Meta-QTL analysis for mining of candidate genes and constitutive gene network development for fungal disease resistance in maize(Zea mays L.)

The development of resistant maize cultivars is the most effective and sustainable approach to combat fungal diseases.Over the last three decades,many quantitative trait loci(QTL)mapping studies reported numerous QTL for fungal disease resistance(FDR)in maize.However,different genetic backgrounds of germplasm and differing QTL analysis algorithms limit the use of identified QTL for comparative studies.The meta-QTL(MQTL)analysis is the meta-analysis of multiple QTL experiments,which entails broader allelic coverage and helps in the combined analysis of diverse QTL mapping studies revealing common genomic regions for target traits.In the present study,128(33.59%)out of 381 reported QTL(from 82 studies)for FDR could be projected on the maize genome through MQTL analysis.It revealed 38 MQTL for FDR(12 diseases)on all chromosomes except chromosome 10.Five MQTL namely 1_4,2_4,3_2,3_4,and 5_4 were linked with multiple FDR.Total of 1910 candidate genes were identified for all the MQTL regions,with protein kinase gene families,TFs,pathogenesis-related,and disease-responsive proteins directly or indirectly associated with FDR.The comparison of physical positions of marker-traits association(MTAs)from genome-wide association studies with genes underlying MQTL interval verified the presence of QTL/candidate genes for particular diseases.The linked markers to MQTL and putative candidate genes underlying identified MQTL can be further validated in the germplasm through marker screening and expression studies.The study also attempted to unravel the underlying mechanism for FDR resistance by analyzing the constitutive gene network,which will be a useful resource to understand the molecular mechanism of defense-response of a particular disease and multiple FDR in maize.

Identification and expression analysis of sugar transporter family genes reveal the role of ZmSTP2 and ZmSTP20 in maize disease resistance

Sugar is an indispensable source of energy for plant growth and development, and it requires the participation of sugar transporter proteins(STPs) for crossing the hydrophobic barrier in plants. Here, we systematically identified the genes encoding sugar transporters in the genome of maize(Zea mays L.), analyzed their expression patterns under different conditions, and determined their functions in disease resistance. The results showed that the mazie sugar transporter family contained 24 members, all of which were predicted to be distributed on the cell membrane and had a highly conserved transmembrane transport domain. The tissue-specific expression of the maize sugar transporter genes was analyzed, and the expression level of these genes was found to be significantly different in different tissues. The analysis of biotic and abiotic stress data showed that the expression levels of the sugar transporter genes changed significantly under different stress factors. The expression levels of Zm STP2 and Zm STP20 continued to increase following Fusarium graminearum infection. By performing disease resistance analysis of zmstp2 and zmstp20 mutants, we found that after inoculation with Cochliobolus carbonum, Setosphaeria turcica, Cochliobolus heterostrophus, and F. graminearum, the lesion area of the mutants was significantly higher than that of the wild-type B73 plant. In this study, the genes encoding sugar transporters in maize were systematically identified and analyzed at the whole genome level. The expression patterns of the sugar transporter-encoding genes in different tissues of maize and under biotic and abiotic stresses were revealed, which laid an important theoretical foundation for further elucidation of their functions.

Identification and Evaluation of Insect and Disease Resistance in Transgenic Cry1Ab13-1 and NPR1 Maize

PCR detection,quantitative real-time PCR(q-RTPCR),outdoor insect resistance,and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations(T2,T3,and T4)in transgenic maize germplasms(S3002 and 349)containing the bivalent genes(insect resistance gene Cry1Ab13-1 and disease resistance gene NPR1)and their corresponding wild type.Results indicated that the target genes Cry1Ab13-1 and NPR1 were successfully transferred into both germplasms through tested generations;q-PCR confirmed the expression of Cry1Ab13-1 and NPR1 genes in roots,stems,and leaves of tested maize plants.In addition,S3002 and 349 bivalent gene-transformed lines exhibited resistance to large leaf spots and corn borer in the field evaluation compared to the wild type.Our study confirmed that Cry1Ab13-1 and NPR1 bivalent genes enhanced the resistance against maize borer and large leaf spot disease and can stably inherit.These findings could be exploited for improving other cultivated maize varieties.

