Gene therapy for spinal muscular atrophy:perspectives on the possibility of optimizing SMN1 delivery to correct all neurological and systemic perturbations

Spinal muscular atrophy(SMA)is a genetic condition that results in selective lower motor neuron loss with concomitant muscle weakness and atrophy.The genetic cause of SMA was understood in 1995 when loss or impairment of the survival motor neuron 1(SMN1)gene was identified as the main contributing factor(Lefebvre et al.,1995).This,in combination with the discovery that humans have a“back-up”gene,SMN2,which can produce low levels(approximately 10%)of the full-length functional SMN protein,has led to the generation of SMA-specific gene therapies.SMA was traditionally classified according to age of symptom onset and developmental milestones achieved,with life expectancy and severity varying between individuals.Now,SMN2 copy number is used as a proxy for the prediction of disease severity,with higher SMN2 copy number typically being associated with reduced severity of SMA,although this relationship is not absolute:some individuals with low SMN2 copy number have less severe SMA phenotypes and vice versa.Additionally,the etiology of SMA is further complicated by other factors,such as non-typical nucleotide variants and SMN2-independent modifiers of disease severity.

Development and molecular cytogenetic identification of a new wheat-rye 6RL ditelosomic addition and 1R(1B)substitution line with powderymildew resistance

Powdery mildew is a serious disease caused by Blumeria graminis f.sp.tritici(Bgt)that critically threatens the yield and quality of wheat(Triticum aestivum L.).Using effective powdery mildew resistance genes is the optimal method for controlling this disease.Against the background of high genetic homogeneity among the modern commercial cultivars that are mainly derived from conventional interbreeding,the resistance genes from wheat relatives have especially prominent advantages.Octoploid triticale,produced from common wheat and rye(Secale cereale L.)through distant hybridization,is a new synthetic species and valuable gene donor for wheat improvement.In this study,we developed the wheat-rye line YT5 through the hybridization of octaploid triticale and two wheat lines.YT5 was confirmed to be a 6RL ditelosomic addition and 1R(1B)substitution line using genomic in situ hybridization(GISH),multicolor fluorescence in situ hybridization(mc-FISH),multicolor GISH(mc-GISH)and molecular marker analysis.Genetic analysis showed that the powdery mildew resistance in YT5 was derived from the rye chromosome arm 6RL.After inoculation with different Bgt isolates at the seedling stage,YT5 had compound reaction patterns with both obvious spores and hypersensitivity,and it gradually became highly resistant until the adult-plant stage,thus showing a resistance response significantly different from the reported Pm genes from rye chromosome 6RL.YT5 also showed promising agronomic performance,so it is expected to be an elite resistance donor for wheat improvement.To promote the transfer of the chromosome arm 6RL of YT5 in marker-assisted selection(MAS)breeding,we selected and verified two 6RL-specific kompetitive allelespecific PCR(KASP)markers that can be applied to efficiently detect this chromosome arm in different wheat backgrounds.

The gene encoding flavonol synthase contributes to lesion mimic in wheat

Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection,and is characterized by a hypersensitive-response(HR)that closely linked to plant disease resistance.Despite this,only a few lesion mimic genes have been identified in wheat.In this investigation,a lesion mimic wheat mutant named je0297 was discovered,showing no alteration in yield components when compared to the wild type(WT).Segregation ratio analysis of the F_(2)individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297.Using Bulked segregant analysis(BSA)and exome capture sequencing,we mapped the lesion mimic gene designated as lm6 to chromosome 6BL.Further gene fine mapping using 3315 F_(2)individuals delimited the lm6 within a 1.18 Mb region.Within this region,we identified 16 high-confidence genes,with only two displaying mutations in je0297.Notably,one of the two genes,responsible for encoding flavonol synthase,exhibited altered expression levels.Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6.Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis,including flavone and flavonol biosynthesis,isoflavonoid biosynthesis,and flavonoid biosynthesis pathways.Furthermore,more than 30 pathogen infection-related(PR)genes exhibited upregulation in the mutant.Corresponding to this expression pattern,the flavonoid content in je0297 showed a significant decrease in the 4^(th)leaf,accompanied by a notable accumulation of reactive oxygen,which likely contributed to the development of lesion mimic in the mutant.This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.

