Shoot zinc (Zn) concentration varies widely within Brassica oleracea L. and is affected by soil Zn and phosphorus (P) levels

Summary

The low availability of zinc (Zn) in soils and crops affects dietary Zn intake worldwide. This study sought to determine if the natural genetic variation in shoot Zn concentrations ([Zn]shoot) is sufficient to pursue a crop improvement breeding strategy in a leafy vegetable crop. The gene-pool of Brassica oleracea L. was sampled using a large (n = 376) diversity foundation set (DFS), representing almost all species-wide common allelic variation, and 74 commercial varieties (mostly F₁). The DFS genotypes were grown at low and high soil phosphorus (P) levels under glasshouse and field conditions, and also in a Zn-deficient soil, with or without Zn-fertilisation, in a glasshouse. Despite the large variation in [Zn]shoot among genotypes, environment had a profound effect on [Zn]shoot. The heritability of [Zn]shoot was significant, but relatively low, among 90 doubled-haploid (DH) lines from a mapping population. While several quantitative trait loci (QTL) associated with [Zn]shoot occurred on chromosomes C2, C3, C5, C7, and C9, these were generally weak and conditional upon growth conditions. Breeding for [Zn]shoot in B. oleracea is therefore likely to be challenging. Shoot P concentrations increased substantially in all genotypes under low soil Zn conditions. Conversely, only some genotypes had increased [Zn]shoot at low soil P levels. Sufficient natural genetic variation may therefore exist to study some of the interactions between Zn and P nutrition.     

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

Background

Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes.

Results

We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner? system we were able to detect mutations in heterozygous and homozygous states for both genes.

Conclusions

Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M₃ seed of Brassica rapa from the RevGenUK TILLING service.