The advances in genomics in recent years have increased the accuracy and efficiency of breeding programs for many crops. Nevertheless, the adoption of genomic enhancement for several other crops essential in developing countries is still limited, especially for those that do not have a reference genome. These crops are more often called orphans. This is the first report to show how the results provided by different platforms, including the use of a simulated genome called the mock genome, can generate in population structure and genetic diversity studies, especially when the intention is to use this information to support the formation of heterotic groups, choice of testers and genomic prediction of single-crosses. For that, we used a method to assemble a reference genome to perform the SNP calling without needing an external genome. Thus, we compared the analysis results using the mock genome with the standard approaches (Array and GBS). The results showed that the GBS-Mock presented similar results to the standard methods of genetic diversity studies, division of heterotic groups, the definition of testers, and genomic prediction. These results showed that a mock genome constructed from the population's intrinsic polymorphisms to perform the SNP calling is an effective alternative for conducting genomic studies of this nature in orphan crops, especially those that do not have a reference genome.
Article
On the usefulness of mock genomes to define heterotic pools, testers, and hybrid predictions in orphan crops.
https://doi.org/10.21203/rs.3.rs-2358482/v1
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Genotyping-by-sequencing
SNP-array
formation of heterotic groups
genomic prediction of single-crosses
minor crops
underused crops
simulated genome
Molecular markers have been used to develop genomic tools to improve economically important crops (Mammadov et al., 2012; Thomson, 2014). Currently, SNP (Single Nucleotide Polymorphism) markers are the most used in genomic studies (Fritsche-Neto et al., 2021), as they provide higher resolution due to their frequent occurrence and uniformity throughout the genome (Gupta et al., 2008). Rapid advances in next-generation sequencing (NGS) technologies, combined with high levels of diversity in SNP, have made it possible to develop high-throughput genotyping platforms (Bachlava et al., 2012).
There are several genotyping platforms for obtaining SNPs throughout the genome, which have provided an infinity of sequencing information with remarkable improvements in coverage, time, and costs, making it possible to genotype thousands of samples with many markers (Bevan and Uauy, 2013), with SNP-array and NGS platforms being the most appropriate for this purpose (Rasheed et al., 2017). There are many array-based genotyping platforms available in major crops such as maize (Unterseer et al., 2014), wheat (Winfield et al., 2016), rice (Singh et al., 2015), and soybean (Lee et al., 2015). These platforms have many advantages, such as fast scans with high call rates and density. However, they present an investigation bias when the set of individuals does not faithfully represent the genetic diversity explored in the study panel. Furthermore, it has a high cost, inaccessible to small breeding programs (Messing and Dooner, 2006; Frascaroli et al., 2013), especially those of unprofitable crops.
Beyond crop-specific SNP arrays, NGS-based platforms are adaptable to various crops, regardless of prior knowledge of genomics, genome size, organization, or ploidy (Rasheed et al., 2017). Genotyping-by-sequencing (GBS) appears as an alternative to overcome the verification bias since it is based on sequencing and, therefore, allows the discovery of alleles in the diversity panel analyzed, in addition to having a lower cost compared to SNP-array (Heslot et al., 2013). However, GBS generates many low-quality markers with a high rate of lost data (Heslot et al., 2013).
The advances in genomics in recent years have increased the accuracy and efficiency of breeding programs for many crops. However, the adoption of genomic enhancement for several other staple crops essential in developing countries is still limited, especially for traits under complex genetic control, which are crucial to crop performance (Varshney et al., 2012). This is because most studies use the array-based and GBS-based SNP markers approach, which depends on a reference genome for SNP discovery. And crops that do not have a reference genome cannot take advantage of biotechnological tools to improve their genetic gain and develop modern cultivars faster (Armstead et al., 2009).
There are many crops of unique relevance to developing countries, essential for the food, nutritional and economic security of these countries, which still do not have a reference genome (Baldermann et al., 2016; Hendre et al., 2019). These crops are more often called orphans. The term “orphan” is derived from the condition of neglect and helplessness of these crops by the scientific community, leading to the designation of such species as underused, neglected, or minor crops (Tadele and Assefa, 2012). GBS also appears as an option for genomic studies in these crops, especially when they do not have a reference genome (Sabadin et al., 2022). With these data, it is possible to build a mock reference to perform the SNP calling, where the discovery of polymorphisms will be intrinsic to the study population without using an external genome (Melo et al., 2016). This pipeline has already been successfully used in several genomic studies (Holloway et al., 2018; Munjal et al., 2018; Adhikari et al., 2018; Matias et al., 2019; Sabadin et al., 2022). Adopting this technology in poorly studied crops has a tangible impact on the progress of the breeding process (Ye and Fan, 2021).
Recent studies have compared the performance of genotyping platforms and how this choice affects genomic studies regarding genetic diversity studies (Elbasyoni et al., 2018; Darrier et al., 2019), GWAS (Negro et al., 2019), and genomic prediction (Chu et al., 2020). Only one report compares the performance of the mock reference pipeline with standard genotyping approaches in genomic prediction studies (Sabadin et al., 2022). However, this study used a relatively small germplasm panel. It did not consider the effect of genotyping platforms on population structure, the formation of heterotic groups, and the choice of testers, which is fundamental for a breeding program that wants to synthesize single-crosses.
