Advanced Strategies in Bulked Segregant Analysis and its Applications

Bulked segregant analysis (BSA) is a technique used to identify genetic markers associated with a mutant phenotype and is a quick method for identifying markers in particular genome regions. The paper focussed on Advanced methods which escape the requirement of genotyping all the individuals of the mapping population and generation of high-density linkage maps for mapping of the gene for the trait of interest. With the emergence of re-sequencing techniques, quick mapping of genes has become possible with reduced time and cost by using advanced methodologies like MutMap, MutMap+, MutMap-Gap, QTL-Seq, RNAseq BSA, NGS BSA and QTG seq. The procedure for various advanced BSA strategies has been described.

Riboswitch-mediated inducible expression of an astaxanthin biosynthetic operon in plastids

The high-value carotenoid astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione) is one of the most potent antioxidants in nature. In addition to its large-scale use in fish farming, the pigment has applications as a food supplement and an active ingredient in cosmetics and in pharmaceuticals for the treatment of diseases linked to reactive oxygen species. The biochemical pathway for astaxanthin synthesis has been introduced into seed plants, which do not naturally synthesize this pigment, by nuclear and plastid engineering. The highest accumulation rates have been achieved in transplastomic plants, but massive production of astaxanthin has resulted in severe growth retardation. What limits astaxanthin accumulation levels and what causes the mutant phenotype is unknown. Here we addressed these questions by making astaxanthin synthesis in tobacco (Nicotiana tabacum) plastids inducible by a synthetic riboswitch. We show that, already in the uninduced state, astaxanthin accumulates to similarly high levels as in transplastomic plants expressing the pathway constitutively. Importantly, the inducible plants displayed wild-type–like growth properties and riboswitch induction resulted in a further increase in astaxanthin accumulation. Our data suggest that the mutant phenotype associated with constitutive astaxanthin synthesis is due to massive metabolite turnover, and indicate that astaxanthin accumulation is limited by the sequestration capacity of the plastid.

Folding of cohesin’s coiled coil is important for Scc2/4-induced association with chromosomes

Cohesin’s association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being ‘clamped’ by Scc2 and ATP-dependent engagement of cohesin’s Smc1 and Smc3 head domains. Scc2’s replacement by Pds5 abrogates cohesin’s ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1’s ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin’s coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1’s hinge that alters how Scc2 and Pds5 interact with Smc1’s hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin’s hinge, which in turn requires coiled coil folding.

A Single Nucleotide Substitution of GSAM Gene Causes Massive Accumulation of Glutamate 1-Semialdehyde and Yellow Leaf Phenotype in Rice

Background Tetrapyrroles play indispensable roles in various biological processes. In higher plants, glutamate 1-semialdehyde 2,1-aminomutase (GSAM) converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA), which is the rate-limiting step of tetrapyrrole biosynthesis. Up to now, GSAM genes have been successively identified from many species. Besides, it was found that GSAM could form a dimeric protein with itself by x-ray crystallography. However, no mutant of GSAM has been identified in monocotyledonous plants, and no experiment on interaction of GSAM protein with itself has been reported so far. Result We isolated a yellow leaf mutant, ys53, in rice (Oryza sativa). The mutant showed decreased photosynthetic pigment contents, suppressed chloroplast development, and reduced photosynthetic capacity. In consequence, its major agronomic traits were significantly affected. Map-based cloning revealed that the candidate gene was LOC_Os08g41990 encoding GSAM protein. In ys53 mutant, a single nucleotide substitution in this gene caused an amino acid change in the encoded protein, so its ALA-synthesis ability was significantly reduced and GSA was massively accumulated. Complementation assays suggested the mutant phenotype of ys53 could be rescued by introducing wild-type OsGSAM gene, confirming that the point mutation in OsGSAM is the cause of the mutant phenotype. OsGSAM is mainly expressed in green tissues, and its encoded protein is localized to chloroplast. qRT-PCR analysis indicated that the mutation of OsGSAM not only affected the expressions of tetrapyrrole biosynthetic genes, but also influenced those of photosynthetic genes in rice. In addition, the yeast two-hybrid experiment showed that OsGSAM protein could interact with itself, which could largely depend on the two specific regions containing the 81th–160th and the 321th–400th amino acid residues at its N- and C-terminals, respectively. Conclusions We successfully characterized rice GSAM gene by a yellow leaf mutant and map-based cloning approach. Meanwhile, we verified that OsGSAM protein could interact with itself mainly by means of the two specific regions of amino acid residues at its N- and C-terminals, respectively.

