Genemarker v2

QF-PCR is a method used for detecting chromosome copy number by amplification of polymorphic DNA markers (short tandem repeats) specific to the chromosomes of interest (commonly 13, 18, 21, X, and Y). Following PCR using fluorescent primers, the amplification products are separated by size using a capillary electrophoresis system, and the amount of DNA present in each fragment is automatically quantified.
All samples with a normal female karyotype were subjected to QF-PCR testing for maternal cell contamination by comparison of multiple microsatellite markers in maternal blood versus cell culture and/or fetal sample. The IVD QF-PCR Devyser (Devyser AB, Stockholm, Sweden) prenatal kit, testing for aneuploidies of chromosomes 21, 18, 13, X, and Y, as well as the extended kit for chromosomes 15, 16, and 22 were used. DNA purification was performed using Promega Wizard™ Genomic (Promega, Madison, WI, USA). The amplicons were migrated on the ABI3730xl platform (Applied Biosystems, Foster City, CA, USA), and data were analyzed using GeneMarker v2.2 software (SoftGenetics, State College, PA, USA).

Four cpDNA fragments (trnH-psbA, trnL-F, matK, and rbcL) were amplified and sequenced across all E. nutans samples. These fragments were amplified using primers previously reported in the literature, known for showcasing polymorphisms in other Elymus species (Xiong et al., 2022 (link)). Amplification was conducted in a 30 μL reaction volume, comprising of 30 ng of genomic DNA, 1.5 μL of each primer, and 15 μL of 2× Es Taq MasterMix (CoWin Biosciences, Beijing, China), using a C1000 Touch Thermal Cycler (BIO-RAD, Foster City, CA, USA). The polymerase chain reaction (PCR) conditions throughout the experiment were consistent with those described by Shaw et al (Shaw et al., 2005 (link)), and the resulting products were sequenced by Tsingke Biotech (Beijing, China). The genotypes of all 361 samples were determined using nine pairs of EST-SSR primers developed for E. nutans in our previous study based on transcriptomic data, and the PCR procedure followed the same protocol as we described previously (Li et al., 2023 (link)). The PCR products were subjected to capillary electrophoresis in an ABI 3730xl DNA analyzer (Applied Biosystems, Foster, CA, USA) and analyzed using GeneMarker v. 2.2 software (SoftGenetics, Pennsylvania, USA).

Products were separated on an ABI PRISM 3130 × l genetic analyzer (Life Tech, Grand Island, NY 14072, USA). 1 μl of PCR product was mixed with 0.5 μl of MapMarker ROX 1000 (Bioventures, Murfreesboro, TN, USA) and 9 μl of HiDi Formamide (Life Tech, Grand Island, NY 14072, USA). The mixture was denatured at 95°C for 5 min then loaded onto the 3130 × l genetic analyzer. Fragment size was analyzed using GeneMarker V2.6 (Softgenetics, State College, PA, USA).

For microsatellite analysis, walking legs of specimens were fixed in 80% ethanol prior to extraction of nuclear DNA with the Blood & Cell Culture DNA Kit (Genomic Tips) from Qiagen (Hilden, Germany). A total of five microsatellite primer pairs were tested. Four of them were originally designed for P. clarkii (PclG-02, PclG-04, PclG-08, PclG-48) (Belfiore and May, 2000 (link)) and one pair (PclG-26) was designed for marbled crayfish based on the P. clarkii sequences (Vogt et al., 2008 (link)). The same microsatellite loci were additionally investigated in P. alleni and P. clarkii. PCR was carried out using a Primus 96 Cycler (Peqlab Biotechnologie, Erlangen, Germany). Fragment analysis was performed on a Beckman Coulter CEQ 8000 eight capillary sequencer (Beckman Coulter, Krefeld, Germany) using the Beckman Coulter DNA Size Standard Kit 400 bp. Loci were scored with GeneMarker, v.2.6 (SoftGenetics, State College, PA, USA).

GeneMarker^® V2.7.0 software (SoftGenetics, LLC State College, PA, USA) was used for peak identification and allele size determination. Each peak was considered as an allele, and the genotype of each individual at each locus was determined and exported to Excel for statistical analysis. Observed number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity of each locus (H) [52 (link)], Shannon information index (I), total gene diversity (Ht), within-population gene diversity (Hs) and coefficient of gene differentiation (Gst) were calculated using POPGENE ver 1.32. [53 ].
To examine the genetic relationship among the 43 populations, Nei’s measure of genetic distance was calculated, and a dendrogram was constructed using the unweighted pair group method with arithmetic mean (UPGMA) using MEGA7 software [54 (link),55 ]. To evaluate the differentiation among groups of populations, among populations and variation within populations, an analysis of molecular variance (AMOVA) was conducted using ARLEQUIN ver 3.5 software [56 (link)].