Identification of KASP markers and putative genes for pre-harvest sprouting resistance in common wheat(Triticum aestivum L.)

Common wheat(Triticum aestivum L.)is the most important crop in the world and a typical allopolyploid with a large and complex genome.Pre-harvest sprouting(PHS)leads to a significant reduction in grain quality worldwide.PHS is a complex trait with related QTL located on different chromosomes.However,the study of markers and genes related to PHS resistance is limited especially for whitegrained wheat.Four pairs of near isogenic lines(NILs)from a white-grained wheat cross of Chara
DM5637B*8 targeting a major QTL for PHS resistance(Qphs.ccsu-3A.1)on wheat chromosme 3AL were genotyped using the 90K SNP Illumina iSelect array.Ten SNPs were identified,with a 75%-100%consistency between genotype and phenotype in the resistant or susceptible isolines.The 10 SNPs were converted to cost-effective kompetitive allele-specific PCR(KASP)markers.Screening of 48 wheat cultivars with different phenotypes of PHS identified four KASP markers with 81.3%-85.4%conformity between genotype and phenotype.Further investigation revealed that the four SNPs(BS00022245_51,Kukri_c49927_151,BS00022884_51 and BS00110550_51)corresponding to the four validated KASP markers are residing in three independent genes(TraesCS3A03G1072800,TraesCS3A03G1072400,TraesCS3A03G1071800)close to each other with a distance of 4.28-4.48 Mb to the targeted QTL.These three annotated genes have potential functions related to PHS resistance.Our study revealed that combined use of NILs and the 90K SNP chip is a powerful approach for developing KASP markers and mining functional genes in wheat.The KASP markers for PHS resistance on chromosome 3AL are useful for high-throughput evaluation and marker-assisted selection,and the three identified genes could lead to a better understanding of the genetic pathways controlling PHS.

Fine-mapping of a candidate gene for web blotch resistance in Arachis hypogaea L.

Peanut(Arachis hypogaea L.)is a globally important oil crop.Web blotch is one of the most important foliar diseases affecting peanut,which results in serious yield losses worldwide.Breeding web blotch-resistant peanut varieties is the most effective and economically viable method for minimizing yield losses due to web blotch.In the current study,a bulked segregant analysis with next-generation sequencing was used to analyze an F2:3 segregating population and identify candidate loci related to web blotch resistance.Based on the fine-mapping of the candidate genomic interval using kompetitive allele-specific PCR(KASP)markers,we identified a novel web blotch resistance-related locus spanning approximately 169 kb on chromosome 16.This region included four annotated genes,of which only Arahy.35VVQ3 had a non-synonymous single nucleotide polymorphism in the coding region between the two parents.Two markers(Chr.16.12872635 and Chr.16.12966357)linked to this gene were shown to be co-segregated with the resistance of peanut web blotch by 72 randomly selected recombinant inbred lines(RIL),which could be used in marker-assisted breeding of resistant peanut varieties.

Genomic location of Gb1, a unique gene conferring wheat resistance to greenbug biotype F

Greenbug(Schizaphis graminum, Rondani) is a serious insect pest in many wheat growing regions and has been infesting cereal crops in the USA for over a century. Continuous occurrence of new greenbug biotypes makes it essential to explore all greenbug resistant sources available to manage this pest. Gb1, a recessive greenbug resistance gene in DS28A, confers resistance to several economically important greenbug biotypes and is the only gene found to be resistant to greenbug biotype F. A set of 174 F_(2:3)lines from the cross DS28A × Custer was evaluated for resistance to greenbug biotype F in 2020 and 2022. Selective genotyping of the corresponding F_(2) population using single nucleotide polymorphism(SNP) markers generated by genotyping-by-sequencing(GBS) led to the identification of a candidate genomic region for Gb1. Thus, SSR markers previously mapped in this region were used to genotype the entire F2population,and kompetitive allele specific PCR(KASP) markers were also developed from SNPs in the target region.Gb1 was placed in the terminal region of the short arm of chromosome 1A, and its location was confirmed in a second population derived from the cross DS28A × PI 697274. The combined data analysis from the two mapping populations delimited Gb1 to a < 1 Mb interval between 13,328,200 and 14,241,426 bp on1AS.