We conducted a full study on this topic with a robust germplasm panel. For this, we compared different genotyping scenarios from the beginning of the breeding process with the approach of genetic diversity and population structure, advancing to the formation of heterotic groups and choice of testers to the synthesis and prediction of single-crosses.
Although some reports are available, there still needs to be a consensus on how different platforms provide the results. Thus, we conducted a full study on this topic with a robust germplasm panel. For this, we compared different genotyping scenarios from the beginning of the breeding process with the approach of genetic diversity and population structure, advancing to the formation of heterotic groups and choice of testers to the synthesis and prediction of single-crosses. Thus, this information will be valuable to leverage genomic studies and accelerate the development of cultivars in minor crops without a reference genome. Therefore, our goals were to verify whether the source of SNP can influence the assessment of the population structure of parental lines, to ascertain if the source of SNP can affect the determination of heterotic groups and the prediction of single-crosses performance, and check if the GBS and the mock genome efficiently performs the SNP calling in orphan crops (without reference genome).
Maize was used as a model species in this study because it is already well established in terms of SNP-array, with several array options available (Ganal et al., 2011; Unterseer et al., 2014; Xu et al., 2017). And GBS protocols are also well-established for this species (Crossa et al., 2013; Li et al., 2015; Wang et al., 2020). In addition, maize has a diverse, complex, and dynamic genome (Schnable et al., 2009), which is suitable for this study. We used a public panel of tropical maize germplasm containing 360 parental lines (Yassue, Carvalho, et al., 2021). The genomic and phenotypic information about this panel is available on the Mendeley platform (https://data.mendeley.com/datasets/5rtc89t7v5/1).
Phenotypic data
The phenotypic dataset consists of 903 maize single-crosses (Fritsche-Neto et al., 2019) derived from a diallel cross between 49 parental lines to a public tropical maize diversity, selected based on nitrogen use efficiency (Mendonça et al., 2017). Field trials were carried out in Anhembi (22º50'51''S, 48º01'06''W) and Piracicaba (22º42'23''S, 47º38'12''W), in the State of São Paulo, during the second growing season, from January to June 2016 and 2017. Single-crosses were evaluated in an augmented block design, where each block consisted of 16 single-crosses and two checks (commercial single-crosses). In both locations and years, the single-crosses were evaluated under two nitrogen (N) conditions, low N with 30 kg N ha-1 and ideal N with 100 kg N ha-1. Each location x year x N level combination was defined as an environment.
Each plot consisted of 7 m rows spaced 0.50 m apart. Conventional fertilization and weed and pest control were carried out. The traits evaluated were grain yield (GY, mg ha-1), plant height (PH, cm), and ear height (EH, cm). The plots were harvested manually, and the grain yield was corrected for 13% moisture. More details on the phenotypic dataset's experimental design and cultivation practices were previously reported by Fritsche-Neto et al. (2018) and Galli et al. (2020).
Genetic-statistical model for obtaining BLUEs
The joint analysis of each trait was performed to estimate the means of the single-crosses across the environments. Thus, an equation was adjusted to obtain the Best Linear Unbiased Estimator (BLUE) for each genotype, and later, the adjusted means of these across the environments evaluated by the following mixed model were estimated:
where y is the vector of phenotypic values of single-crosses and checks; l the vector of fixed effects of the environment (site x year x N level combination); b is the vector of random effect of block nested within environments, where b ~N (0, Iσ²b); c is the vector of fixed effects of checks; g is the vector of fixed effects of single-crosses; i is the vector of fixed effects of the interaction checks x environments; Ɛ is the vector of random residual effects, where ε ~ N (0, De). An unstructured covariance matrix across environments was assumed for the residual term (De) due to the contrasting doses of N. Q, S, T, U, and V are the incidence matrices for l, b, c, g, and i. The analysis was performed using the ASReml-R (Butler et al., 2018).
Genotypic data and analysis
The 360 lines of tropical maize belonging to the diversity mentioned above panel were genotyped using two high SNP density genotyping platforms: 1) Affymetrix® Axiom Maize Genotyping Array (SNP-array), and 2) Genotyping-by-sequencing (GBS) method following the protocol described by Poland et al. (2012). In this last method, individual samples of genomic DNA were digested by two restriction enzymes, PstI and MseI, to reduce the genome complexity uniformly. Subsequently, the samples were included in a sequencing plate, performed on the Illumina NextSeq 500 platform (Illumina Inc., San Diego, CA, United States).
The raw GBS data were used for two purposes: the first was to perform the SNP calling using the B73 line of temperate germplasm as a reference genome. The second purpose was to build a simulated reference genome (mock genome), according to the GBS-SNP-CROP pipeline proposed by Melo et al. (2016), and use it to perform the SNP calling. More details about the mock reference can found in (Sabadin et al., 2022).