Genetic Improvement of Japonica Rice Variety Wuyujing 3 for Stripe Disease Resistance and Eating Quality by Pyramiding Stv-b^i and Wx-mq

Japonica rice variety Kanto 194 as the donor of resistance gene Stv-b^i and Iow-amylose content gene Wx-mq was used to improve the resistance and eating quality of Wuyujing 3 by the breeding strategy of backcross. In continuous backcross and selfcross generations, the related molecular markers with Stvobi and Wx-mq genes were utilized for genotypic detection by associated with resistance identification of rice stripe disease and agronomic traits selection. Finally, 10 improved lines with homozygous genotype Stv-b^i Stv-b^iWx-mqWx-mq were obtained from BC3F4 generation. The results of comparative analysis indicated that most characters of these lines were consistent with those of recipient parent Wuyujing 3, except for the improved resistance, appearance and eating quality. By evaluation of the comprehensive performance of them, two excellent lines K01 and K04 were selected for further experiments.

Development of a 50K SNP Array for Japanese Flounder and Its Application in Genomic Selection for Disease Resistance

Single nucleotide polymorphism(SNP)armays are a powerful genotyping tool used in genetic research and genomic breeding programs.Japanese flounder(Paralichthys olivaceus)is an economically-important aquaculture flatfish in many countries.However,the lack of high-efficient genotyping tools has impeded the genomic breeding programs for Japanese flounder.We developed a 50K Japanese flounder SNP array,"Yuxin No.1,"and report its utility in genomic selection(GS)for disease resistance to bacterial pathogens.We screened more than 42,.2 million SNPs from the whole-genome resequencing data of 1099 individuals and selected 48697 SNPs that were evenly distributed across the genome to anchor the array with Affymetrix Axiom genotyping technology.Evaluation of the array performance with 168 fishs howed that 74.7%of the loci were successfully genotyped with high call rates(>98%)and that the poly-morphic SNPs had good cluster separations.More than 85%of the SNPs were concordant with SNPs obtained from the whole-genome resequencing data.To validate"Yuxin No.1"for GS,the arrayed geno-typing data of 27 individuals from a candidate population and 931 individuals from a reference popula-tion were used to calculate the genomic estimated breeding values(GEBVs)for disease resistance toEdwardsiella tarda.There was a 21.2%relative increase in the accuracy of GEBV using the weighted geno-mic best linear unpiased prediction(wGBLUJP),compared to traditional pedigree-based best linear unbi-ased prediction(ABLUP),suggesting good performance of the'Yuxin No.1"SNP array for GS.In summary,we developed the"Yuxin No.1"50K SNP array,which provides a useful platform for high-quality geno-typing that may be beneficial to the genomic selective breeding of Japanese flounder.

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-b＇ and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region （SCAR） marker tightly linked with Stv-b＇ and a cleaved amplified polymorphic sequence （CAPS） marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.

Breeding of Selectable Marker-Free Transgenic Rice Lines Containing AP1 Gene with Enhanced Disease Resistance

In order to obtain marker-free transgenic rice with improved disease resistance, the AP1 gene of Capsicum annuum and hygromycin-resistance gene （HPT） were cloned into the two separate T-DNA regions of the binary vector pSB130, respectively, and introduced into the calli derived from the immature seeds of two elite japonica rice varieties, Guangling Xiangjing and Wuxiangjing 9, mediated by Agrobacterium-mediated transformation. Many cotransgenic rice lines containing both the AP1 gene and the marker gene were regenerated and the integration of both transgenes in the transgenic rice plants was confirmed by either PCR or Southern blotting technique. Several selectable marker-free transgenic rice plants were subsequently obtained from the progeny of the cotransformants, and confirmed by both PCR and Southern blotting analysis. These transgenic rice lines were tested in the field and their resistance to disease was carefully investigated, the results showed that after inoculation the resistance to either bacterial blight or sheath blight of the selected transgenic lines was improved when compared with those of wild type.

