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[学术文献 ] Historical data provide new insights into inheritance of traits important for diploid potato breeding 进入全文
SPRINGER
Key messageUsing a diploid potato diversity panel of 246 breeding lines, a genotyping-by-sequencing and a GWAS approach, we mapped QTL for ten traits important to potato breeders, including two previously unmapped traits: boiled tuber taste and pollen fertility.AbstractPotato breeding at the diploid level has a long history and has gained new impetus recently, when F1 hybrid breeding was made possible with the discovery of a dominant gene for self-compatibility. Our study deploys a unique diploid diversity panel with a broadened cultivated potato gene pool obtained as a result of introgressing valuable traits from wild potato relatives into the Solanum tuberosum background. Using historical phenotyping data collected between 1979 and 2017 for 246 diploid potato clones and high-density genotyping-by-sequencing, we mapped quantitative trait loci (QTL) for tuber yield, mean tuber weight, tuber shape and regularity, tuber eye depth, purple tuber skin colour, flesh colour, tuber starch content, boiled tuber taste (flavour) and pollen fertility. We found some QTL located in genomic regions described in earlier studies, e.g. the QTL for the tuber flesh colour on chromosome 3 overlapping with the location of beta-carotene hydroxylase gene. We identified novel QTL for mean tuber weight on chromosomes 8, 9 and 11 and for purple tuber skin colour on chromosomes 6, 7 and 8. QTL for boiled tuber taste and pollen fertility estimated by Lactofuchsin staining have not been mapped before. We found two regions on chromosome 10 affecting the boiled tuber taste, and QTL on chromosomes 2, 4, 5, 6, 9, and 12 for pollen fertility. Considering the increased interest in diploid hybrid potato breeding, the results presented here hold greater relevance and provide novel targets for potato breeding and research at the diploid level.
[学术文献 ] Chromosomal domain formation by archaeal SMC, a roadblock protein, and DNA structure 进入全文
Nature Communications
In eukaryotes, structural maintenance of chromosomes (SMC) complexes form topologically associating domains (TADs) by extruding DNA loops and being stalled by roadblock proteins. It remains unclear whether a similar mechanism of domain formation exists in prokaryotes. Using high-resolution chromosome conformation capture sequencing, we show that an archaeal homolog of the bacterial Smc-ScpAB complex organizes the genome of Thermococcus kodakarensis into TAD-like domains. We find that TrmBL2, a nucleoid-associated protein that forms a stiff nucleoprotein filament, stalls the T. kodakarensis SMC complex and establishes a boundary at the site-specific recombination site dif. TrmBL2 stalls the SMC complex at tens of additional non-boundary loci with lower efficiency. Intriguingly, the stalling efficiency is correlated with structural properties of underlying DNA sequences. Our study illuminates a eukaryotic-like mechanism of domain formation in archaea and a role of intrinsic DNA structure in large-scale genome organization.
[学术文献 ] biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at the ACTB locus 进入全文
Synthetic Biology
Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multiscale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution and the visualization of macromolecular complexes in situ, especially if these molecules are expressed with appropriately engineered and easily detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our system comprises a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion. As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express six fluorescently tagged proteins and simultaneously visualize five different subcellular compartments. Our protocol leads from the in silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.
[学术文献 ] Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome 进入全文
nature communications
The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5’ UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.
[学术文献 ] The Silencing of the StPAM16-1 Gene Enhanced the Resistance of Potato Plants to the Phytotoxin Thaxtomin A 进入全文
MDPI
Potato common scab (CS) caused by Streptomyces scabiei is a severe disease that threatens tuber quality and its market value. To date, little is known about the mechanism regulating the resistance of potato to CS. In this study, we identified a presequence translocase-associated motor 16 gene from potato (designated StPAM16-1) that is involved in the response to the phytotoxin thaxtomin A (TA) secreted by S. scabiei. The StPAM16-1 protein was localized in the mitochondria, and the expression of the gene was upregulated in potato leaves treated with TA. The suppression of StPAM16-1 in potato led to enhanced resistance to TA and S. scabiei. Protein interaction analyses revealed that StPAM16-1 interacted with the subunit 5b of the COP9 signalosome complex (StCSN5). Similar to that of StPAM16-1, the expression levels of StCSN5 significantly increased in potato leaves treated with TA. These results indicated that StPAM16-1 acted as a negative regulator and was functionally associated with StCSN5 in the immune response of potato plants against CS. Our study sheds light on the molecular mechanism by which PAM16 participates in the plant immune response. Furthermore, both StPAM16-1 and StCSN5 could be potential target genes in the molecular breeding of potato cultivars with increased resistance to CS.
[学术文献 ] Computational design of serine hydrolases 进入全文
science
The design of enzymes with complex active sites that mediate multistep reactions remains an outstanding challenge. With serine hydrolases as a model system, we combined the generative capabilities of RFdiffusion with an ensemble generation method for assessing active site preorganization to design enzymes starting from minimal active site descriptions. Experimental characterization revealed catalytic efficiencies (kcat/Km) up to 2.2x105 M−1 s−1 and crystal structures that closely match the design models (Cα RMSDs < 1 Å). Selection for structural compatibility across the reaction coordinate enabled identification of new catalysts in low-throughput screens with five different folds distinct from those of natural serine hydrolases. Our de novo approach provides insight into the geometric basis of catalysis and a roadmap for designing enzymes that catalyze multistep transformations.