Molecular cytogenetic analyses of two new wheat-rye 6RL translocation lines with resistance to wheat powdery mildew

Rye(Secale cereale)is a valuable gene donor for wheat improvement,especially for its resistance to diseases.Developing rye-derived resistance sources is important for wheat breeding.In the present study,two wheat-rye derivatives,designated JS016 and JS110,were produced by crossing common wheat cultivar Yangmai 23 with Pakistani rye accession W2A.Using sequential genomic in situ hybridization(GISH)and multicolor fluorescence in situ hybridization(mc-FISH),JS016 and JS110 were identified as a T6BS.6RL translocation line and a T6BS.6BL6RL translocation line,respectively.Ten newly 6RL chromosome arm-specific markers were developed and used to confirm the 6RL translocation.The wheat 55K single-nucleotide polymorphism(SNP)array further verified the molecular cytogenetic identification results above and clarified their breakpoints at 430.9 and 523.0 Mb of chromosome 6B in JS016 and JS110,respectively.Resistance spectrum and allelism test demonstrated that JS016 and JS110 possessed novel powdery mildew resistance gene(s)that was derived from the 6RL translocation but differed from Pm20.Moreover,JS016 and JS110 had better agronomic traits than the previously reported 6RL translocation line carrying Pm20.To efficiently transfer and detect the 6RL translocation from JS016 and JS110,one 6RL-specific Kompetitive allele specific PCR(KASP)marker was developed and validated in high throughput marker-assisted selection(MAS).

Identification of a pleiotropic QTL cluster for Fusarium head blight resistance,spikelet compactness,grain number per spike and thousand-grain weight in common wheat

Simultaneously improving Fusarium head blight(FHB)resistance and grain yield is challenging in wheat breeding.The correlations between spikelet compactness(SC),grain number per spike(GNS),thousandgrain weight(TGW)and FHB resistance remains unclear in common wheat.Identification of major quantitative trait loci(QTL)conferring FHB resistance and yield components,and development of breeder-friendly markers for the QTL are prerequisites for marker-assisted selection(MAS).Here,a recombinant inbred line(RIL)population derived from a cross between a resistant cultivar Yangmai 12(YM12)and a susceptible cultivar Yanzhan 1(YZ1)was used to map QTL for FHB resistance and yield components.A total of 22 QTL were identified;among these,six are likely to be new for corresponding traits.A QTL cluster(Qclu.yas-2D)for FHB type II resistance,SC,GNS,and TGW was detected on chromosome 2D.Breeder-friendly kompetitive allele-specific PCR(KASP)markers flanking the interval of Qclu.yas-2D were developed and validated in a diverse panel of 166 wheat cultivars and advanced lines.The YM12 alleles of Qclu.yas-2D significantly increased FHB resistance,SC,and GNS but decreased TGW in the validation population.The KASP markers developed for Qclu.yas-2D have great potential for breeding high-yielding wheat cultivars with enhanced FHB resistance.

QTL mapping for pre-harvest sprouting in a recombinant inbred line population of elite wheat varieties Zhongmai 578 and Jimai 22

Pre-harvest sprouting(PHS)is one of the serious global issues in wheat production.Identification of quantitative trait loci(QTL)and closely-linked markers is greatly helpful for wheat improvement.In the present study,a recombinant inbred line(RIL)population derived from the cross of Zhongmai 578(ZM578)/Jimai 22(JM22)and parents were phenotyped in five environments and genotyped by the wheat 50 K single-nucleotide polymorphism(SNP)array.Two QTL of germination index(GI),QGI.caas-3A and QGI.caas-5A,were detected,explaining 4.33%–5.58%and 4.43%–8.02%of the phenotypic variances,respectively.The resistant effect of QGI.caas-3A was contributed by JM22,whereas that of QGI.caas.5A was from ZM578.The two QTL did not correspond to any previously identified genes or genetic loci for PHSrelated traits according to their locations in the Chinese Spring reference genome,indicating that they are likely to be new loci for PHS resistance.Four kompetitive allele-specific PCR(KASP)markers K_AX-109605367and K_AX-179559687 flanking QGI.caas-3A,and K_AX-111258240 and K_AX-109402944flanking QGI.caas-5A,were developed and validated in a natural population of 100 wheat cultivars.The distribution frequency of resistance alleles at Qphs.caas-3A and Qphs.caas-5A loci were 82.7%and57.1%,respectively,in the natural population.These findings provide new QTL and tightly linked KASP markers for improvement of PHS resistance in wheat.

