Decide to try source, DNA removal, and genome sequencing
Right here from the entire genome sequencing out-of 55 honey bees and also by constructing a leading quality recombination chart inside the honey-bee, i found that crossovers try regarding the GC content, nucleotide variety, and you can gene density. I plus confirmed the former tip one genetics conveyed during the employee minds have oddly highest CO costs. Our very own study support the check one variation out of employee choices, but not protected setting, are a driver of your own higher crossing-more speed from inside the bees. We discover no facts the crossing-more than rates is actually followed closely by a high NCO speed.
Strategies and information
Five territories off honeybees (Apis mellifera ligustica Spin) was in fact amassed away from a great bee ranch during the Zhenjiang, Asia. For each colony consisted of that queen, dozens of drones, and you may a huge selection of pros. Bees of about three colonies were picked to have whole genome sequencing.
The fresh DNA of every individual is actually removed playing with phenol/chloroform/isoamyl alcoholic beverages approach. To attenuate the possibility of microbial contaminants, brand new stomachs of bees have been got rid of in advance of DNA extraction. In the 3 ?g away from DNA away from per try were used for entire genome resequencing once the remaining DNA is actually left having PCR and you will Sanger sequencing. Structure of your DNA libraries and you may Illumina sequencing were performed on BGI-Shenzhen. From inside the short-term, paired-stop sequencing libraries having insert size of 500 bp were constructed for every single test with respect to the maker’s information. Up coming dos ? a hundred bp matched-end checks out had been generated to your IlluminaHiSEq 2000. The brand new queens were sequenced during the everything 67? coverage normally, drones during the whenever 35? visibility, and you may experts on up to 29? publicity (Dining table S1 for the Most document dos). The fresh sequences had been transferred throughout the GenBank databases (accession zero. SRP043350).
SNP contacting and you may marker character
Honeybee reference genome was downloaded regarding NCBI . Brand new sequencing checks out was in fact very first mapped to source genome which have bwa right after which realigned having stampy . Then regional realignment to indels are performed because of the Genome Studies Toolkit (GATK) , and you will variants had been named because of the GATK UnifiedGenotyper.
As a result of the down reliability out of calling indel variations, just recognized SNPs are used due to the fact indicators. First, 920,528 so you can 960,246 hetSNPs was named for the per king (Table S2 inside Additional file 2). Then, up to twenty two% of them had been got rid of because web sites are hetSNPs inside a minumum of one haploid drone (this might echo low-allelic succession alignments because of CNVs, sequencing error, or lowest sequencing quality). Equivalent dimensions of the latest hetSNPs together with have been observed in person jizz sequencing . In the end, 671,690 so you can 740,763 reputable hetSNPs in the for each and every nest were utilized just like the indicators to locate recombination situations (Dining table S2 inside the Most 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 ‘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.