Molecular Fingerprinting and Hybridity Authentication in Cowpea Using Single Nucleotide Polymorphism Based Kompetitive Allele-Specific PCR Assay

Abstract

Optimization of a breeding program for increased genetic gain requires quality assurance (QA) and quality control (QC) at key phases of the breeding process. One vital phase in a breeding program that requires QC and QA is the choice of parents and successful hybridizations to combine parental attributes and create variations. The objective of this study was to determine parental diversity and confirm hybridity of cowpea F1 progenies using KASP (Kompetitive Allele-Specific PCR)-based single nucleotide polymorphism (SNP) markers. A total of 1,436 F1 plants were derived from crossing 220 cowpea breeding lines and landraces to 2 elite sister lines IT99K-573-1-1 and IT99K-573-2-1 as male parents, constituting 225 cross combinations. The progenies and the parents were genotyped with 17 QC SNP markers via high-throughput KASP genotyping assay. The QC markers differentiated the parents with mean efficiency of 37.90% and a range of 3.4–82.8%, revealing unique fingerprints of the parents. Neighbor-Joining cladogram divided the 222 parents into 3 clusters. Genetic distances between parents ranged from 0 to 3.74 with a mean of 2.41. Principal component analysis (PCA) depicted a considerable overlap between parents and F1 progenies with more scatters among parents than the F1s. The differentiation among parents and F1s was best contributed to by 82% of the markers. As expected, parents and F1s showed a significant contrast in proportion of heterozygous individuals, with mean values of 0.02 and 0.32, respectively. KASP markers detected true hybridity with 100% success rate in 72% of the populations. Overall, 79% of the putative F1 plants were true hybrids, 14% were selfed plants, and 7% were undetermined due to missing data and lack of marker polymorphism between parents. The study demonstrated an effective application of KASP-based SNP assay in fingerprinting, confirmation of hybridity, and early detection of false F1 plants. The results further uncovered the need to deploy markers as a QC step in a breeding program.

DNA-Based Tools to Certify Authenticity of Rice Varieties—An Overview

“…With the current resequencing efforts, it is expected that the use of SNPs will rise due to the above-discussed advantages they show over other molecular markers. In fact, SNP strategies are already being implemented for authentication in other cereal species [124][125][126].…”

“…With the current resequencing efforts, it is expected that the use of SNPs will rise due to the above-discussed advantages they show over other molecular markers. In fact, SNP strategies are already being implemented for authentication in other cereal species [124][125][126]. The identification of polymorphisms generated by insertion or deletion (InDel) of small sequences in target genes has also been used in the identification of varieties, particularly for aromatic/basmati rice (Figure 4) [58,114].…”

A medium density DArTag single nucleotide polymorphism panel for genetic dissections and deployment in cowpea improvement

“…For this analysis, a neighbor-joining method (Nei & Saitou, 1987) was used to generate the genetic distances and phylogenetic tree using archaeopteryx in TASSEL v.5.2.79 to visualize the clustering among parents. This was followed by analysis of marker polymorphism between every pair of parents used in making the 30 F 1 s as previously described (Ongom et al, 2021). Markers that were found to be polymorphic between the parental pairs were then used to assess the level of hybridity among the F1s.…”

“…Markers that were found to be polymorphic between the parental pairs were then used to assess the level of hybridity among the F1s. Hybridity was expressed as a ratio of the number of polymorphic markers that detected a particular F 1 as being heterozygous to the total number of polymorphic markers between the parents of that cross (Ongom et al, 2021). Further, SNP marker efficiency was assessed by determining how frequent a marker was polymorphic across the 30 pairs of parents (Ongom et al, 2021), and this allowed us to identify a set of SNPs that are suitable for hybridity testing and parental fingerprinting.…”

“…For instance, in a breeding program, identification of genetic relatedness among parents is key in prioritizing crosses that will maximize diversity for eventual increased genetic gain (Ongom et al, 2021). Also, association studies and linkage analyses without accounting for the increased relatedness due to population genetic structure could lead to spurious associations (Pritchard et al, 2000b).…”