Dneasy 96 plant kit

Ninty-three accessions belonging to 92 cultivated C. arietinum desi (39 accessions) and kabuli (53), and one C. reticulatum wild accession (ICC 17160) were utilized (Supplementary Table S1) for genome-wide discovery and genotyping of SNPs employing GBS assay. Ninty-two desi and kabuli accessions of these, with significant phenotypic [seed yield (g)/plant] and genotypic diversity (>80% diversity of total germplasm lines evaluated) were selected from available chickpea germplasm collections (16,991, including 211 minicore and 300 reference core germplasm lines) (Upadhyaya and Ortiz, 2001 (link); Upadhyaya et al., 2001 (link), 2008 (link)) following the methods of Kujur et al. (2014 (link)). An additional wild accession (ICC 17160) was included in GBS assay for understanding its molecular diversity and phylogenies with cultivated desi and kabuli chickpea. The genomic DNA was isolated from the young leaf samples of 93 accessions using a QIAGEN DNeasy 96 Plant Kit (QIAGEN, CA, USA) following the manufacturer's instructions.

Genomic DNA was extracted from young freeze-dried leaves harvested from 79 accessions, using the QIAGEN DNeasy 96 Plant Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s protocols with small modifications: 1.6% PVP40 (Sigma Aldrich) was added to the AP1 buffer and samples were incubated at 65 °C for 5 minutes; DNA was eluted in milliQ water at 65 °C. DNA quantification was performed using Quant-iT™ PicoGreen™ dsDNA Assay Kit (ThermoFisher Scientific) by Synergy2 Fluorometer (Biotek); DNA quality was checked both on an Agilent 2200 Tapestation (Agilent Technologies, CA), using the DNA genomic ScreenTape (Agilent Technologies) for DNA integrity detection, and NanoDrop 8000 Spectrophotometer (Thermo Scientific, MA), for 260/230 and 260/280 ratios evaluation.

For the DArT markers, DNA was extracted from 94 genotypes of each of the six genetic mapping families using the QIAGEN DNeasy 96 Plant Kit (QIAGEN, Crawley, UK). These samples were processed on the high-density Lolium/Festuca DArT arrays at Diversity Arrays Technology Pty Ltd, Canberra, Australia and segregating markers were identified. Additionally, the six families were screened with 48 simple sequence repeat (SSR) markers with known genetic map positions in L. perenne in order to anchor the DArT maps to existing linkage groups (LGs).

We homogenized 10–20 mg dry weight of sporophyte material using the gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) before extracting DNA with the NucleoSpin^® Plant II kit (Machery-Nagel, Düren, Germany). We followed the standard protocol (using Lysis Buffer PL1) based on the established CTAB procedure [37 (link)] with a couple of changes: the lysis step was incubated at room temperature for one hour, and the DNA was eluted into RNase-free water.
DNA samples provided by UiB were homogenized using the Qiagen TissueLyser II (Qiagen, Hilden, Germany) and extracted with either the Qiagen DNeasy^® 96 Plant Kit (Qiagen, Hilden, Germany) or the NucleoMag^® Plant kit (Machery-Nagel, Düren, Germany) [38 ]. The extraction was carried out following the protocols, with the following few exceptions: for the Qiagen DNeasy Plant Kit procedure, the centrifugation steps were prolonged, and when applying the NucleoMag Plant Kit, the modifications suggested by Fort and colleagues [39 (link)] were followed, meaning that the lysis of samples was performed for 2 h at 56 °C with the addition of 20 μL of 1 mg/mL of proteinase K (Sigma-Aldrich P6556) and 3 μL of RNAse A (provided). After lysis, samples were centrifugated for 15 min at 4 °C (instead of room temperature), and the supernatant was used for the rest of the DNA extraction.

A total of 94 different Cicer accessions belonging to one annual cultivated C. arietinum (12 accessions), five annual wild species, namely C. reticulatum (16), C. echinospermum (8), C. judaicum (22), C. bijugum (19) and C. pinnatifidum (16) and one perennial species C. microphyllum (Table 1,Table S1) were used for large-scale validation and genotyping of microsatellite and SNP markers. The genomic DNA was isolated from the young leaf samples of 94 accessions using QIAGEN DNeasy 96 Plant Kit (QIAGEN, USA) following the manufacturer's instructions. Despite of wide availability of wild chickpea accessions in different Genebanks, poor viability, germination and regeneration efficiency largely limits their utilization. Henceforth, we were able to use only 82 accessions belonging to six annual and perennial wild chickpea species for understanding their diversity and domestication patterns.

Young leaves were harvested from 242 plants and DNA was extracted using the Qiagen DNeasy 96 Plant Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. A NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Inc. Wilmington, DE, USA) was used for measuring DNA concentration and quality. The extracted DNA was submitted to the University of Minnesota Genomics Center for processing and sequencing using the GBS protocol according to Elshire et al. [67 (link)]. Briefly, genomic DNA (100 ng) was digested with 10 units of ApeKI (NEB) and incubated at 75 °C for 2 h. Phased adaptors with a three-base overhang on the 5′ ends of the bottom strand were ligated with digested DNA and 200 units of T4 ligase (NEB) at 22 °C for 1 h and heat-inactivated. The ligated samples were purified, and bar codes were added by 18 cycles with 2X NEB Taq Master Mix. Lastly, pooled libraries were size-selected for the 300 to 744 bp library region (156 to 600 DNA inserts). The final pool was then diluted to 1 nM and sequenced on the Illumina NovaSeq 6000 using single-end 1 × 100 reads on a single lane of an SP variant flowcell.

In 2009 we collected leaf material for DNA extraction from G. sericea adults, seedlings and seeds. To increase the power of our analyses we only included in our final data set individuals with a minimum of 8 typed loci. We thus analysed 181 adults and 178 juveniles (between 20 cm and 1 m height) at six sites and 595 seeds (from 24 mother trees) at five sites, see Table 1. Leaf material was immediately dried and stored in silica gel. DNA was extracted from the leaves using the QIAGEN DNeasy 96 Plant Kit, following the manufacturer’s protocol. All samples were screened at a total of ten nuclear microsatellite loci, details of which are described in [27] . Fragment analysis was conducted using an ABI3730 sequencer and genotyped using Genemapper 3.5 software (Applied Biosystems). There was no evidence for linkage disequilibrium for any pair of loci and no evidence of null alleles after Bonferroni corrections, see [27] .