Bison

To compare the efficacy of the SCR to other commonly used library preparation approaches in ancient DNA, we prepared DNA extracts from 5 previously characterized ancient bones (4 bison and one horse) that varied in DNA concentration, average fragment length, and deamination frequency (Supplemental Table 1). We powdered each bone using a MM 400 ball mill (Retsch) and performed 4 extractions, each with 100–120 mg of bone powder, from each sample following the silica column-based method described in Dabney et al (Dabney et al. 2013a (link)). We eluted DNA from the column using 50 µL of EBT buffer (10 mm Tris–HCl, 0.05% Tween-20) and pooled the 4 extracts from each sample into a single tube. We then quantified the DNA extraction pools with a Qubit 1X dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA) using 5 µL of DNA extract and a Qubit 4 Fluorometer (Invitrogen). Using these data, we calculated pmols/µL of dsDNA in each pooled extract using an estimated average length of 90 bp for all samples, and pmols/µL of ssDNA or dsDNA ends by multiplying the dsDNA pmol/µL value by 2.

Eight bones were selected to span the ¹⁴C timescale (back to 50,000 BP) at a range of preservation typical for archaeological bones. Collagen extracts from bones R-EVA 124, R-EVA 123 and R-EVA 1489 were previously dated using both graphite targets and the gas ion source in Fewlass, et al.^{29 (link)}. R-EVA 124 was previously labelled as a bison bone but recent aDNA analysis has identified it as belonging to a woolly rhinoceros^{56 (link)}. R-EVA 548 and R-EVA 570 are two faunal long bones from Teixoneres, Spain. R-EVA 1860 is a faunal long bone excavated from the site of Ranis, Germany and R-EVA 1905 is a predominantly trabecular fragment of horse bone excavated from Pietraszyn, Poland. R-EVA 1753 is a well-preserved cave bear rib known to date beyond the ¹⁴C timescale based on repeated measurements. As standard practice, an aliquot of this bone is extracted and dated alongside every batch of samples to monitor contamination introduced during sample preparation and is used in the age correction of the unknown samples. This is the referred to in the text as the ‘background bone’.

The LRRs alignments within the TLR family were made for TLR1 from four species (human [Q15399, Q5FWG5, Q6FI64, Q32MK3], mouse [Q9EPQ1], pig [Q4LDR7, Q59HI9], Takifugu rubripes [Q5H727]); TLR2 from 17 species (human [O60603], mouse [Q9QUN7, Q8K3D9, Q811T5], pig [Q59HI8, Q5DX20, Q76L24], chicken [Q9DD78 (TLR2.1), Q9DGB6 (TLR2.2)], bovine [Q95LA9], rat [Q6YGU2], dog [Q689D1], rabbit [AAM50059], goat [ABI31733], horse [AAR08196], hamster [Q9R1F8], Cynomolgus monkey [Q95M53], domestic water buffalo [Q2PZH4], Nilgai [Q2V897], Takifugu rubripes [Q5H725], zebrafish [Q6TS42], Japanese flounder [Q76CT8]); TLR3 from 9 species (human [O15455, Q4VAL2, Q504W0], mouse [Q99MB1, Q3TM31, Q499F3], bovine [Q5TJ58, Q5TJ59], rat [Q7TNI8], buffalo [Q1G1A3], Rhesus macaque [Q3BBY1], Takifugu rubripes [Q5H721], zebrafish [Q6IWL5, Q32PW5], Japanese flounder [Q76CT7, Q76CT9]; TLR4 from 17 species (human [O00206, Q5VZI7, Q5VZI8, Q5VZI9], mouse [Q9QUK6, Q5RGT4, Q8K2T5], pig [Q68Y56, Q2TNK4, Q5F4K7, Q401C7], bovine [Q9GL65, Q6WCD5, Q8SQ55], rat [Q9QX05], hamster [Q9WV82], cat [P58727], lowland gorilla [Q8SPE8], horse [Q9MYW3], Pygmy chimpanzee [Q9TTN0], olive baboon [Q9TSP2], orangutan [Q8SPE9], Nilgai [Q2V898], American bison [Q3ZD70], dog [Q8SQH3], rabbit [AAM50060]; zebrafish [Q6NV08, Q6TS41(TLR4b)]; TLR5 from 8 species (human [O60602], pig [Q59HI7], mouse [Q9JLF7], bovine [Q2LDA0], chicken [Q4ZJ82], Japanese house mouse [Q1ZZX0], Takifugu rubripes [Q5H720, Q5H716(TLRS5)], rainbow trout [Q7ZT81]); TLR6 from 5 species (human [Q9Y2C9], mouse [Q9EPW9, Q7TPC5], rat [Q6P690], pig [Q59HI6, Q76L23], bovine [Q704V6, Q706D2]; TLR7 from 4 species (human [Q9NYK1], mouse [P58681, Q548J0], dog [Q2L4T3], Takifugu rubripes [Q5H719]); TLR8 from 4 species ((human [Q9NR97, Q495P4, Q495P6, Q495P7], mouse [P58682], pig [Q865R7], Takifugu rubripes [Q5H718]); TLR9 from 12 species (human [Q9NR96[, mouse [Q9EQU3], pig [Q5I2M3, Q865R8], bovine [Q5I2M5, Q866B2], dog [Q5I2M8], cat [Q5I2M7], Japanese flounder [Q2ABQ3], horse [Q2EEY0], sheep Q5I2M4], Ma's night monkey [Q56R09], Gilthead sea bream [Q3L273, Q3L274], Takifugu rubripes [Q5H717]]; TLR10 from two species (human [Q9BXR5, Q5FWG4, Q32MI7, Q32MI8], pig [Q4LDR6, Q59HI5]); TLR11 from mouse [Q6R5P0, Q32ME8]; TLR12 from mouse [Q6QNU9]; TLR13 from mouse [Q6R5N8]; TLR14 from Takifugu rubripes [Q5H726] and zebrafish [XP_687315]; TLR15 from chicken [ABB71177], TLR21 from Takifugu rubripes [NP_001027751], TLR22 from Takifugu rubripes [Q5H723], TLR23 from Takifugu rubripes [AAW70378], and TLR from rainbow trout [Q6KCC7, Q4LBC9], Atlantic salmon [Q2A132], goldfish [Q801F9]), Japanese lamprey [Q33E92, Q33E93] and green puffer (Fragment) [Q4S0D3]).