Effects of Nano Chinese Herbal Medicine Feed Additive on Growth Performance,Meat Quality and Disease Resistance of Chickens

[Objectives]To explore the effects of nano compound Chinese herbal medicine feed additive on growth performance,meat quality and disease resistance of chickens.[Methods]Chickens were fed with nano compound Chinese herbal medicine feed additive developed by Hunan Polytechnic of Environment and Biology,including 120 chickens in the treatment group and 120 chickens in the control group(CK).The growth performance indices(body weight gain,feed to gain ratio and slaughter index),meat quality indices(pH value,color,drip loss,shear force)and disease resistance indices(morbidity and mortality)of the chickens in the treatment and CK groups were recorded and determined,respectively.[Results]The inclusion of 2%nano compound Chinese herbal medicine feed additive in the diet significantly increased the growth rate,reduced the feed-to-gain ratio and improved the meat quality of the chickens.Supplementing Chinese herbal medicine could increase the pH value and reduce the drip loss and shear force of chicken meat.At the same time,the body's immune function,antioxidant level and resistance against diseases of the chickens fed with nano compound Chinese herbal medicine feed additive were improved.[Conclusions]The inclusion of nano compound Chinese herbal medicine feed additive in the diet can improve the growth performance,meat quality and disease resistance of chickens.

Marker-Assisted Breeding of Thermo-Sensitive Genic Male Sterile Line 1892S for Disease Resistance and Submergence Tolerance

Rice line 1892S is an elite thermo-sensitive genic male sterile(TGMS)line for two-line hybrid rice production.However,1892S is susceptible to rice blast,bacterial blight and submergence.Here we reported the introduction of blast resistance(R)gene Pi9,bacterial blight R gene Xa21 and submergence tolerance gene Sub1A into 1892S genetic background through backcrossing and marker-assisted selection.The improved TGMS line 31892S and its hybrids conferred disease resistance to rice blast and bacterial blight,and showed submergence tolerance for over 14 d without significant loss of viability.The sterility-fertility conversion of 31892S was similar to that of 1892S.31892S and its derived hybrid rice had similar agronomic traits and grain quality with 1892S and the control hybrid rice,respectively.The newly developed 31892S provided an improved TGMS line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight,and submergence tolerance with no yield penalty or change in grain quality.

Improvement of three popular Indian groundnut varieties for foliar disease resistance and high oleic acid using SSR markers and SNP array in marker-assisted backcrossing

Foliar fungal diseases(rust and late leaf spot)incur large yield losses,in addition to the deterioration of fodder quality in groundnut worldwide.High oleic acid has emerged as a key market trait in groundnut,as it increases the shelf life of the produce/products in addition to providing health benefits to consumers.Marker-assisted backcrossing(MABC)is the most successful approach to introgressing or pyramiding one or more traits using traitlinked markers.We used MABC to improve three popular Indian cultivars(GJG 9,GG 20,and GJGHPS 1)for foliar disease resistance(FDR)and high oleic acid content.A total of 22 BC3F4 and 30 BC2F4 introgression lines(ILs)for FDR and 46 BC3F4 and 41 BC2F4 ILs for high oleic acid were developed.Recurrent parent genome analysis using the 58 K Axiom_Arachis array identified several lines showing upto 94%of genome recovery among second and third backcross progenies.Phenotyping of these ILs revealed FDR scores comparable to the resistant parent,GPBD 4,and ILs with high(~80%)oleic acid in addition to high genome recovery.These ILs provide further opportunities for pyramiding FDR and high oleic acid in all three genetic backgrounds as well as for conducting multi-location yield trials for further evaluation and release for cultivation in target regions of India.

Genome Editing Strategies Towards Enhancement of Rice Disease Resistance

The emerging pests and phytopathogens have reduced the crop yield and quality, which hasthreatened the global food security. Traditional breeding methods, molecular marker-based breedingapproaches and use of genetically modified crops have played a crucial role in strengthening the foodsecurity worldwide. However, their usages in crop improvement have been highly limited due to multiplecaveats. Genome editing tools like transcriptional activator-like effector nucleases and clustered regularlyinterspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 (CRISPR/Cas9) haveeffectively overcome limitations of the conventional breeding methods and are being widely accepted forimprovement of crops. Among the genome editing tools, the CRISPR/Cas9 system has emerged as themost powerful tool of genome editing because of its efficiency, amicability, flexibility, low cost andadaptability. Accumulated evidences indicate that genome editing has great potential in improving thedisease resistance in crop plants. In this review, we offered a brief introduction to the mechanisms of differentgenome editing systems and then discussed recent developments in CRISPR/Cas9 system-based genomeediting towards enhancement of rice disease resistance by different strategies. This review also discussed thepossible applications of recently developed genome editing approaches like CRISPR/Cas12a (formerlyknown as Cpf1) and base editors for enhancement of rice disease resistance.