Rosaceae phylogenomic studies provide insights into the evolution of new genes

Rosa banksiae,known as Lady Banks'rose,is a perennial ornamental crop and a versatile herb in traditional Chinese medicine.Given the lack of genomic resources,we assembled a Hi Fi and Nanopore sequencing-derived 458.58 Mb gap-free telomere-to-telomere high-quality R.banksiae genome with a scaffold N50=63.90 Mb.The genome of R.banksiae exhibited no lineage-specific whole-genome duplication compared with other Rosaceae.The phylogenomic analysis of 13 Rosaceae and Arabidopsis through a comparative genomics study showed that numerous gene families were lineage-specific both before and after the diversification of Rosaceae.Some of these genes are candidates for new genes that have evolved from parental genes through fusion events.Fusion genes are divided into three types:Type-I and Type-II genes contain two parental genes that are generated by duplication,distributed in the same and different regions of the genome,respectively;and Type-III can only be detected in one parental gene.Here,Type-I genes are found to have more relaxed selection pressure and lower Ks values than Type-II,indicating that these newly evolved Type-I genes may play important roles in driving phenotypic evolution.Functional analysis exhibited that newly formed fusion genes can regulate the phenotype traits of plant growth and development,suggesting the functional significance of these genes.This study identifies new fusion genes that could be responsible for phenotype evolution and provides information on the evolutionary history of recently diverged species in the Rosa genus.Our data represents the major progress in understanding the new fusion genes evolution pattern of Rosaceae and provides an invaluable resource for phylogenomic studies in plants.

Direct somatic embryogenesis and related gene expression networks in leaf explants of Hippeastrum ‘Bangkok Rose’

Hippeastrum, a highly diverse genus in the Amaryllidaceae family, is a valuable ornamental bulbous flowering plant. Somatic embryogenesis(SE) is an efficient method for mass production of Hippeastrum plantlets. Previous studies have been devoted to the in vitro propagation of Hippeastrum, but the SE and its regulatory networks are rarely reported. In this study, we established a direct SE method of Hippeastrum Bangkok Rose' using leaf bases as explants. MS supplemented with 1.00 mg·L^(-1)NAA +1.00 mg·L^(-1)KT + 0.25 mg·L^(-1)TDZ was the optimal medium for SE. Histological observations showed that the bipolar somatic embryo originated from the epidermal cell layer and underwent initiation,globular, scutellar and coleoptile stages. During SE, endogenous hormones of IAA, CTK, ABA, and SA were highly accumulated. Transcriptomic analysis revealed the genes encoding auxin biosynthesis/metabolic enzymes and efflux carriers were induced, while the auxin receptor of TIR1 and ARF transcriptional repressor of Aux/IAA were down-regulated and up-regulated, respectively, leading to suppression of auxin signaling. In contrast, cytokine signaling was promoted at the early stage of SE, as biosynthesis, transport, and signaling components were up-regulated.Various stress-related genes were up-regulated at the early or late stages of SE. Chromatin remodeling could also be dynamically regulated via distinct expression enzymes that control histone methylation and acetylation during SE. Moreover, key SE regulators, including WOXs and SERKs were highly expressed along with SE. Overall, the present study provides insights into the SE regulatory mechanisms of the Hippeastrum.

Pangenome and multi-tissue gene atlas provide new insights into the domestication and highland adaptation of yaks

Background The genetic diversity of yak,a key domestic animal on the Qinghai-Tibetan Plateau(QTP),is a vital resource for domestication and breeding efforts.This study presents the first yak pangenome obtained through the de novo assembly of 16 yak genomes.Results We discovered 290 Mb of nonreference sequences and 504 new genes.Our pangenome-wide presence and absence variation(PAV)analysis revealed 5,120 PAV-related genes,highlighting a wide range of variety-specific genes and genes with varying frequencies across yak populations.Principal component analysis(PCA)based on binary gene PAV data classified yaks into three new groups:wild,domestic,and Jinchuan.Moreover,we pro-posed a‘two-haplotype genomic hybridization model'for understanding the hybridization patterns among breeds by integrating gene frequency,heterozygosity,and gene PAV data.A gene PAV-GWAS identified a novel gene(Bos-Gru3G009179)that may be associated with the multirib trait in Jinchuan yaks.Furthermore,an integrated transcrip-tome and pangenome analysis highlighted the significant differences in the expression of core genes and the muta-tional burden of differentially expressed genes between yaks from high and low altitudes.Transcriptome analysis across multiple species revealed that yaks have the most unique differentially expressed m RNAs and lnc RNAs(between high-and low-altitude regions),especially in the heart and lungs,when comparing high-and low-altitude adaptations.Conclusions The yak pangenome offers a comprehensive resource and new insights for functional genomic studies,supporting future biological research and breeding strategies.