Further analysis was performed considering three SNP datasets: 1) SNP-array; 2) GBS with SNP call using B73 as the reference genome (GBS-B73) and 3) GBS with the simulated genome being used as the reference genome (GBS-Mock). For GBS datasets, SNPs were scored from raw data using the TASSEL 5.0 GBSv2 pipeline (Glaubitz et al., 2014) according to standard parameters. Using the BWA aligner (Li and Durbin, 2009), the tags were aligned against the reference genome (GBS-B73 and GBS-Mock).
As two genotyping (SNP-array and GBS) were performed, the parental lines that showed a very contrasting genotypic profile between the two platforms were removed from the analysis to obtain a fair comparison. Thus, between sequencing errors and divergences in genotypic profiles between platforms, 330 parental lines remained, among which 45 parental lines make up the diallel, which generated 751 single-crosses. The number of markers concerning to the raw data was 18,413 (SNP-array), 131,350 (GBS-B73), and 46,9126 (GBS-Mock). All sets of SNPs underwent quality control, in which low call rate (<90%) and non-biallelic markers were removed from the datasets. The remaining missing data were imputed by the Beagle 5.0 algorithm (Browning et al., 2018). Pairwise linkage disequilibrium was calculated as the correlation of allele frequencies squared (r²), and values greater than 0.99 were removed from the datasets using the SNPRelate package (Zheng et al., 2012), resulting in 12,704 (SNP-array), 11,153 (GBS-B73, and 4,935 (GBS-Mock) SNP markers.
Subsequently, new quality control was performed, in which heterozygous loci in at least one individual were removed, and high-quality polymorphic SNPs from the parental lines were combined (in silico) to build an artificial single-crosses genomic matrix. In addition, duplicate markers between chromosomes were removed to avoid overparameterization caused by multicollinearity. Finally, markers with Minor Allele Frequency (MAF) < 0.05 were removed from the single-crosses genomic matrices, resulting in 11,884 (SNP-array), 10,361 (GBS-B73, and 4,801 (GBS-Mock) SNP markers to perform the remaining analysis.
Analysis of population structure and genetic diversity
The three SNP datasets (SNP-array, GBS-B73, and GBS-Mock) from the 330 parental lines were used to assess the population structure of the panel. In these analyses, precisely, heterozygous loci and rare variants (MAF < 0.05) were considered to capture all diversity and variability to perform principal component analysis (PCA) and determine the relatedness between parental lines.
K-means clustering was applied, using the total Within-cluster Sum of Square (WSS) method to determine the optimal number of clusters so that the total intra-cluster variation is minimized (Kassambara, 2017). The factoextra package (Kassambara and Mundt, 2020) was used for this. Subsequently, the Kendall method determined the coincidence in forming clusters among the different datasets (Kendall, 1938). Kendall's tau correlation coefficient was tested at a probability level of 0.01. PCA was performed, and biplots were constructed to assess population structure.
The genetic distances between the parental lines were calculated for each SNP dataset using the Rogers distance (Rogers, 1972). Subsequently, to measure the correlation among the kinship matrices, the Mantel correlation test (Mantel, 1967) was applied to detect significance. The Mantel correlation test is non-parametric and computes the significance of the correlation similarity measures using 1000 permutations of the rows and columns of one distance matrices. The heatmaps of the genetic distance matrices were obtained using the superheat R package (Barter and Yu, 2018). Correlations were obtained using the vegan package (Oksanen et al., 2019), and each analysis was performed for each SNP dataset scenario.
Full diallel genomic analysis
To find out how diversity and population structure can influence the formation of heterotic groups, it was necessary to construct in silico genome of the 751 single-crosses from parental lines. So, at this stage, we combined phenotypic and genotypic information from these individuals to estimate general (GCA) and specific combining abilities (SCA). For this, the following diallel model was adjusted:
y = ZPgP + ZHh + Ɛ
where, y is the adjusted phenotypic data vector of the single-crosses for the trait; gP is the random effect vector of the GCA captured by the markers of the parental lines, and h is the random effect vector of the SCA that denotes the interaction effects across the parental lines. ZP e ZH are incidence matrices that relate y to gP and h to gp ~N (0, σ²pGp) and h ~N (0, σ²HH), where σ²P and σ²H are variance components associated with GCA and SCA, respectively. And GP and H are relationship matrices for the parental lines and single-crosses, respectively. Finally, Ɛ ~ N (0, σ²Ɛ I), where σ²Ɛ is the variance associated with the residuals.
The GP relationship matrix was calculated using the SNP markers according to (VanRaden, 2008), where WP is the matrix of centered and patterned markers. So, GP = 
(Technow et al., 2014; Lopez-Cruz et al., 2015), where p is the number of markers. This resulted in an average diagonal Gp value of ~ 1; therefore, σ²p was defined on the same scale as σ² Ɛ. The elements of the H matrix were obtained directly from the GP (Bernardo, 2002; Technow et al., 2014). The matrix H for all possible crosses was obtained with the Kronecker product between GP’s, H = Gp ⊗ Gp (Covarrubias-Pazaran, 2016). A model was built with their respective kernels for each SNP marker source. Analyses were performed using the ASReml-R package (Butler et al., 2018).
