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

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.

Potential of SNP markers for the characterization of Brazilian cassava germplasm.

KEY MESSAGE: High-throughput markers, such as SNPs, along with different methodologies were used to evaluate the applicability of the Bayesian approach and the multivariate analysis in structuring the genetic diversity in cassavas. The objective of the present work was to evaluate the diversity and genetic structure of the largest cassava germplasm bank in Brazil. Complementary methodological approaches such as discriminant analysis of principal components (DAPC), Bayesian analysis and molecular analysis of variance (AMOVA) were used to understand the structure and diversity of 1,280 accessions genotyped using 402 single nucleotide polymorphism markers. The genetic diversity (0.327) and the average observed heterozygosity (0.322) were high considering the bi-allelic markers. In terms of population, the presence of a complex genetic structure was observed indicating the formation of 30 clusters by DAPC and 34 clusters by Bayesian analysis. Both methodologies presented difficulties and controversies in terms of the allocation of some accessions to specific clusters. However, the clusters suggested by the DAPC analysis seemed to be more consistent for presenting higher probability of allocation of the accessions within the clusters. Prior information related to breeding patterns and geographic origins of the accessions were not sufficient for providing clear differentiation between the clusters according to the AMOVA analysis. In contrast, the F ST was maximized when considering the clusters suggested by the Bayesian and DAPC analyses. The high frequency of germplasm exchange between producers and the subsequent alteration of the name of the same material may be one of the causes of the low association between genetic diversity and geographic origin. The results of this study may benefit cassava germplasm conservation programs, and contribute to the maximization of genetic gains in breeding programs.

Cross-species amplification of microsatellite loci developed for Passiflora edulis Sims. in related Passiflora Species

The aim of this study was to evaluate the selected 41 SSR markers developed for yellow passion fruit (Passiflora edulis f. flavicarpa Sims.) for their transferability to 11 different Passiflora species. Twenty-one SSR were successfully amplified in 10 wild species of passion fruit producing 101 bands. All the markers were amplifiable for at least one species. The mean transferability was 68,8%, ranging from 15,4% (primer PE11) to 100 % (PE13, PE18, PE37, PE41 and PE88). Transferability was higher for the species from the Passiflora subgenus than for those from the Decaloba and Dysosmia subgenus. The results indicated a high level of nucleotide sequence conservation of the primer regions in the species evaluated, and consequently, they could potentially be used for the establishment of molecular strategies for use in passion fruit breeding and genetics.