Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice

Background: Classical genetic crosses in malaria parasites involve isolation, genotyping, and phenotyping of multiple progeny parasites, which is time consuming and laborious. Bulk segregant analysis (BSA) offers a powerful and efficient alternative to identify loci underlying complex traits in the human malaria parasite, Plasmodium falciparum. Methods: We have used BSA, which combines genetic crosses using humanized mice with pooled sequencing of progeny populations to measure changes in allele frequency following selection with antimalarial drugs. We used dihydroartemisinin (DHA) drug selection in two genetic crosses (Mal31xKH004 and NF54xNHP1337). We specifically investigated how synchronization, cryopreservation, and the drug selection regimen of progeny pools impacted the success of BSA experiments. Findings: We detected a strong and repeatable quantitative trait locus (QTL) at chr13 kelch13 locus in both crosses, but did not detect QTLs at ferredoxin (fd), the apicoplast ribosomal protein S10 (arps10), multidrug resistance protein 2 (mdr2). QTLs were detected using synchronized, but not unsynchronized pools, consistent with the stage-specific action of DHA. We also successfully applied BSA to cryopreserved progeny pools. Interpretation: Our results provide proof-of-principal of the utility of BSA for rapid, robust genetic mapping of drug resistance loci. Use of cryopreserved progeny pools expands the utility of BSA because we can conduct experiments using archived progeny pools from previous genetic crosses. BSA provides a powerful approach that complements traditional QTL methods for investigating the genetic architecture of resistance to antimalarials, and to reveal new or accessory loci contributing to artemisinin resistance.

Genetic control of rhizosheath formation in pearl millet

The rhizosheath, the layer of soil that adheres strongly to roots, influences water and nutrients acquisition. Pearl millet is a cereal crop that plays a major role for food security in arid regions of sub Saharan Africa and India. We previously showed that root-adhering soil mass is a heritable trait in pearl millet and that it correlates with changes in rhizosphere microbiota structure and functions. Here, we studied the correlation between root-adhering soil mass and root hair development, root architecture, and symbiosis with arbuscular mycorrhizal fungi and we analysed the genetic control of this trait using genome wide association (GWAS) combined with bulk segregant analysis and gene expression studies. Root-adhering soil mass was weakly correlated only to root hairs traits in pearl millet. Twelve QTLs for rhizosheath formation were identified by GWAS and bulk segregant analysis on a biparental population further validated five of these QTLs. Combining genetics with a comparison of global gene expression in the root tip of contrasted inbred lines revealed candidate genes that might control rhizosheath formation in pearl millet. Our study indicates that rhizosheath formation is under complex genetic control in pearl millet and suggests that it is mainly regulated by root exudation.

Refining Bulk Segregant Analyses: Ontology-Mediated Discovery of Flowering Time Genes in Brassica oleracea

Bulk segregant analysis (BSA) can help identify quantitative trait loci (QTLs), but this may result in substantial bycatch of functionally irrelevant genes. Here we develop a Gene Ontology-mediated approach to zoom in on specific markers implicated in flowering time from among QTLs identified by BSA of the giant woody Jersey kale phenotyped in four bulks of flowering onset. Our BSA yielded tens of thousands of candidate genes. We reduced this by two orders of magnitude by focusing on genes annotated with terms contained within relevant subgraphs of the Gene Ontology. A further enrichment test led to the pathway for circadian rhythm in plants. The genes that enriched this pathway are attested from previous research as regulating flowering time. Some of these genes were also identified as having functionally significant variation compared to Arabidopsis. We validated and confirmed our ontology-mediated results through a more targeted, homology-based approach. However, our ontology-mediated approach produced additional genes of putative importance, showing that the approach aids in exploration and discovery. We view our method as potentially applicable to the study of other complex traits and therefore make our workflows available as open-source code and a reusable Docker container.

