Legume Genomics and Genetics 2026, Vol.17, No.1, 1-13 http://cropscipublisher.com/index.php/lgg 9 (2) y=μ+Xrτr+Xcτc+Xtτt+Xpup+Xaua+Xgug+Xgauga+Xbub+ε Where: y : Observed value for the analyzed trait, μ : constant associated with all observations, Xrτr : vector of replicate fixed effect, Xcτc: vector of maturity group fixed effect, Xtτt: vector of checks or test fixed effect, Xpup: vector of population effects the six cultivars (random), b~N(0, Iσp 2), Xaua: vector of environment effects the years (random), a~N(0, Iσa 2) , Xgug : vector of progenies effect (random), g~N(0, Iσg 2) , Xgauga : vector of interaction effect progenies × environment (random), ga~N(0, FA1⊗ Iσga 2 ) , Xbub : vector of block effect aligned with replications (random) b~N(0,Iσb 2), and ε: vector of effect of associated errors (random), ε~N(0, N(0, j ⊕ j=1 Iσε 2)). The residual variance-covariance matrix was modeled with a diagonal structure to account for heterogeneity identified within the dataset. Subsequently, the variance-covariance structure for the genotype-by-environment interaction was modeled using an extended form of the factor analytic (FA) approach. The most suitable model was selected based on the Bayesian Information Criterion (BIC) and the Akaike Information Criterion (AIC). Progeny, population and total heritability were estimated. Accuracy was calculated at three levels: population, progeny, and overall accuracy. Additionally, the coefficient of variation of the experimental error was determined for each evaluated variable to assess the precision and consistency of the data. 4.4 DNA extraction, library preparation and sequencing The seeds of the 288 genotypes were sown in trays and placed in a growth chamber maintained at 25°C. The seeds were grown until the emergence of the first trifoliate and six leaf punches were collected from each genotype. The samples were immediately frozen and stored at -80°C. Subsequently, the samples were lyophilized (freeze-dried) to remove moisture. DNA extraction and genotyping were conducted using the KLEARGENE commercial kit (LGC Group, Teddington, England). A customized Ion AgriseqTM target GBS panel was used to genotype 1329 single nucleotide polymorphism (SNP) markers. The library construction was performed using ThermoFisher’s protocol which uses 5 µL AmpliSeq Panel, 2 µL 2x Ion AgriSeq Amplification Mix, and gDNA (10 ng/rxn). The target amplification depends on the of the number of targets for this panel size, 15 cycles were used, consisting of 99C for 15 seconds of denaturation and 60C for 8 minutes forannealing and extension. Barcode Reaction Mix from ThermoFisher was used with 1 µL of barcode enzyme and 2 µL of barcoding buffer that was used with IonCode™ Barcode Adapters. The barcode ligation was performed running the program 22C for 30 minutes and 72C for 10 minutes. Samples pools were created, and library cleanup and normalization were performed. A two-round AMPure clean-up was done. First, 1.5X AMPure clean-up was added to remove previous residual reagents, 2X 70% Ethanol washes and Elute in Low TE. Then a second round was performed with 1.2X AMPure clean-up, 2X 70% Ethanol washes and elute in normalization master mix and 9-cycle PCR 98C for 15 seconds of denature and 64C for 1 minute for annealing and extension. Normalization clean-up brings all libraries to the same concentration (200 pM) so they can be pooled 1:1. The purified and normalized libraries were prepared for loading into the Ion Chef system, which automates template preparation and chip loading for the ION S5 sequencer. The final step was sequencing those libraries on Ion GeneStudioTMS5 Prime, which can deliver up to 80 million reads per run. Bioinformatic analysis was then performed using Torrent Suite™ Software, which processed the sequencing data on a computer connected to the Ion Torrent™ server v.5.12.3. The outcome of this analysis was a matrix, listing the genotypes alongside their corresponding SNP markers. A quality control filter analysis was performed using call rate below 75% per markers and genotypes and MAF below 0.05 using the function qc.filtering from ASRgenomics R package (Gezan et al., 2022). 4.5 Selection, imputation and coverage of SNPs Monomorphic SNPs and those with a Minor Allele Frequency below 5% were removed. Genotypes with a call rate below 75% were excluded. The final dataset comprised 605 selected SNPs and 267 genotypes. For the
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