Qubit fluorometer instrument

Green wood cuttings of A. arguta cv. ‘Longcheng No.2’ were picked and grown in a tissue culture incubator at Anhui Agricultural University, Anhui Province, China, under 25 °C, 12-/12-h days. Fresh young healthy leaves were collected from 3-week-old branches, quickly frozen with liquid nitrogen and then stored at –80 °C for PacBio HiFi and Hi-C sequencing. High molecular weight genomic DNA (gDNA) was extracted separately from each leaf tissue sample using a slightly modified cetyltrimethylammonium bromide (CTAB) method (Allen et al. 2006 (link)). The quality and quantity of the isolated gDNAs were evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) and a Qubit fluorometer instrument (Thermo Fisher Scientific, MA, USA), respectively. For PacBio HiFi sequencing, a standard SMRTbell library was prepared with 50 μg gDNA using the SMRTbell Express Template Prep Kit 2.0 according to the manufacturer's instructions. SMRTbell libraries were then sequenced on the PacBio Sequel II system (Pacific Biosciences, CA, USA). The Hi-C sequencing library was prepared and sequenced based on a previously published protocol (Rao et al. 2014 (link)).

“Midao 31”, a hybrid between A. eriantha “White” (female) and A. eriantha “MHX-1” (male), was used in this study. High-quality genomic DNA was extracted from fresh young leaf tissue from “Midao 31”, growing in Hefei, Anhui Province, China, and separately packaged for PacBio HiFi, ONT ultra-long, and Hi-C sequencing. Tissue materials from leaves, stems, and fruits were used for RNA-seq and genomic annotation.
The kiwifruit genomic DNA (Allen et al. 2006 (link)) was prepared using a modified CTAB method and evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit fluorometer instrument (Thermo Fisher Scientific, MA, USA). The library for ONT sequencing was constructed using the 1D ligation sequencing kit (SQK-LSK108, ONT, UK) and sequenced on the ONT PromethION platform. The Hi-C libraries were then constructed as a standard procedure, including chromatin extraction and digestion, DNA ligation, and purification. For PacBio HiFi sequencing, a standard SMRTbell library was prepared with 50 μg of gDNA by using the SMRTbell Express Template Prep Kit 2.0, according to the manufacturer’s instructions. SMRTbell libraries were then sequenced on a PacBio Sequel II system (Pacific Biosciences, CA, USA).

High-molecular-weight genomic DNAs (gDNAs) were separately extracted from each leaf tissue sample by using a slightly modified cetyltrimethylammonium bromide (CTAB) method [39 (link)]. The quality and quantity of the isolated gDNAs were evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) and a Qubit fluorometer instrument (Thermo Fisher Scientific, MA, USA), respectively. For PacBio HiFi sequencing, a standard SMRTbell library was prepared with 50 μg of gDNA by using the SMRTbell Express Template Prep Kit 2.0, according to the manufacturer’s instructions. SMRTbell libraries were then sequenced on a PacBio Sequel II system (Pacific Biosciences, CA, USA). For ONT ultra-long sequencing, the gDNAs with larger sizes were selected with Short Read Eliminator XL (Circulomics, MD, USA) following the protocol provided by ONT Community. The library was prepared with the Oxford Nanopore SQK-LSK109 kit according to the manufacturer’s instructions, and then sequenced on a PromethION flow cell. Finally, the Hi-C sequencing library was prepared and sequenced based on a previously published protocol [40 (link)].