The detection and characterization of sex chromosome sequences is particularly important for major pest families,like the Tephritidae,whereas alternative pest management approaches,mainly involving male-only release p...The detection and characterization of sex chromosome sequences is particularly important for major pest families,like the Tephritidae,whereas alternative pest management approaches,mainly involving male-only release programs,rely on the ability to target and manipulate sex-specific genomic regions,particularly those of the Y chromosome.However,resolving and detecting X and Y chromosome sequences at the chromosome level requires careful consideration of algorithmic outputs,especially in species where extensive sex chromosome markers are not available.Here,we present R-CQ and KAMY,two computational methods developed for the detection of sex chromosomelinked sequences through sex-specific short-read DNA sequencing datasets.We evaluate their performance on newly generated chromosome-level assemblies of four important Tephritid pest species:Ceratitis capitata,Bactrocera dorsalis,Bactrocera zonata,and Anastrepha ludens.By combining algorithmic predictions with a manual curation process,we assess the strengths and limitations of each method and provide a robust dataset of curated X-and Y-linked sequences.Overall,our results establish a framework for studying poorly characterized sex chromosome lineages and identifying sex-specific genomic regions,supporting the broader development of sex chromosome-based pest managements systems.展开更多
The Bactrocera genus includes highly invasive fruit and vegetable pest species such as Bactrocera dorsalis,Bactrocera correcta,and Bactrocera oleae.The sterile insect technique(SIT)is a biological control method used ...The Bactrocera genus includes highly invasive fruit and vegetable pest species such as Bactrocera dorsalis,Bactrocera correcta,and Bactrocera oleae.The sterile insect technique(SIT)is a biological control method used to suppress populations of the invasive Bactrocera fruit flies by releasing sterilized male insects that compete with wild males for mates,reducing reproduction and eventually pest numbers.The effectiveness of the SIT against insect pests can be enhanced through male-only releases,achieved via genetic sexing strains(GSS)that enable early-stage sex separation.To overcome limitations faced when developing a new GSS through the classical genetic approach,a novel“neoclassical approach”has been proposed,focusing on the identification of genetic markers,the induction of desired phenotypes through genome editing,and the linkage of selectable markers to male sex.In this study,we evaluated the white pupae gene as a selectable marker for GSS development in 3 Bactrocera species.The white pupae orthologous genes have been identified,and,through CRISPR/Cas9 mutagenesis,the 3rd exon of the white pupae gene was knocked out resulting in white pupae lines in Bactrocera dorsalis,Bactrocera correcta,and Bactrocera oleae species.These results demonstrate the applicability of CRISPR/Cas9-mediated disruption of the conserved white pupae gene as a selectable marker in multiple Bactrocera species,supporting the development of genetic sexing systems for SIT-based pest management.展开更多
Bactrocera zonata is a highly invasive agricultural pest that causes extensive damage to fruit crops.The Sterile Insect Technique(SIT),a species-specific and environmentally friendly pest control method,significantly ...Bactrocera zonata is a highly invasive agricultural pest that causes extensive damage to fruit crops.The Sterile Insect Technique(SIT),a species-specific and environmentally friendly pest control method,significantly benefits from the availability of Genetic Sexing Strains(GSSs)that enable efficient mass production of males for sterile release.However,no GSS currently exists for B.zonata limiting SIT applications targeting this important invasive pest.Here,we report two key advancements toward GSS development in this species.First,we present a high-quality,chromosome-level genome assembly from male B.zonata,identifying two scaffolds derived from the Y chromosome,which represent potential targets for future male-specific genetic engineering.Second,we demonstrate the feasibility of CRISPR/Cas9 genome editing in B.zonata by generating stable,homozygous white-eye mutants through targeted disruption of the conserved white-eye gene.This visible,recessive phenotype serves as a proof-of-concept for developing selectable markers in this species.Together,these results provide foundational genomic and genetic tools to support the development of GSSs in B.zonata,advancing the potential for sustainable,genetics-based pest control strategies.展开更多
基金supported by the International Atomic Energy Agency research contract no.23378 as part of the Coordinated Research Project“Generic approach for the development of genetic sexing strains for SIT applications.”This research was supported by the European Union’s Horizon Europe Research and Innovation Programme REACT(Grant agreement 101059523)the three postgraduate programs of the Department of Biochemistry and Biotechnology of the University of Thessaly(“Advanced Experimental&Computational Biosciences,”“Biotechnology-Quality Assessment in Nutrition and the Environment”and“Applications of Molecular Biology-Genetics-Diagnostic Biomarkers”).