Heterotic groups and testers
The determination of heterotic groups was performed based on SCA estimates for each trait. These estimates corresponded to a matrix of genetic distances. According to (Falconer and Mackay, 1996), the genetic distance between parents positively affects heterosis. This association depends on dominance effects or differences in the frequency of the alleles that control the trait considered (Falconer, 1960). Burstin et al. (1994) also found that SCA variance is an indicator for predicting hybrid performance by genetic distance between parents. According to this information, it was assumed that the higher the SCA estimates, the greater the distance between the parents and the more significant the heterosis. From this, the 45 lines were divided in two heterotic groups.
The SCA estimates were submitted to a clustering algorithm, K-means, which grouped them according to the SCA estimates. To estimate the correlation between the heterotic groups formed for the different genotyping methods, Pearson's correlation was applied and tested at a probability level of 0.01 by Student's t-test. Subsequently, the identification of the best tester in each group was performed according to the GCA estimates. The best tester of a given group was the line that showed the highest GCA with the other group. Based on this, the coincidence of testers between the scenarios was evaluated.
Obtaining single-crosses combinations and genomic prediction
After the parental lines were divided into heterotic groups, only the single-crosses corresponding to interpopulation crosses via North Carolina II (NCII) design were considered for the following analyses. The number of single-crosses changed according to the configuration of heterotic groups for each trait in the three SNP scenarios.
For the genomic prediction of the single-crosses, an additive-dominant Genomic Best Linear Unbiased Prediction (GBLUP) model was used, as described below:
where, 
is the adjusted means vector of the single-crosses for the trait; µ is the mean (intercept); a is the vector of additive genetic effects of individuals, where a ~ N (0, Gaσ²a); d is the vector of dominance effects, where d ~ N (0, Gdσ²d); and Ɛ is the random effects vector of the residuals, where Ɛ ~ N (0, I σ²Ɛ). Z is the incidence matrix for a and d. σ²a is the additive genomic variance, σ²d is the dominance genomic variance, and σ² Ɛ is the residual variance. Ga and Gd are the additive and dominance genomic relationship matrices of the single-crosses, where 
, where pi is the frequency of an allele at locus i and W is the matrix incidence of markers (VanRaden, 2008). The WA matrix was encoded as 0 for A1A1, 1 for A1A2 heterozygote, and 2 for A2A2 homozygote. For WD, genotypes were coded as 0 for both homozygotes and 1 for the heterozygote. The genomic relationship matrices were built using the snpReady package (Granato et al., 2018). And the genomic prediction models were performed using the sommer package (Covarrubias-Pazaran, 2016). It is worth noting that all three sets of markers were used to build the kernels. The Mantel correlation test (Mantel, 1967) was applied to detect significance between the additive and dominance genomic relationship matrices.
To evaluate the model performance, we used the CV-α cross-validation with five folds and four replicates (Yassue, Sabadin, et al., 2021). The predictive ability was estimated by Pearson's correlation between predicted genotypic and observed values from the validation set. Correspondence between phenotypic and genotypic selection was calculated for each set of markers through the percentage of common genotypes selected by their adjusted means from the phenotypic analysis and their Genomic Estimated Breeding Values (GEBV) from the genomic prediction model concerning different intensities of selection (1%, 10%, 20%, 30%, and 40%). The heritability in the broad-sense (H²) and the narrow-sense (h²) was also estimated by the equations below:
Genetic diversity and population structure
According to the WSS method, for all datasets, the optimal number of clusters among the 330 parental lines that minimized within-group variance and maximized between-group variance was six (Fig. S1). Subsequently, the K-means clustering method showed a remarkable similarity in the arrangement of clusters among datasets (Fig. 1). This similarity is confirmed by the coincidence values in the clustering (Table S1), with correlation coefficients above 0.95.
Concerning principal component analysis (PCA), the SNP datasets showed similar performances regarding the variance explained by the principal components. The first principal components hold the highest percentage of explained variance (Fig. S2a). When considering the first ten main components, SNP-array showed the highest value of cumulative explained variance (27.3%). At the same time, GBS-B73 and GBS-Mock presented discounts of 24.1% and 16.8%, respectively (Fig. S2b).
In general, PCA revealed that the first eigenvectors exhibited similar patterns of variance in all combinations between datasets, supported by the coefficient of determination (R²). However, the other eigenvectors showed less similarity between the captured variance patterns (Fig. 2). The first four eigenvectors of SNP-array and GBS-B73 showed high values of R² (Fig. 2a). In contrast, for SNP-array and GBS-Mock, the highest values of R² were concentrated in the first three eigenvectors (Fig. 2b). For GBS-B73 and GBS-Mock all eigenvectors showed high magnitude R², the former being slightly higher than the others (Fig. 2c).
Biplots were constructed to visualize the spatial distribution of lines in all SNP datasets (Fig. 3, Fig. S3, Fig. S4). For this, the first three PCs were used, together with the information obtained by the K-means clustering method (Fig. 1). All datasets showed the same pattern of dispersion among the lines, in agreement with the cluster analysis, which suggests that the SNP datasets capture similar patterns of variance (Fig. 3).