Fine mapping and characterization of stripe rust resistance gene YrAYH in near-isogenic lines derived from a cross involving wheat landrace Anyuehong

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is a devastating disease in wheat worldwide.Discovering and characterizing new resistance genes/QTL is crucial for wheat breeding programs.In this study,we fine-mapped and characterized a stripe rust resistance gene,YRAYH,on chromosome arm 5BL in the Chinese wheat landrace Anyuehong(AYH).Evaluations of stripe rust response to prevalent Chinese Pst races in near-isogenic lines derived from a cross of Anyuehong and Taichung 29 showed that YrAYH conferred a high level of resistance at all growth stages.Fine mapping using a large segregating population of 9748 plants,narrowed the YRAYH locus to a 3.7 Mb interval on chromosome arm 5BL that included 61 annotated genes.Transcriptome analysis of two NIL pairs identified 64 upregulated differentially expressed genes(DEGs)in the resistant NILs(NILs-R).Annotations indicated that many of these genes have roles in plant disease resistance pathways.Through a combined approach of fine-mapping and transcriptome sequencing,we identified a serine/threonine-protein kinase SRPK as a candidate gene underlying YrAYH.A unique 25 bp insertion was identified in the NILs-R compared to the NILs-S and previously published wheat genomes.An InDel marker was developed and co-segregated with YrAYH.Agronomic trait evaluation of the NILs suggested that YrAYH not only reduces the impact of stripe rust but was also associated with a gene that increases plant height and spike length.

Population genomic analysis reveals key genetic variations and the driving force for embryonic callus induction capability in maize

Genetic transformation has been an effective technology for improving the agronomic traits of maize.However,it is highly reliant on the use of embryonic callus(EC)and shows a serious genotype dependence.In this study,we performed genomic sequencing for 80 core maize germplasms and constructed a high-density genomic variation map using our newly developed pipeline(MQ2Gpipe).Based on the induction rate of EC(REC),these inbred lines were categorized into three subpopulations.The low-REC germplasms displayed more abundant genetic diversity than the high-REC germplasms.By integrating a genome-wide selective signature screen and region-based association analysis,we revealed 95.23 Mb of selective regions and 43 REC-associated variants.These variants had phenotypic variance explained values ranging between 21.46 and 49.46%.In total,103 candidate genes were identified within the linkage disequilibrium regions of these REC-associated loci.These genes mainly participate in regulation of the cell cycle,regulation of cytokinesis,and other functions,among which MYB15 and EMB2745 were located within the previously reported QTL for EC induction.Numerous leaf area-associated variants with large effects were closely linked to several REC-related loci,implying a potential synergistic selection of REC and leaf size during modern maize breeding.

J-family genes redundantly regulate flowering time and increase yield in soybean

Soybean(Glycine max)is a short-day crop whose flowering time is regulated by photoperiod.The longjuvenile trait extends its vegetative phase and increases yield under short-day conditions.Natural variation in J,the major locus controlling this trait,modulates flowering time.We report that the three J-family genes influence soybean flowering time,with the triple mutant Guangzhou Mammoth-2 flowering late under short days by inhibiting transcription of E1-family genes.J-family genes offer promising allelic combinations for breeding.

Genes controlling grain chalkiness in rice

With rising living standards,there is an increasing demand for high-quality rice.Rice quality is mainly defined by milling quality,appearance quality,cooking and eating quality,and nutrition quality.Among them,chalkiness is a key trait for appearance quality,which adversely affects cooking and eating quality,head rice yield,and commercial value.Therefore,chalkiness is undesirable,and reducing chalkiness is a major goal in rice quality improvement.However,chalkiness is a complex trait jointly influenced by genetic and environmental factors,making its genetic study and precision improvement a huge challenge.With the rapid development of molecular techniques,much knowledge has been gained about the genes and molecular networks involved in chalkiness formation.The present review describes the major environmental factors affecting chalkiness and summarizes the quantitative trait loci(QTL)associated with chalkiness.More than 150 genes related to chalkiness formation have been reported.The functions of the genes regulating chalkiness,primarily those involved in starch synthesis,storage protein synthesis,transcription regulation,organelle development,grain shape regulation,and hightemperature response,are described.Finally,we identify the challenges associated with genetic improvement of chalkiness and suggest potential strategies.Thus,the review offers insight into the molecular dynamics of chalkiness and provides a strong basis for the future breeding of high-quality rice varieties.

Multi-genome evolutionary study of the ABC1 gene family and identification of the pleiotropic effects of OsABC1-13 in rice development

In four rice genomes,85 ABC1-family genes were identified by comparative genomics,evolution,genetics,and physiology.One,OsABC1-13,was shown by knockdown and knockout experiments to affect plant height,grain size,and photosynthetic capability.

