Genetic diversity, QTL mapping for essential yield components and aluminum tolerance in hexaploid wheat (Triticum aestivum L.) by Next-Generation Sequencing

Abstract

Next Generation Sequencing (NGS) has revolutionized research in plants and animals by developing new high throughput genotyping methods to greatly accelerate the study of genome composition and function. Bread wheat is one of the world's three major cereal crops, with a complex genome whose study requires the use of NGS tools. In addition, It’s has been reported that wheat production in Cameroon faces several constraints such as low grain yields and acidic soils with aluminum toxicity. This thesis project was investigated to characterize the genetic diversity and select high-yielding and tolerant wheat varieties to aluminum toxicity. By evaluating the genetic diversity in the 17 wheat cultivars collected in Cameroon, 11 microsatellite markers revealed 77 alleles allowing to discriminate these cultivars. The number of alleles per locus ranged from 2 to 13 with an average of 7. The results obtained in our study provided new information on the genetic relationships in Cameroonian wheat cultivars. The set of microsatellite markers used showed a high level of polymorphism and necessaries information to discriminate these cultivars of hexaploid wheat. Thus, the Cameroonian wheat varieties have been grouped into 5 main groups according to their geographical origin, showing a level of genetic diversity moderately high but insufficient to cope with the biotic and abiotic constraints. In order to increase the level of diversity in Cameroonian cultivars, several wheat varieties have been collected from the major world producers and characterized using the Genotyping-by-Sequencing (GBS) protocol, which is an approach based on the sequencing of short DNA segments (~100-150pb) scattered throughout the genome. The resulting DNA sequences (~ 2.4 million per line) were analyzed using the FastGBS pipeline. This allowed to identify 96,667 SNP markers distributed across all 21 wheat chromosomes. The analysis of genetic diversity revealed the presence of six distinct groups in this international collection, with strong genetic structuring. Very high degrees of molecular diversity have been observed among the populations studied, with a low level of material exchanges between Cameroon and other countries. After the introduction of exotic cultivars, the level of wheat genetic diversity in Cameroon was improved. Using a subset of 71 lines for which chip data (90K SNP Wheat Infinium) were available, we found a high level of agreement (> 95%) between the two genotyping technologies. In order to discover the genomic regions controlling seed size variation, a Genome-Wide Association Study (GWAS) was conducted using the GWAS-GBS approach. A total of 7 SNPs was associated with both traits (grain length and width), identifying 2 quantitative trait loci (QTLs) located on the chromosomes 1D and 2D. In the vicinity of the SNP peak on the chromosome 2D, we found a promising candidate gene (D11), whose homolog had previously been involved in the regulation of grain size in rice. The D11 gene has been reported to encode a novel cytochrome P450 with homology to the enzymes involved in the biosynthesis of brassinosteroids. In addition, 102 high yielding wheat varieties were selected. In order to select varieties for aluminum tolerance, a total of 45 bread wheat lines were phenotyped in a greenhouse experiment and genotyped by GBS. Citrate efflux of excised root apices was measured on individual seedlings using as few as four apices and its concentration was estimated with coupled enzyme assays that detect the production or consumption of NADH. The root growth rate of all genotypes was reduced with the addition of Al to the pots and Al-susceptible and Al-tolerant wheat genotypes were clearly identified. Correlations between root length and plant height, fresh biomass of shoots / roots, shoot / root dry biomass, released citrate, or tolerance indices in non-acid soil versus acid soil conditions were very significant (P <0.01). Thus, 17 wheat varieties were selected for acidic soil tolerance under aluminum toxicity conditions; Finally, we were able to identify seven (7) high yielding wheat varieties, which are also tolerant to aluminum toxicity (having both MATE1B and D11 functional genes). The present project has made it possible to highlight the level of genetic diversity available in Cameroonian wheat cultivars and in an international collection of wheat lines, selecting high-yielding wheat varieties tolerant to aluminum toxicity, which could be useful to boost wheat productivity in breeding programs