Reflection of P53 Phenotype in Tumor Tissue by Genotypic Variants in Glutathione S-Transferases: An Association with DNA Damage in Lung Adenocarcinoma

Background: Genetic deficiency of glutathione S-transferase (GST) enzymes results in accumulating carcinogens from tobacco smoke. Consequently, the elevated risk of lung carcinoma (LC) is due to increased DNA damage and mutational frequency of the P53 caretaker gene.Objective: To determine GST gene variants association with LUAD susceptibility and the relative risk of these variations developing the P53 mutation and DNA damageMethods: We analyzed genetic polymorphisms in GST-M1 (+/-), T1 (+/-), and P1 (Ile 105 Val) genes using restriction fragment length PCR. The P53 phenotype was categorized as the mutant type (>50% and 2-3 + intensity in IHC) in M1 (+/-), T1 (+/-), and P1 (Ile105Val) variants. Lymphocytic DNA damage was assessed using the comet assay. Results: The M1 (-/-) and P1 Val/Val (GG) genotypes were significantly associated with the risk of the P53 mutant phenotype (RR: 1.81, p=0.03) and (RR: 2.23, p=0.05), respectively. In contrast, GSTT1 was not associated with the P53 phenotype. GSTM1 (-/-)/ GSTP1 Ile/Ile (AA) combined genotypes showed a significant synergistic effect on the P53 phenotype (RR: 8.50, p=0.01). DNA damage was significantly associated with GST-M1 (-/-), T1 (-/-), and P1 Val/Val (GG) genotypes and smoking (p=0.001) as compared to GST-T1 (+/+)/M1 (+/+)/P1 Ile/Ile (AA) genotypes. However, P53 expression was not associated with DNA damage. Conclusion: The GSTM1 (-/-) and GSTT1 (-/-) null genotypes act as risk modifiers for LUAD and increase DNA damage. The GSTM1 (-/-)/ GSTP1 Ile/Ile (AA) combined genotype amplified 8-fold the risk of a P53 mutant phenotype.

A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host-range mutants within infant rabbits infected with a mixture of wild type and OmpU mutant strains. ICP2 host-range mutants, that can now infect OmpU mutant strains, had missense mutations in putative tail fiber gene gp25 and putative adhesin gp23. Using site-specific mutagenesis we show that single or double mutations in gp25 are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild type V. cholerae cells. The strength of binding of host-range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first, gp25 mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication. Importance Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to a renewed interest in phage biology and their potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular co-evolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host-range mutants within infant rabbits infected with a mixture of wild type and OmpU mutant strains. ICP2 host-range mutants had missense mutations in putative tail fiber gene gp25 and putative adhesin gp23. Using site-specific mutagenesis we show that single or double mutations in gp25 are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild type V. cholerae cells. The strength of binding of host-range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first, gp25 mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication.ImportanceConcern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to a renewed interest in phage biology and their potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies a ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular co-evolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine

The initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc+ in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.

Genetic mapping and transcriptomic characterization of a new fuzzless-tufted cottonseed mutant

Fiber mutants are unique and valuable resources for understanding the genetic and molecular mechanisms controlling initiation and development of cotton fibers that are extremely elongated single epidermal cells protruding from the seed coat of cottonseeds. In this study, we reported a new fuzzless-tufted cotton mutant (Gossypium hirsutum) and showed that fuzzless-tufted near-isogenic lines (NILs) had similar agronomic traits and a higher ginning efficiency compared to their recurrent parents with normal fuzzy seeds. Genetic analysis revealed that the mutant phenotype is determined by a single incomplete dominant locus, designated N5. The mutation was fine mapped to an approximately 250-kb interval containing 33 annotated genes using a combination of bulked segregant sequencing, SNP chip genotyping, and fine mapping. Comparative transcriptomic analysis using 0–6 days post-anthesis (dpa) ovules from NILs segregating for the phenotypes of fuzzless-tufted (mutant) and normal fuzzy cottonseeds (wild-type) uncovered candidate genes responsible for the mutant phenotype. It also revealed that the flanking region of the N5 locus is enriched with differentially expressed genes (DEGs) between the mutant and wild-type. Several of those DEGs are members of the gene families with demonstrated roles in cell initiation and elongation, such as calcium-dependent protein kinase and expansin. The transcriptome landscape of the mutant was significantly reprogrammed in the 6 dpa ovules and, to a less extent, in the 0 dpa ovules, but not in the 2 and 4 dpa ovules. At both 0 and 6 dpa, the reprogrammed mutant transcriptome was mainly associated with cell wall modifications and transmembrane transportation, while transcription factor activity was significantly altered in the 6 dpa mutant ovules. These results imply a similar molecular basis for initiation of lint and fuzz fibers despite certain differences.