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR(TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35 S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.

Development of a MaizeGerm50K array and application to maize genetic studies and breeding

Genotyping arrays based on single nucleotide polymorphisms(SNPs)provide a low-cost,highthroughput platform.The development of a SNP array that fully reflects the genetic diversity of maize(Zea mays L.)germplasm and is applicable to molecular breeding programs is desirable.In this study,we developed a MaizeGerm50K array comprising 50,852 SNPs selected from the resequencing data of 1604 maize inbred lines and other markers.A genome-wide association study using a landrace panel genotyped with the array permitted mapping of several known genes.We also verified a candidate gene,RNA-binding motif protein 24-like 1(ZmRBM24L1),delaying flowering through overexpression lines.Genomic selection for yield and agronomic traits showed high prediction accuracy.The MaizeGerm50K array is thus a valuable genomic tool for maize genetic studies and breeding.

One bird,multiple stones:The race to find a gene of dominant negative effect in wheat

In the current issue of The Crop Journal,Chen et al.[1]reports map-based cloning of a wheat gene that showed temperaturedependent pleiotropic effects on multiple traits including plant height,leaf shape,spike and grain morphology,and accordingly was named WPA1 for Wheat Plant Architecture 1.The mutant was first observed among EMS-treated plants and repeatedly appeared in multiple occasions.

Mutations in the WUSCHEL-related homeobox1 gene cause an increased leaflet number in soybean

WUSCHEL-related homeobox(WOX)transcription factors play a crucial role in lateral organ development in several plant species;however,their precise functions in soybean(Glycine max[L.]Merr.)were unclear.Here,we identified two independent multi-leaflet soybean mutants,mlw48-8 and mlw48-161,from a CRISPR/Cas9-engineered mutant library in the Williams 82 background.Both mutants exhibited irregular leaf margins,and the upper leaves were narrow and almost lanceolate at maturity.Molecular analysis revealed that these are allelic mutants with independent mutations in the WUSCHEL-related homeobox1(GmWOX1A)gene.A transcriptome analysis demonstrated that GmWOX1A modulates the expression of auxin-and leaf development–related genes.Yeast two-hybrid and split-luciferase complementation imaging assays revealed that GmWOX1A interacts with the YABBY family protein GmYAB5,providing further evidence of its potential involvement in leaf development.Notably,the mlw48-161 mutant showed an increased seed number per plant.Consequently,our study not only provides valuable insights into the role of GmWOX1A in soybean leaf development but also offers a potential strategy for high-yield breeding.

Genetic analysis and fine mapping of a grain size QTL in the smallgrain sterile rice line Zhuo201S

The development and application of the small-grain rice sterile line Zhuo201S(Z201S)has demonstrated its potential for mechanized hybrid rice seed production,leading to significant cost reductions.However,the molecular mechanism responsible for the small-grain size characteristic of Z201S remains unclear.In this study,we conducted a genetic analysis using near-isogenic lines constructed from Z210S,a small-grain rice sterile line,and R2115,a normal-grain variety.The results revealed that the small-grain trait in Z201S is governed by a single partially dominant gene which also enhances grain number.Through mapping,we localized the causal gene to the short arm of chromosome 2,within a 113 kb physical region delimited by the molecular markers S2-4-1 and LB63.Transgenic analysis and gene expression assays indicated LOC_Os02g14760 as the most likely candidate gene,suggesting that the small-grain size trait of Z201S is controlled by a novel locus that has not been previously identified.

Aβ-Carotene Ketolase Gene NfcrtO from Subaerial Cyanobacteria Confers Drought Tolerance in Rice