Rogers distance matrices (GD) from all SNP datasets sampled similar groups and subgroups, with slight differences between them (Fig. S5). Regarding the Mantel correlations between the GDs, high magnitude correlations (> 0.83) were observed involving different scenarios (Table 1).
Variance components, genomic heritability, and genomic relationship matrices (GRMs)
Broad and narrow heritabilities were higher for EH, followed by PH and GY (Table S2). For all SNP datasets, GY showed broad-sense heritability, on average, 36% higher than narrow-sense heritability. This difference is significantly smaller for the other traits, 15%, and 6%, for PH and EH, respectively. For GY, the narrow-sense heritability for all SNP datasets was practically the same. As for PH, there was a slight difference in SNP-array, and GBS-Mock presented heritability slightly higher than GBS-B73. For EH traits, narrow-sense heritability varied little among SNP datasets, with GBS-Mock and SNP-array showing the highest heritabilities. The heritabilities in the broad-sense (H²) followed the same tendency.
Regarding the additive genomic relationship matrices (Ga) across the single-crosses, SNP-array, GBS-B73, and GBS-Mock showed high Mantel correlations (Table 1, Fig. S6a, b, c). On the other hand, the genomic dominance relationship matrices (Gd) showed lower correlations than Ga. The correlations between the dominant relationship matrices (Gd) were lower but still from medium to high. GBS-Mock stands out with a correlation of 0.72 with GBS-B73.
Heterotic groups and testers
The 45 parental lines were divided into heterotic groups based on SCA estimates as the genetic distance between them for the evaluated traits, GY, PH, and EH. Accordingly, two heterotic groups were formed for all SNP datasets (Fig. 4). The formation of heterotic groups among the SNP datasets was quite similar, with high correlations, higher than 0.94 for GY and 0.87 for PH and EH (Table S3). There was, at most, a change in the allocation of two parental lines between heterotic groups in different SNP datasets. Likewise, the SCA correlations of the parental lines among the SNP datasets were higher than 0.96 (Table S4).
GCA estimates from each parental line, trait, and SNP dataset were used to choose the best tester in each group (Table 2). Thus, the testers matched among SNP datasets in the respective heterotic groups for each trait. The tester chosen, based on GY, for heterotic group one (HP1) was L023, and for heterotic group two (HP2), it was L006. As for PH and EH, L001 was elected as the HP1 tester, and L003 as the HP2 tester. The correlations between the GCAs confirm this result, with maximum correlations (Table S4).
Genomic prediction
The predictive ability estimated by the additive-dominant model for all traits and following the same trend as the other results did not vary significantly among SNP datasets (Fig. 5). The mean values of PA were 0.58 for GY, 0.64 for PH and 0.83 for EH. The coincidence between selected individuals based on the adjusted means of the phenotypic analysis and the GEBVs of the genomic prediction model was generally satisfactory. It increased with rising selection intensity (Fig. 6). Although GY is considered the most complex, the selection coincidence levels of this one were similar to the other traits. SNP-array showed slightly higher coincidence values for almost all selection intensities. However, the different datasets showed approximate coincident values.
Recent advances in crop genetics and genomics have gained remarkable attention and offered genotyping technologies (Chakradhar et al., 2017). Various genotyping platforms are available to meet the most diverse needs regarding costs per sample and different marker densities (Thomson, 2014). GBS, in particular, has emerged as a cost-effective strategy for genome-wide SNP discovery and population genotyping due to the simple library preparation and the robust approach to genome reduction (Elshire et al., 2011).
All this progress, including well-characterized genes and vast collections of genetic and genomic resources, focuses on a small group of crops (Tester and Langridge, 2010) to the detriment of smaller agricultural species, considered orphans, historically poorly researched (Mayes et al., 2012), in that the large majority do not have a reference genome. Sabadin et al. (2022) showed that using mock genomes could be a worthy strategy that permits using SNP markers for genomic selection in orphan crops. However, orphan crop breeding programs focused on single-crosses development must also determine heterotic groups to maximize the heterosis. Our study aims to go forward and verify the usefulness of mock genomes as a method to permit reliable heterotic group clustering.
Influence of genotyping methods on population structure and diversity
The study of the characterization of genetic diversity, population structure, and genetic relationships among elite parents of germplasm, based on the use of molecular markers, can accelerate genetic gains in breeding programs (Romay et al., 2013; Adu et al., 2019). This study helps understand how the germplasm is organized in selecting parents that present effective contributions and in the designation of heterotic groups (Wu et al., 2016). Thus, genomic data not only allows the estimation of genetic diversity but also combines them with phenotypic information to find new functional genes and build prediction models (Milner et al., 2019). However, in this topic, the focus is on whether, with the simulated reference genome, there is the discovery of the same polymorphisms and how it reflects on the population structure of the lines.