Combining conventional QTL analysis and whole-exome capture-based bulk-segregant analysis provides new genetic insights into tuber sprout elongation and dormancy release in a diploid potato population

Tuber dormancy and sprouting are commercially important potato traits as long-term tuber storage is necessary to ensure year-round availability. Premature dormancy release and sprout growth in tubers during storage can result in a significant deterioration in product quality. In addition, the main chemical sprout suppressant chlorpropham has been withdrawn in Europe, necessitating alternative approaches for controlling sprouting. Breeding potato cultivars with longer dormancy and slower sprout growth is a desirable goal, although this must be tempered by the needs of the seed potato industry, where dormancy break and sprout vigour are required for rapid emergence. We have performed a detailed genetic analysis of tuber sprout growth using a diploid potato population derived from two highly heterozygous parents. A dual approach employing conventional QTL analysis allied to a combined bulk-segregant analysis (BSA) using a novel potato whole-exome capture (WEC) platform was evaluated. Tubers were assessed for sprout growth in storage at six time-points over two consecutive growing seasons. Genetic analysis revealed the presence of main QTL on five chromosomes, several of which were consistent across two growing seasons. In addition, phenotypic bulks displaying extreme sprout growth phenotypes were subjected to WEC sequencing for performing BSA. The combined BSA and WEC approach corroborated QTL locations and served to narrow the associated genomic regions, while also identifying new QTL for further investigation. Overall, our findings reveal a very complex genetic architecture for tuber sprouting and sprout growth, which has implications both for potato and other root, bulb and tuber crops where long-term storage is essential.

Predicting the Genomic Resolution of Bulk Segregant Analysis

Bulk segregant analysis (BSA) is a technique for identifying the genetic loci that underlie phenotypic trait differences. The basic approach of this method is to compare two pools of individuals from the opposing tails of the phenotypic distribution, sampled from an interbred population. Each pool is sequenced and scanned for alleles that show divergent frequencies between the pools, indicating potential association with the observed trait differences. BSA has already been successfully applied to the mapping of various quantitative trait loci in organisms ranging from yeast to maize. However, these studies have typically suffered from rather low mapping resolution, and we still lack a detailed understanding of how this resolution is affected by experimental parameters. Here, we use coalescence theory to calculate the expected genomic resolution of BSA. We first show that in an idealized interbreeding population of infinite size, the expected length of the mapped region is inversely proportional to the recombination rate, the number of generations of interbreeding, and the number of genomes sampled, as intuitively expected. In a finite population, coalescence events in the genealogy of the sample reduce the number of potentially informative recombination events during interbreeding, thereby increasing the length of the mapped region. This is incorporated into our theory by an effective population size parameter that specifies the pairwise coalescence rate of the interbreeding population. The mapping resolution predicted by our theory closely matches numerical simulations. Furthermore, we show that the approach can easily be extended to modifications of the crossing scheme. Our framework enables researchers to predict the expected power of their mapping experiments, and to evaluate how their experimental design could be tuned to optimize mapping resolution.

Molecular tagging of Rf genes for the fertility restoration of WA-CMS system by bulk segregant analysis in rice

The process of screening for fertility restoration trait involves test crossing with a set of cytoplasmic male sterile (CMS) lines and evaluation of F1 hybrids for pollen and spikelet fertility. In the present study, F2 mapping population derived from a cross, APMS 6A × RP 5933-123 was utilized to map Rf genes. The F2 population was also genetically analysed for pollen and spikelet fertility percentage. Chisquare (?2) analysis to showed that the fertility restoration trait followed expected digenic ratio. By bulk segregant analysis (BSA) likely Rf genes containing regions were located on chromosome 10. The SSR markers viz., RM304, RM258 located on chromosome 10 and RM23958 located on chromosome 9 showed clear polymorphism between two groups of fertile and sterile bulks. Based on BSA linkage analysis and F2 population, pollen and spikelet fertility analysis along with molecular screening results of Rf linked markers, it is concluded that Rf4 gene located on chromosome 10 is playing major role and contributing to 90% of fertility restoration trait of newly derived restorer line RP5933 along with minor effect genes from chromosome 9. The findings may be useful for rice hybrid breeding.