Comparative transcriptome profiling of two maize near-isogenic lines differing in the allelic state for bacterial brown spot disease resistance

The bacterial brown spot disease（BBS）, caused primarily by Pseudomonas syringae pv. syringae van Hall（Pss）, reduces plant vigor, yield and quality in maize. To reveal the nature of the defense mechanisms and identify genes involved in the effective host resistance, the dynamic changes of defense transcriptome triggered by the infection of Pss were investigated and compared between two maize near-isogenic lines（NILs）. We found that Pss infection resulted in a sophisticated transcriptional reprogramming of several biological processes and the resistant NIL employed much faster defense responses than the susceptible NIL. Numerous genes encoding essential components of plant basal resistance would be able to be activated in the susceptible NIL, such as PEN1, PEN2, PEN3, and EDR1, however, in a basic manner, such resistance might not be sufficient for suppressing Pss pathogenesis. In addition, the expressions of a large number of PTI-, ETI-, PR-, and WRKY-related genes were pronouncedly activated in the resistant NIL, suggesting that maize employ a multitude of defense pathways to defend Pss infection. Six R-gene homologs were identified to have significantly higher expression levels in the resistant NIL at early time point, indicating that a robust surveillance system（gene-to-gene model） might operate in maize during Pss attacks, and these homolog genes are likely to be potential candidate resistance genes involved in BBS disease resistance. Furthermore, a holistic group of novel pathogen-responsive genes were defined, providing the repertoire of candidate genes for further functional characterization and identification of their regulation patterns during pathogen infection.

Immunogenomics for identification of disease resistance genes in pigs: a review focusing on Gram-negative bacilli

Over the past years, infectious disease has caused enormous economic loss in pig industry. Among the pathogens, gram negative bacteria not only cause inflammation, but also cause different diseases and make the pigs more susceptible to virus infection. Vaccination, medication and elimination of sick pigs are major strategies of controlling disease. Genetic methods, such as selection of disease resistance in the pig, have not been widely used. Recently, the completion of the porcine whole genome sequencing has provided powerful tools to identify the genome regions that harboring genes controlling disease or immunity. Immunogenornics, which combines DNA variations, transcriptorne, immune response, and QTL mapping data to illustrate the interactions between pathogen and host immune system, will be an effective genomics tool for identification of disease resistance genes in pigs. These genes will be potential targets for disease resistance in breeding programs. This paper reviewed the progress of disease resistance study in the pig focusing on Gram-negative bacilli. Major porcine Gram-negative bacilli and diseases, suggested candidate genes/pathways against porcine Gram-negative bacilli, and distributions of QTLs for immune capacity on pig chromosomes were summarized. Some tools for immunogenomics research were described. We conclude that integration of sequencing, whole genome associations, functional genomics studies, and immune response information is necessary to illustrate molecular mechanisms and key genes in disease resistance.

Molecular Detection and Disease Resistance Identification of Transgenic Wheat with pti5-vp16 Gene

The immature embryos of wheat cultivars Liaochun10, Tiechun1 and Fengqiang3 were bombarded with gold particles coated with pti5 vp16 by gene gun and disease resistant regenerated plants were attained. In order to confirm that the plants are genuine transformed ones, a series of molecular tests were conducted as follows. Firstly, transient GUS expression test on embryos two days after bombardment was done. There were many obvious blue spots produced on the surface of bombarded embryos after GUS staining, in which the maximum reached to 85 blue spots per embryo. Secondly, PCR test was performed with DNA from the regenerated plants obtained after double selection with ppt. 6 plants were found PCR test positive. At last, further verification analysis using dot hybridization and southern blotting was carried out on those PCR positive plants and the strong hybridization signals appeared as expected. All the above tests were uniformly indicated that the disease resistant regenerated plants were true transgenic plants. When inoculated with Blumeria graminis, transgenic wheat plants of PCR positive results were mostly resistant(R) after 7 days, and resis tant, moderate resistant(MR), moderate susceptible(MS) at 14 days respectively. The disease severity of them was distinctively lighter than that of control.