The WSS method indicated the optimal number of clusters by locating a curve on the plot, generally considered an indicator of the optimal number of groups (Kassambara, 2017). With this information and the results of the K-means clustering, the parental lines were partitioned into subpopulations, where the SNP datasets showed similar behavior (Fig. S1, Fig. 1, Table S1), in agreement with the spatial distributions obtained in the biplot graphs (Fig. 3), in which all SNP datasets showed the same dispersion pattern between lines. This suggests that the SNP datasets capture similar patterns of variance, despite the difference in the number of markers between them, where GBS-Mock has a lower number and the difference in the genotyping platform itself (array and GBS). Thus, SNP-array, GBS-B73, and GBS-Mock revealed similar performances concerning genetic diversity and the population structure of parental lines. Darrier et al. (2019) compared the performance of SNP-array and GBS to investigate the extent and pattern of genetic variance in barley and observed that the two methodologies selectively access the informative polymorphism in different portions of the genome. Despite this, their results showed a strong positive correlation between the matrices of both genotyping approaches, supporting their similarity and validity.
PCA shows that these variance patterns captured by the SNP datasets are more similar concerning the first eigenvectors (Fig. 2). However, the captured variance is more consistent when comparing GBS-B73 and GBS-Mock (Fig. 2c). This can be explained by the verification bias existing in the SNP-array since this bias arises when the markers are not obtained from a random sample of the polymorphisms of the population of interest, since the matrix is constructed using temperate maize lines (Frascaroli et al., 2013; HeslotHeslop et al., 2013; Unterseer et al., 2014), and the lines in the study are from tropical germplasm.
The matrices of genetic distances among the parental lines revealed similar patterns, showing the formation of subpopulations between the lines (Fig. S5). When using wheat as a model species to test for verification bias and investigate its impact on genetic diversity estimates, Chu et al. (2020) observed a tendency for SNP array, leading to an underestimation of molecular diversity within the population. These results agree with a previous study on wheat lines (Elbasyoni et al., 2018) and maize lines (Frascaroli et al., 2013). Despite the verification bias mentioned above and the difference between the reference genome used, the temperate B73 genome, or the Mock genome, the population structure between the lines did not show a significant difference, as the correlations between the matrices of genetic distances were of high magnitude. Even though GBS-Mock uses a different reference genome from SNP-array and GBS-B73, the correlation between them was high (Table 1). Elbasyoni et al. (2018), investigating the influence of SNPs from different genotyping platforms on genomic prediction, observed a high correlation (r = 0.77) between SNP-array and GBS genetic distance matrices. These high-magnitude correlations suggest that the broad sampling of diversity is well represented by the approaches used in the study. This is supported by the GWAS study by Darrier et al. (2019). They indicated that methods using SNP-array and GBS could detect markers closely associated with genes that control key phenotypic traits.
Influence of genotyping methods in the determination of heterotic groups in the choice of testers
Heterosis is a fundamental phenomenon in obtaining superior single-crosses. Establishing heterotic groups to exploit them effectively throughout the breeding cycles is necessary. These, in turn, are made up of genetically related parental lines, which generate little or no heterosis when crossed with each other. Crossing with lines from another heterotic group tends to result in vigorous single-crosses (Lee, 1995). Therefore, genetic diversity among heterotic groups tends to increase the level of heterosis detected in hybrid combinations (Falconer and Mackay, 1996; Fu et al., 2014). Badu-Apraku et al. (2011) reported in their diallel study between maize lines that their genetic diversity was small and, because of this, distinct heterotic groups could not be identified. Significant genetic diversity was found in a similar study with other maize lines, and two clear heterotic groups were identified. The type of predominant gene action in the parents under investigation is another factor that affects heterotic clustering. When additive and non-additive effects are significant, and there is a predominance of additive gene action over non-additive gene action, heterotic groups are easily identified (Badu-Apraku et al., 2015; Badu-Apraku, Fakorede, Talabi, et al., 2016; Badu-Apraku, Fakorede, Gedil, et al., 2016).
The PH and EH traits showed higher proportions of additive variance captured by the Ga matrices than GY (Table S2). Although these traits have polygenic inheritance, GY is the most complex trait and most influenced by dominance deviations (Fischer et al., 2008; Hallauer, 2010). According to Hallauer (2010), most of the loci involved with GY in maize are due to the occurrence of dominance. This is reflected in a greater difference between H² and h² for GY than for the other traits, confirming the greater influence of dominance deviations on this trait. The additive genomic relationship matrices of the single-crosses (Ga) showed high correlations among SNP-array, GBS-B73, and GBS-Mock, indicating that these approaches capture similar additive variance patterns. GBS-Mock captures additive relationships in single-crosses similar to standard procedures, SNP-array, and GBS-B73 (Table 1, Fig. S6a, b, c). On the other hand, the correlations between the dominant relationship matrices (Gd) were lower but still from medium to high. In both Ga and Gd, the correlations between SNP-array and GBS-Mock were lower, which can be explained by the fact that these SNP datasets use different reference genomes to perform SNP calling.