文摘The detection and characterization of sex chromosome sequences is particularly important for major pest families,like the Tephritidae,whereas alternative pest management approaches,mainly involving male-only release programs,rely on the ability to target and manipulate sex-specific genomic regions,particularly those of the Y chromosome.However,resolving and detecting X and Y chromosome sequences at the chromosome level requires careful consideration of algorithmic outputs,especially in species where extensive sex chromosome markers are not available.Here,we present R-CQ and KAMY,two computational methods developed for the detection of sex chromosomelinked sequences through sex-specific short-read DNA sequencing datasets.We evaluate their performance on newly generated chromosome-level assemblies of four important Tephritid pest species:Ceratitis capitata,Bactrocera dorsalis,Bactrocera zonata,and Anastrepha ludens.By combining algorithmic predictions with a manual curation process,we assess the strengths and limitations of each method and provide a robust dataset of curated X-and Y-linked sequences.Overall,our results establish a framework for studying poorly characterized sex chromosome lineages and identifying sex-specific genomic regions,supporting the broader development of sex chromosome-based pest managements systems.
基金supported by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture.Additional funding was provided by the European Union’s Horizon Europe Research and Innovation Program REACT(grant agreement number 101059523 to MFS and KDM)Further support was provided by the two postgraduate programs of the Department of Biochemistry and Biotechnology of the University of Thessaly(“Biotechnology-Quality Assessment in Nutrition and the Environment”and“Applications of Molecular Biology-Genetics-Diagnostic Biomarkers”)+2 种基金also by the project“Synthetic Biology:from omics technologies to genomic engineering(OMIC-ENGINE)”(MIS 5002636)which is implemented under the Action“Reinforcement of the Research and Innovation Infrastructure,”funded by the Operational Programme“Competitiveness,Entrepreneurship and Innovation”(NSRF 2014-2020)co-financed by Greece and the European Union(European Regional Development Fund)(to M-EG,and KM)Publication costs for this study were provided by the International Atomic Energy Agency as part of the Coordinated Research Project“Generic approach for the development of genetic sexing strains for SIT applications”。
文摘The Bactrocera genus includes highly invasive fruit and vegetable pest species such as Bactrocera dorsalis,Bactrocera correcta,and Bactrocera oleae.The sterile insect technique(SIT)is a biological control method used to suppress populations of the invasive Bactrocera fruit flies by releasing sterilized male insects that compete with wild males for mates,reducing reproduction and eventually pest numbers.The effectiveness of the SIT against insect pests can be enhanced through male-only releases,achieved via genetic sexing strains(GSS)that enable early-stage sex separation.To overcome limitations faced when developing a new GSS through the classical genetic approach,a novel“neoclassical approach”has been proposed,focusing on the identification of genetic markers,the induction of desired phenotypes through genome editing,and the linkage of selectable markers to male sex.In this study,we evaluated the white pupae gene as a selectable marker for GSS development in 3 Bactrocera species.The white pupae orthologous genes have been identified,and,through CRISPR/Cas9 mutagenesis,the 3rd exon of the white pupae gene was knocked out resulting in white pupae lines in Bactrocera dorsalis,Bactrocera correcta,and Bactrocera oleae species.These results demonstrate the applicability of CRISPR/Cas9-mediated disruption of the conserved white pupae gene as a selectable marker in multiple Bactrocera species,supporting the development of genetic sexing systems for SIT-based pest management.
基金Funding was provided by the European Union’s Horizon Europe Research and Innovation Program(REACTgrant agreement number 101059523 to PAP,GP and KM)the US-Israel Binational Agricultural Research and Development Fund(BARD-grant agreement number IS-5590-23 to PAP,GP,AH)+3 种基金the Hebrew University of Jerusalem and Zelman Cowen Academic Initiatives(ZCAI Joint Project 2021 number 0456 to PAP and SWB)Initial support was generously provided in the form of an International Fellowship to FK from the Research Fund for International Cooperation,Robert H.Smith Faculty of Agriculture,Food and Environment,HUJI and HUJI startup funds to PAP.Genome sequencing was financially supported by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture,the German Research Foundation through the Middle East Cooperation project 491548882(PAP)the Canada Foundation for Innovation grants 40104 and 35444(to Ioannis Ragousis)Publication cost for this study was provided by the International Atomic Energy Agency as part of the Coordinated Research Project“Generic approach for the development of genetic sexing strains for SIT applications”。
文摘Bactrocera zonata is a highly invasive agricultural pest that causes extensive damage to fruit crops.The Sterile Insect Technique(SIT),a species-specific and environmentally friendly pest control method,significantly benefits from the availability of Genetic Sexing Strains(GSSs)that enable efficient mass production of males for sterile release.However,no GSS currently exists for B.zonata limiting SIT applications targeting this important invasive pest.Here,we report two key advancements toward GSS development in this species.First,we present a high-quality,chromosome-level genome assembly from male B.zonata,identifying two scaffolds derived from the Y chromosome,which represent potential targets for future male-specific genetic engineering.Second,we demonstrate the feasibility of CRISPR/Cas9 genome editing in B.zonata by generating stable,homozygous white-eye mutants through targeted disruption of the conserved white-eye gene.This visible,recessive phenotype serves as a proof-of-concept for developing selectable markers in this species.Together,these results provide foundational genomic and genetic tools to support the development of GSSs in B.zonata,advancing the potential for sustainable,genetics-based pest control strategies.