Bioinformatics Analysis of Disease Resistance Gene PR1 and Its Genetic Transformation in Soybeans and Cultivation of Multi-resistant Materials

In agricultural production,a single insect-resistant and disease-resistant variety can no longer meet the demand.In this study,the expression vector pCAMBIA-3301-PR1 containing the disease-resistant gene PR1 was constructed by means of genetic engineering,and the PR1 gene was genetically transformed to contain the PR1 gene through the pollen tube method.In CryAb-8Like transgenic high-generation T7 receptor soybean,a new material that is resistant to insects and diseases is obtained.For T2 transformed plants,routine PCR detection,Southern Blot hybridization,fluorescence quantitative PCR detection,indoor and outdoor pest resistance identification and indoor disease resistance identification were performed.The results showed that there were 9 positive plants in the routine PCR test of T2 generation.In Southern Blot hybridization,both PR1 and CryAb-8Like genes are integrated in soybeans in the form of single copies.Fluorescence quantitative PCR showed that the expression levels of PR1 and CryAb-8Like genes are different in different tissues.The average expression levels of PR1 gene in plant roots,stems,and leaves are 2.88,1.54,and 5.26,respectively.CryAb-8Like genes are found in roots,stems,and leaves.The average expression levels were 1.36,1.39,and 4.25,respectively.The insectivorous rate of the CryAb-8Like gene in outdoor plants with positive insect resistance identification was 3.78%.The disc partition method was used indoors for pest resistance identification,and the bud length of transformed plants increased significantly.The average mortality rate of untransformed plants in indoor disease resistance identification was as high as 56.66%,and the average mortality rate of plants transformed with PR1 gene was 10.00%,and disease resistance was significantly improved.Therefore,a new material with resistance to diseases and insects is obtained.

Transgenic Cotton and Disease Resistance Genes

Success in conventional breeding for resistance to mycotoxin-producing or other phytopathogenic fungi is dependent on the availability of resistance gene(s) in the germplasm.Even when it is available,breeding for disease-resistant crops is very time consuming,especially in perennial crops such

Plant cell wall-mediated disease resistance:Current understanding and future perspectives

Beyond their function as structural barriers,plant cell walls are essential elements for the adaptation of plants to environmental conditions.Cell walls are dynamic structures whose composition and integrity can be altered in response to environmental challenges and developmental cues.These wall changes are perceived by plant sensors/receptors to trigger adaptative responses during development and upon stress perception.Plant cell wall damage caused by pathogen infection,wounding,or other stresses leads to the release of wall molecules,such as carbohydrates(glycans),that function as damage-associated molecular patterns(DAMPs).DAMPs are perceived by the extracellular ectodomains(ECDs)of pattern recognition receptors(PRRs)to activate pattern-triggered immunity(PTI)and disease resistance.Similarly,glycans released from the walls and extracellular layers of microorganisms interacting with plants are recognized as microbe-associated molecular patterns(MAMPs)by specific ECD-PRRs triggering PTI responses.The number of oligosaccharides DAMPs/MAMPs identified that are perceived by plants has increased in recent years.However,the structural mechanisms underlying glycan recognition by plant PRRs remain limited.Currently,this knowledge is mainly focused on receptors of the LysM-PRR family,which are involved in the perception of various molecules,such as chitooligosaccharides from fungi and lipo-chitooligosaccharides(i.e.,Nod/MYC factors from bacteria and mycorrhiza,respectively)that trigger differential physiological responses.Nevertheless,additional families of plant PRRs have recently been implicated in oligosaccharide/polysaccharide recognition.These include receptor kinases(RKs)with leucine-rich repeat and Malectin domains in their ECDs(LRR-MAL RKs),Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE group(CrRLK1L)with Malectin-like domains in their ECDs,as well as wall-associated kinases,lectin-RKs,and LRR-extensins.The characterization of structural basis of glycans recognition by these new plant receptors will shed light on their similarities with those of mammalians involved in glycan perception.The gained knowledge holds the potential to facilitate the development of sustainable,glycan-based crop protection solutions.