Most commonly preferred genetic diversity parameters such as nucleotide
diversity ( $\mathit{\pi }$ ), haplotype diversity ( $H_{\mathrm{d}}$ ), number of haplotypes ( $h$ ),
number of polymorphic sites (NPS), number of monomorphic sites (NMS) and
average number of nucleotide differences ( $k$ ), as well as $F_{\mathrm{ST}}$ fixation
index, were calculated by DnaSP v.6 (Rozas et al., 2017) with default
settings. The MitoToolPy program (Peng et al., 2015) was run in a Python
environment in order to detect haplogroups in each sample via pre-set
options such as species (cattle) and region (dloop). Haplogroup
classification of the detected haplotypes was visualised by median-joining
network (MJ) analysis run in PopART 1.7 software (Leigh and Bryant, 2015)
with default parameters. Analysis of molecular variance (AMOVA) was carried
out to evaluate total genetic variation among and within populations via
Arlequin 3.5.2.2 (Excoffier and Lischer, 2010) with 1000 permutations. The
detailed information on the detected haplotypes and haplogroups in breed and
sampling locations is summarised in File S1 in the Supplement.
A total of 125, 70 and 3 sequences belonging to cosmopolitan taurine
(Aberdeen Angus, Brown Swiss, Hereford, Holstein Friesian, Jersey and
Simmental), indicine (Bachaur, Gangatiri, Kenkatha, Kherigarh, Purnea,
Shahabadi, Bhutanese, Myanmar, Iranian, Iraqi and Nellore) and outgroup
species (bison, goat and sheep) were retrieved from GenBank database
(Hiendleder, 1998; Steinborn et al., 2002; Hansen et al., 2003; Pietro et
al., 2003; Slate and Phua, 2003; Lin et al., 2007; Achilli et al., 2008;
Hiendleder et al., 2008; Ginja et al., 2010; Seroussi and Yakobson, 2010;
Sharma et al., 2015; Lwin et al., 2018) and summarised in File S2. The geographic origins of taurine samples were Argentina (ANG and HER),
Israel (ANG, HER, HF, SIM), Canada (BS, HF and JER), New Zealand (HF and
JER) and USA (JER). In contrast, indicine samples originated from India
(Bachaur, Gangatiri, Kenkatha, Kherigarh, Purnea and Shahabadi), Myanmar,
Iran, and Iraq. These sequences were combined with our 62 sequences and
optimised at 450 bp via the CLC Sequence Viewer 8 program
(https://www.qiagenbioinformatics.com, last access: 20 January 2023) to explain the genetic origin and breeding
history of native Turkish cattle breeds via phylogenetic relationship
analyses. Neighbour-joining (NJ) and MJ analyses were utilised to visualise
the distribution of the Anatolian cattle samples into taurine, indicine and
outgroup clades. NJ tree analysis was conducted per individual and breed
levels based on the Jukes–Cantor method with 1000 bootstrap replicates via
MEGA 11 software (Kumar et al., 2008). MJ analysis was performed by PopART
1.7 software (Leigh and Bryant, 2015) with default parameters. DnaSP v.6
(Rozas et al., 2017) was used to perform haplotype classification to deepen
knowledge of the genetic background of Anatolian cattle by comparison of
each sequence with the haplotypes reported in 6 taurine and 11
indicine cattle breeds simultaneously. The level of admixture among taurine,
indicine and Anatolian cattle breeds was estimated based on the observed
proportion of shared haplotypes. The detailed information on the unique and
shared sequences between Anatolian and other cattle (taurine and indicine)
breeds is summarised in File S3.