diversification regarding employee decisions, however immune function, are a drivers of one’s higher crossing-more than rate into the bees. We find zero facts your crossing-over rate is followed by a premier NCO speed.

Measures and product

Four colonies away from honeybees (Apis mellifera ligustica Twist) have been gathered regarding a beneficial bee ranch within the Zhenjiang, Asia. For each nest contained you to definitely king, all those drones, and you can countless experts. Bees of three territories were chosen getting whole genome sequencing.

The new DNA of each and every personal is actually extracted using phenol/chloroform/isoamyl liquor approach. To reduce the risk of microbial toxic contamination, the brand new stomachs regarding bees had been eliminated ahead of DNA removal. On step three ?g regarding DNA out of for each and every decide to try were utilized having whole genome resequencing while the leftover DNA is kept to own PCR and you can Sanger sequencing. Design of your own DNA libraries and you can Illumina sequencing was indeed did in the BGI-Shenzhen. From inside the temporary, paired-prevent sequencing libraries with input size of five hundred bp was indeed developed for every single test with regards to the maker’s guidelines. Upcoming dos ? one hundred bp coordinated-avoid checks out had been produced towards the IlluminaHiSEq 2000. The brand new queens had been sequenced within around 67? exposure typically, drones at the everything 35? publicity, and you may workers at the whenever 30? visibility (Desk S1 during the Even more file 2). Brand new sequences was basically deposited regarding GenBank database (accession no. SRP043350).

SNP getting in touch with and you may marker identification

Honeybee source genome is downloaded of NCBI . This new sequencing checks out was basically basic mapped on to reference genome with bwa immediately after which realigned having stampy . Following regional realignment as much as indels are did of the Genome Studies Toolkit (GATK) , and variants was titled from the GATK UnifiedGenotyper.

Because of the lower precision regarding getting in touch with indel variants, simply recognized SNPs are utilized because indicators. Earliest, 920,528 to help you 960,246 hetSNPs were entitled inside per queen (Desk S2 into the Even more file 2). Next, up to 22% of them was basically got rid of due to the fact that the websites are hetSNPs within the at least one haploid drone (this could reflect non-allelic succession alignments caused by CNVs, sequencing error, or lowest sequencing top quality). Equivalent size of new hetSNPs as well as had been seen in person sperm sequencing . Fundamentally, 671,690 so you’re able to 740,763 reputable hetSNPs in for each nest were used since indicators so you can locate recombination occurrences (Dining table S2 into the Even more 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.