Nostoc flagelliforme is a terrestrial cyanobacterium that can resist many types of stressors,including drought,ultraviolet radiation,and extreme temperatures.In this study,we identified the drought tolerance gene NfcrtO,which encodes aβ-carotene ketolase,through screening the transcriptome of N.flagelliforme under water loss stress.Prokaryotic expression of NfcrtO under 0.6 mol/L sorbitol or under 0.3 mol/L NaCl stress significantly increased the growth rate of Escherichia coli.When NfcrtO was heterologously expressed in rice,the seedling height and root length of NfcrtO-overexpressing rice plants were significantly higher than those of the wild type(WT)plants grown on½Murashige and Skoog solid medium with 120 mmol/L mannitol at the seedling stage.Transcriptome analysis revealed that NfcrtO was involved in osmotic stress,antioxidant,and other stress-related pathways.Additionally,the survival rate of the NfcrtO-overexpression lines was significantly higher than that of the WT line under both hydroponic stress(24%PEG and 100 mmol/L H_(2)O_(2))and soil drought treatment at the seedling stage.Physiological traits,including the activity levels of superoxide dismutase,peroxidase,catalase,total antioxidant capacity,and the contents of proline,trehalose,and soluble sugar,were significantly improved in the NfcrtO-overexpression lines relative to those in the WT line under 20%PEG treatment.Furthermore,when water was withheld at the booting stage,the grain yield per plant of NfcrtO-overexpression lines was significantly higher than that of the WT line.Yeast two-hybrid analysis identified interactions between NfcrtO and Dna J protein,E3 ubiquitin-protein ligase,and pyrophosphate-energized vacuolar membrane proton pump.Thus,heterologous expression of NfcrtO in rice could significantly improve the tolerance of rice to osmotic stress,potentially facilitating the development of new rice varieties.

Artificial selection of the Green Revolution gene Semidwarf 1 is implicated in upland rice breeding

Semidwarf breeding has boosted crop production and is a well-known outcome from the first Green Revolution. The Green Revolution gene Semidwarf 1(SD1), which modulates gibberellic acid(GA) biosynthesis, plays a principal role in determining rice plant height. Mutations in SD1 reduce rice plant height and promote lodging resistance and fertilizer tolerance to increase grain production. The plant height mediated by SD1 also favors grain yield under certain conditions. However, it is not yet known whether the function of SD1 in upland rice promotes adaptation and grain production. In this study, the plant height and grain yield of irrigated and upland rice were comparatively analyzed under paddy and dryland conditions. In response to dryland environments, rice requires a reduction in plant height to cope with water deficits. Upland rice accessions had greater plant heights than their irrigated counterparts under both paddy and dryland conditions, and appropriately reducing plant height could improve adaptability to dryland environments and maintain high grain yield formation. Moreover, upland rice cultivars with thicker stem diameters had stronger lodging resistance, which addresses the lodging problem. Knockout of SD1 in the upland rice cultivar IRAT104 reduced the plant height and grain yield, demonstrating that the adjustment of plant height mediated by SD1 could increase grain production in dryland fields. In addition, an SD1 genetic diversity analysis verified that haplotype variation causes phenotypic variation in plant height. During the breeding history of rice, SD1 allelic mutations were selected from landraces to improve the grain yield of irrigated rice cultivars, and this selection was accompanied by a reduction in plant height. Thus, five known mutant alleles were analyzed to verify that functional SD1 is required for upland rice production. All these results suggest that SD1 might have undergone artificial positive selection in upland rice, which provides further insights concerning greater plant height in upland rice breeding.

Leaf Morphology Genes SRL1 and RENL1 Co-Regulate Cellulose Synthesis and Affect Rice Drought Tolerance

The morphological development of rice(Oryza sativa L.)leaves is closely related to plant architecture,physiological activities,and resistance.However,it is unclear whether there is a co-regulatory relationship between the morphological development of leaves and adaptation to drought environment.In this study,a drought-sensitive,roll-enhanced,and narrow-leaf mutant(renl1)was induced from a semi-rolled leaf mutant(srl1)by ethyl methane sulfonate(EMS),which was obtained from Nipponbare(NPB)through EMS.Map-based cloning and functional validation showed that RENL1 encodes a cellulose synthase,allelic to NRL1/OsCLSD4.The RENL1 mutation resulted in reduced vascular bundles,vesicular cells,cellulose,and hemicellulose contents in cell walls,diminishing the water-holding capacity of leaves.In addition,the root system of the renl1 mutant was poorly developed and its ability to scavenge reactive oxygen species(ROS)was decreased,leading to an increase in ROS after drought stress.Meanwhile,genetic results showed that RENL1 and SRL1 synergistically regulated cell wall components.Our results revealed a theoretical basis for further elucidating the molecular regulation mechanism of cellulose on rice drought tolerance,and provided a new genetic resource for enhancing the synergistic regulation network of plant type and stress resistance,thereby realizing simultaneous improvement of multiple traits in rice.

Roles of cancer-associated fibroblast functional heterogeneity in shaping the lymphatic metastatic landscape:new insights and therapeutic strategies

Lymph node (LN) metastasis is a process in which cancer cells travel from primary tumors to LNs via the lymphatic system,then proliferate and spread within the LNs. In most cancers,LN metastasis is a major mode of cancer dissemination,and a critical indicator of cancer progression and worsening prognosis1. The occurrence of LN metastasis indicates that the tumor has invaded the lymphatic system.