Identification and validation of mutation points associated with waxy phenotype in cassava

Abstract

Background: The granule-bound starch synthase I (GBSSI) enzyme is responsible for the synthesis of amylose, and therefore, its absence results in individuals with a waxy starch phenotype in various amylaceous crops. The validation of mutation points previously associated with the waxy starch phenotype in cassava, as well as the identification of alternative mutant alleles in the GBSSI gene, can allow the development of molecular-assisted selection to introgress the waxy starch mutation into cassava breeding populations. Results: A waxy cassava allele has been identified previously, associated with several SNPs. A particular SNP (intron 11) was used to develop SNAP markers for screening heterozygote types in cassava germplasm. Although the molecular segregation corresponds to the expected segregation at 3:1 ratio (dominant gene for the presence of amylose), the homozygotes containing the SNP associated with the waxy mutation did not show waxy phenotypes. To identify more markers, we sequenced the GBSS gene from 89 genotypes, including some that were segregated from a cross with a line carrying the known waxy allele. As a result, 17 mutations in the GBSSI gene were identified, in which only the deletion in exon 6 (MeWxEx6-del-C) was correlated with the waxy phenotype. The evaluation of mutation points by discriminant analysis of principal component analysis (DAPC) also did not completely discriminate the waxy individuals. Therefore, we developed Kompetitive Allele Specific PCR (KASP) markers that allowed discrimination between WX and wx alleles. The results demonstrated the non-existence of heterozygous individuals of the MeWxEx6-del-C deletion in the analyzed germplasm. Therefore, the deletion MeWxEx6-del-C should not be used for assisted selection in genetic backgrounds different from the original source of waxy starch. Also, the alternative SNPs identified in this study were not associated with the waxy phenotype when compared to a panel of accessions with high genetic diversity. Conclusion: Although the GBSSI gene can exhibit several mutations in cassava, only the deletion in exon 6 (MeWxEx6-del-C) was correlated with the waxy phenotype in the original AM206-5 source.

Near-infrared spectroscopy for early selection of waxy cassava clones via seed analysis

“…Several studies employ molecular markers to understand the genetic control of the waxy genotype, which guides the crossing planning of accessions, since the waxy phenotype is expressed in the recessive condition ( Aiemnaka et al., 2012 ; Carmo et al., 2020 ). However, despite the development of protocols that allow the use of selection assisted by molecular markers related to the GBSSI (granule-bound starch synthase I) gene derived from the waxy starch source AM206-5, there remain obstacles when the population has a different genetic origin than the AM206-5 source ( Carmo et al., 2020 ). Therefore, using technologies that allow a faster, earlier selection of waxy genotypes is desirable in the most diverse breeding programs.…”

“…Genomic studies have enabled the identification of target genes that control amylose and amylopectin synthesis and enabled the selection of markers associated with these genes with potential use in marker-assisted selection (MAS) ( Aiemnaka et al., 2012 ; Carmo et al., 2020 ). Starch biosynthesis is genetically controlled by target genes, including granule-bound starch synthases (GBSS), soluble starch synthases (SSS), starch branching enzyme (SBE or BE), debranching enzyme (DBE), and protein targeting to starch (PTST) ( Zeeman et al., 2010 ; Bahaji et al., 2014 ; Seung et al., 2015 ; Seung et al., 2017 ).…”

“…GBSSI-related SNP markers have not proven useful for MAS in populations with different genetic backgrounds ( Aiemnaka et al., 2012 ; Carmo et al., 2020 ). Alternatively, the phenotypic identification between waxy and non-waxy genotypes is usually determined by staining the roots with iodine, which is a chemical method.…”

Cassava (Manihot esculenta) dual use for food and bioenergy: A review

“…Genotypes are categorized into four classes based on the levels of amylose: waxy, semi‐waxy, normal‐regular and high, with maximum amounts of 2, 15, 35 and >35% of amylose per total starch respectively (Chisenga, 2021). Most cultivated genotypes fall within the normal‐regular group, but genotypes completely lacking amylose, aka waxy category, have been naturally reported (do Carmo et al, 2020; Toae et al, 2019). Mutants of the granule ‐ bound starch synthase ( GBSS ) gene that is involved in amylose biosynthesis equally results in amylose‐free cassava starch (Bull et al, 2018).…”