Multi-species probiotics enhance survival,growth,intestinal microbiota and disease resistance of rohu(Labeo rohita)larvae

Multi-strain probiotics provide the most dependable approaches to improve health,immune response,and disease resistance in farmed fishes.In the present study,we examined the effects of multi-species probiotics on the survival,growth,immune response,and disease resistance of rohu(Labeo rohita)larvae.Newly hatched larvae from the day of first feeding(average weight of 0.003 g)were reared with multi-species probiotics having a combination of Bacillus subtilis(109 colony forming units(cfu)/mL)and Lactobacillus spp.(Lactobacillus plantarum,Lactobacillus buchneri-1011 cfu/mL)in water containing doses of 0(control-C),0.5 mL/L(treatment 1-T1),and 1.0 mL/L(treatment 2-T2)in triplicates for 90 days.After the experiment,a challenge test was performed to assess the fish's resistance to pathogenic Aeromonas veronii.Significantly higher survival was recorded in larvae of treated groups(87%in T2 and 79%in T1)compared to the control(62%).Significantly higher growth performance(weight gain and specific growth rate—SGR)was shown by the probiotic-treated larval groups compared to the control.Probiotic supplementation resulted in significantly higher counts of total viable colony(TVC)and lactic acid bacteria(LAB)in the intestine.Some immunological parameters(mucosal fold fattening,goblet cell abundance,expansion of lamina propria and enterocytes)of the gut were significantly better in probiotic-treated fish.The liver of treated fish showed irregular shape nuclei turning into regular shape and reducing spaces between the hepatic cells.Probiotic-treated fish had the highest post-challenge survival rate(90%)against A.veronii infection.The erythrocytes of challenged fish treated with probiotics had significantly lower frequencies of various nuclear and cellular abnormalities.These findings suggest that multi-species probiotic supplements could improve the survival,growth,health status,and immune response of rohu in the early stages of its development.

Breeding and Application Techniques of a New Rice Variety Liliang-you 3822 with High Yield and Disease Resistance

Liliangyou 3822 is a novel indica hybrid rice variety that exhibits disease resistance,high yield,lodging resistance,and late maturity.It employs a self-selected two-line sterile line,Li 38S,and a self-selected restorer line,R22.This variety was subjected to a regional test of indica late-maturing groups in the middle and lower reaches of the Yangtze River in 2020.The results demonstrated that the average yield of the variety was 9.95 t/hm 2,which was 10.67%higher than that of the control Fengliangyou 4,indicating a highly significant yield increase.In the continuous test in 2021,the average yield was 9.74 t/hm 2,representing a 6.52%increase over the control,which also exhibited a significant increase.Finally,the average yield of the two years regional test was 9.84 t/hm 2,which was 8.58%higher than that of the control.In the 2021 production test,the average yield of the variety was 9.32 t/hm 2,which was 12.19%higher than that of the control,indicating a remarkably significant yield increase.In 2022,the variety was validated by the National Crop Variety Approval Committee(GSD 20220143).

A chromosome-scale genome assembly of Dasypyrum villosum provides insights into its application as a broad-spectrum disease resistance resource for wheat improvement

Dasypyrum villosum is one of the most valuable gene resources in wheat improvement,especially for disease resistance.The mining of favorable genes from D.villosum is frustrated by the lack of a whole genome sequence.In this study,we generated a doubled-haploid line,91C43^(DH),using microspore culture and obtained a 4.05-GB high-quality,chromosome-scale genome assembly for D.villosum.The assembly contains39727 high-confidence genes,and 85.31% of the sequences are repetitive.Two reciprocal translocation events were detected,and 7VS-4VL is a unique translocation in D.villosum.The prolamin seed storage protein-coding genes were found to be duplicated;in particular,the genes encoding low-molecular-weight glutenin at the Glu-V3 locus were significantly expanded.RNA sequencing(RNA-seq)analysis indicated that,after Blumeria graminearum f.sp tritici(Bgt)inoculation,there were more upregulated genes involved in the pattern-triggered immunity and effector-triggered immunity defense pathways in D.villosum than in Triticum urartu.MNase hypersensitive sequencing(MH-seq)identified two Bgt-inducible MH sites(MHSs),one in the promoter and one in the 3'terminal region of the powdery mildew resistance(Pm)gene NLR1-V.Each site had two subpeaks and they were termed MHS1(MHS1.1/1.2)and MHS2(MHS2.1/2.2).Bgt-inducible MHS2.2 was uniquely present in D.villosum,and MHS1.1 was more inducible in D.villosum than in wheat,suggesting that MHSs may be critical for regulation of NLR1-V expression and plant defense.In summary,this study provides a valuable genome resource for functional genomics studies and wheat-D.villosum introgression breeding.The identified regulatory mechanisms may also be exploited to develop new strategies for enhancing Pm resistance by optimizing gene expression in wheat.