SCA reflects the action of non-additive gene effects, indicating intra-allelic interactions, is one of the most important parameters in identifying superior single-crosses, and is an indicator of genetic distance between parents (Sprague and Tatum, 1942; Carvalho, 1993). Thus, using the SCA estimates as the genetic distance between the lines to identify the panel structure, two heterotic groups were formed, in which the distance between them is maximized. The correlations between the SCA estimates were almost perfect (Table S4). In other words, SNP-array, GBS-B73, and GBS-Mock presented equivalent SCA estimates. Thus, the composition of heterotic groups practically did not change from one SNP dataset to another. Therefore, the determination of heterotic groups was similar regardless of the platform used (Fig. 4, Table S3).
In addition to presenting distinct heterotic groups, a well-established breeding program also offers good testers. When crossed with parental lines, these provide information about the genetic value of the lines when evaluating the ability to combine between them since it is associated with the additive effects of alleles and additive-type epistatic actions (Cruz and Vencovsky, 1989; Albrecht et al., 2014). The correct choice of a tester can have great significance in the expectation of a successful selection process (Miranda Filho, 2018). According to Hallauer and Martinson (1975), a good tester presents simplicity in use, information that correctly classifies the relative merit of the lines, and the potential for maximizing genetic gain. Thus, based on the GCA estimates between the lines, testers were elected for each heterotic group based on the evaluated traits and the SNP datasets. As expected, there were no differences in tester choice between SNP datasets, as the correlations between GCA estimates across rows were perfect (Table 2, Table S5).
Once previous results regarding the study of population structure of parental lines, the genotyping approaches produced very similar results but not the same, it was expected that this would somehow influence the formation of heterotic groups and the choice of testers. However, given the results, the genotyping platform, and, more specifically, the approach that uses the simulated genome as a strategy, the GBS-Mock, produces similar results to the standard procedures.
Influence of genotyping methods on genomic prediction of single-crosses
Assessing the performance of all single-crosses combinations of parental lines that excel in a breeding program is impractical in most cases, given that the number of combinations grows exponentially as the number of elite parents increases. Thus, obtaining estimates of the genetic values of single-crosses not evaluated became viable with the increased availability of molecular markers and genomic prediction models (Hallauer, 2010). Therefore, to accelerate genetic gain with limited resources, the prediction of single-crosses performance is highly important in modern breeding programs (Basnet et al., 2019).
However, few studies still address how genotyping platforms influence single-crosses' prediction and, more specifically, regarding the mock genome as a tool for more sophisticated analyses, such as genomic prediction. Only one recent study shows the mock genome's efficiency in predicting maize single-crosses, which may be an alternative for crops that do not yet have a reference genome (Sabadin et al., 2022). However, our study is more complete and more representative because getting approaches from the population structure phase is crucial for the intended use of germplasm through the division of heterotic groups, the definition of testers, and, finally, the genomic prediction of single-crosses.
GY showed the lowest predictive abilities in all SNP datasets, and EH was the highest in the additive-dominant GBLUP prediction model (Fig. 5, Table S2). Combs and Bernardo (2013) suggested that genomic predictions are more accurate for traits with higher heritability. In the results of Hayes et al. (2010), complex traits controlled by many small effect loci, such as GY, showed lower predictive abilities than less complex traits. Although GBS-Mock has a lower number of markers, this approach presented a similar performance to the other SNP datasets for all traits, corroborating the hypothesis that it is possible to substantially reduce the number of markers and maintain a high predictive ability (Tayeh et al., 2015; Ma et al., 2016; Sousa et al., 2019). Exceptions for long-term breeding cycles without updating the training population that would demand high marker densities (DoVale et al., 2022). In addition, the genetic distance estimates between the SNP datasets were very similar (Fig. S5).
Selection intensity must be chosen thoughtfully, as genetic variability can be drastically reduced with high selection pressure. The choice of appropriate selection intensities depends on the size of the population and the duration of the breeding program, whether short-term or long-term. In general, selection intensities ranging from 10 to 40% are used in plant breeding, the highest being applied at the beginning of a breeding program (Hallauer, 2010). For the coincidence of individuals by phenotypic selection and genomic selection, the SNP datasets showed similar behavior as the selection intensity was increased, being more pronounced from 1 to 10% of selection intensity. From then on, observing the coincidence of selection gains smaller increments (Fig. 6). Our results for predictive ability and coincidence of selection agree with the results of Sabadin et al. (2022). It is valid to consider that those different intensities modify the response rates. Thus, this coincidence between phenotypic and genomic selections is expected to reach a plateau and subsequently decrease.
Despite the apparent differences between SNP datasets, the general message is that these approaches perform comparably in the analyses performed in this study, even accessing different types of genomic sequences. While SNP-array is derived from exome capture and therefore focused on coding sequence variation, the GBS data represent a wider diversity survey in genomic regions associated with low levels of DNA methylation, which may also include many genes and gene regulatory regions (Darrier et al., 2019; Negro et al., 2019). On the other hand, the physical distribution of markers reveals higher frequencies of SNPs at the gene-rich telomeric ends of each of the chromosomes for both approaches, with this frequency being more pronounced in SNP-array (Bayer et al., 2017). The platforms probably capture nearby markers in linkage disequilibrium with QTLs (Quantitative Trait Loci). In this sense, using different platforms can be advantageous, as it allows the identification of additional QTLs.
Possible applications of the Mock genome in plant breeding
Until recently, only the main commercial crops benefited from state-of-the-art technologies. However, the development of the GBS platform emerged as an alternative for using such technologies to be viable for orphan crops. Approaches like this can convert orphan crops into crops rich in genomic resources and substantially reduce the breeding process (Varshney et al., 2009, 2012; Varshney and May, 2012).
Previously, this process was much slower than nowadays. Rice, for example, took almost 20 years to stop being an orphan crop and become a basic model for cereals (Varshney et al., 2009). Introducing these crops into the genomic era also accelerates the identification of genes underlying important agronomic traits and improves our understanding of the evolution of these species (Ye and Fan, 2021). However, many minor crops are becoming rich in genetic resources as a result of investments from various public and private initiatives, such as the African Orphan Crops Consortium (AOCC) (Hendre et al., 2019), which is a global partnership that is generating resources genomics for 101 African orphans. One of the objectives of this Consortium is to create reference genomes for these cultures. Although some efforts are being made to pay greater attention to these crops (Chiurugwi et al., 2019; Gregory et al., 2019; Jamnadass et al., 2020), the ideal is still far from being achieved with a view to several species relevant to local diets around the world that are understudied.
Despite initiatives and investments, not all crops will benefit, so they cannot take advantage of modern breeding tools. While these advances are being consolidated, mock genomes can be an alternative, where the absence of a reference genome presents a barrier to the efficient use of GBS data (Melo et al., 2017; Hale et al., 2018). In the meantime, the present study has shown that using a population-tailored mock reference to perform SNP discovery is a valid alternative. With this approach, it was possible to carry out investigations to outline a breeding program, from studies of diversity and population to genomic prediction studies. However, it is important to emphasize that a population with maximum representativeness must be considered when building a mock reference to capture all the population polymorphisms (Sabadin et al., 2022).
Although these advantages of using a mock genome in genomic studies must consider some caveats, for example, diploid crops with relatively smaller genomes are preferred over cross-pollinated or polyploid orphan crops, as these have genomes that are too complex to be sequenced. However, genome size will become less of a barrier with advances in sequencing technologies and bioinformatics tools (Armstead et al., 2009). Another challenge is in the SNP calling due to the limitations of GBS, which can lead to incorrect identification of homozygotes and heterozygotes because of the low coverage of NGS reads, in addition to a large number of lost and low-quality data (Heslot et al., 2013). According to Sabadin et al. (2022), the mock genomes do not present the physical position of the markers in a constant reference, which hinders studies such as GWAS and candidate gene discovery. Negro et al., (2019) state that SNP-array and GBS are complementary to detect QTLs tagging different haplotypes in association studies. In this sense, using other platforms can be advantageous, as it allows the identification of additional QTLs. However, no studies still demonstrate the performance of mock genomes for these purposes. When looking for these larger effect marks, the results will probably differ from those obtained with the SNP array due to changes in coverage between platforms.
Given what has been shown, it is possible to infer and recommend that a mock genome constructed from the population's polymorphisms to perform the SNP calling is an excellent strategy to support plant breeders in studies of diversity, population structure, the definition of heterotic groups, choice of testers and genomic prediction in species that still do not have a reference genome available, which is an alternative for the rapid advancement of orphan crop improvement. This approach will play a key role in improving the genetic potential of orphan crops and helping develop sustainable food systems.
Acknowledgments: We thank the Federal University of Ceará, “Luiz de Queiroz” College of Agriculture (University of São Paulo), Limagrain Field Seeds, Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - Process: 409096/2021-0 CNPq/MCTI/FNDCT, and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) - Process 2017/24327-0, for the financial support.
Author Contributions: IPM was responsible for writing the report, analysing data, interpreting results, and creating tables/figures. JCDV was responsible for supervising and analyzing data regarding genomic prediction. JFGS contributed to the manipulation of the raw genomic data and mock genome assembly. RFN contributed extracting data and designed the work that led to the submission. JCDV, JFGS and RFN played an important role in interpreting the results and contributed to the report review. All authors approved the final version.
Conflicts of interest: The authors declare no conflict of interest.
Data archiving: The datasets are available in the link: https://data.mendeley.com/datasets/5rtc89t7v5/1.
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Tables 1 and 2 are available in the Supplementary Files section.
There is no duality of interest
- Table1.xlsx
Table 1 Mantel correlation of Rogers genetic distance (GD) matrices for 330 parental lines, and of additive genomic relationship (𝑮𝒂) and dominance genomic relationship (𝑮𝒅) matrices for 751 maize single-crosses estimated from SNP-array, GBS-B73 and GBS-Mock markers.
- Table2.xlsx
Table 2 Best tester per trait, dataset and heterotic group.
- SupplementaryInformation.pdf
Supplementary Information: On the usefulness of mock genomes to define heterotic pools, testers, and hybrid predictions in orphan crops.