Identification and validation of stable quantitative trait loci for yield component traits in wheat

Grain weight and grain number are important yield component traits in wheat and identification of underlying genetic loci is helpful for improving yield.Here,we identified eight stable quantitative trait loci(QTL)for yield component traits,including five loci for thousand grain weight(TGW)and three for grain number per spike(GNS)in a recombinant inbred line population derived from cross Yangxiaomai/Zhongyou 9507 across four environments.Since grain size is a major determinant of grain weight,we also mapped QTL for grain length(GL)and grain width(GW).QTGW.caas-2D,QTGW.caas-3B,QTGW.caas-5A and QTGW.caas-7A.2 for TGW co-located with those for grain size.QTGW.caas-2D also had a consistent genetic position with QGNS.caas-2D,suggesting that the pleiotropic locus is a modulator of trade-off effect between TGW and GNS.Sequencing and linkage mapping showed that TaGL3-5A and WAPO-A1 were candidate genes of QTGW.caas-5A and QTGW.caas-7A.2,respectively.We developed Kompetitive allele specific PCR(KASP)markers linked with the stable QTL for yield component traits and validated their genetic effects in a diverse panel of wheat cultivars from the Huang-Huai River Valley region.KASP-based genotyping analysis further revealed that the superior alleles of all stable QTL for TGW but not GNS were subject to positive selection,indicating that yield improvement in the region largely depends on increased TGW.Comparative analyses with previous studies showed that most of the QTL could be detected in different genetic backgrounds,and QTGW.caas-7A.1 is likely a new QTL.These findings provide not only valuable genetic information for yield improvement but also useful tools for marker-assisted selection.

Identification of genetic loci for grain yield-related traits in the wheat population Zhongmai 578/Jimai 22

The identification of stable quantitative trait locus(QTL)for yield-related traits and tightly linked molecular markers is important for improving wheat grain yield.In the present study,six yield-related traits in a recombinant inbred line(RIL)population derived from the Zhongmai 578/Jimai 22 cross were phenotyped in five environments.The parents and 262 RILs were genotyped using the wheat 50K single nucleotide polymorphism(SNP)array.A high-density genetic map was constructed with 1501 non-redundant bin markers,spanning 2384.95 cM.Fifty-three QTLs for six yield-related traits were mapped on chromosomes 1D(2),2A(9),2B(6),2D,3A(2),3B(2),4A(5),4D,5B(8),5D(2),7A(7),7B(3)and 7D(5),which explained 2.7-25.5%of the phenotypic variances.Among the 53 QTLs,23 were detected in at least three environments,including seven for thousand-kernel weight(TKW),four for kernel length(KL),four for kernel width(KW),three for average grain filling rate(GFR),one for kernel number per spike(KNS)and four for plant height(PH).The stable QTLs QKl.caas-2A.1,QKl.caas-7D,QKw.caas-7D,QGfr.caas-2B.1,QGfr.caas-4A,QGfr.caas-7A and QPh.caas-2A.1 are likely to be new loci.Six QTL-rich regions on 2A,2B,4A,5B,7A and 7D,showed pleiotropic effects on various yield traits.TaSus2-2B and WAPO-A1 are potential candidate genes for the pleiotropic regions on 2B and 7A,respectively.The pleiotropic QTL on 7D for TKW,KL,KW and PH was verified in a natural population.The results of this study enrich our knowledge of the genetic basis underlying yield-related traits and provide molecular markers for high-yield wheat breeding.

Characterization of Rsg2.a3:A new greenbug resistance allele at the Rsg2 locus from wild barley(Hordeum vulgare ssp.spontaneum)

Greenbug(Schizaphis graminum Rondani)is a destructive insect pest that not only damages plants,but also serves as a vector for many viruses.Host plant resistance is the preferred strategy for managing greenbug.Two greenbug resistance genes,Rsg1 and Rsg2,have been reported in barley.To breed cultivars with effective resistance against various greenbug biotypes,additional resistance genes are urgently needed to sustain barley production.Wild barley accession WBDC053(PI 681777)was previously found to be resistant to several greenbug biotypes.In this study,a recombinant inbred line(RIL)population derived from Weskan×WBDC053 was evaluated for response to two greenbug biotypes(E and TX1)and genotyped using genotyping by sequencing(GBS).A set of 3347 high quality GBS-derived single nucleotide polymorphisms(SNPs)were then used to map the greenbug resistance gene in this wild barley accession.Linkage analysis placed the greenbug resistance gene in a 2.35 Mb interval(0-2,354,645 bp)in the terminal region of the short arm of chromosome 2H.This interval harbors 15 genes with leucine-rich-repeat(LRR)protein domains.An allelism test indicated that the greenbug resistance gene in WBDC053,designated Rsg2.a3,is likely allelic or closely linked to Rsg2.GBS-SNPs 2H_1318811and 2H_1839499 co-segregating with Rsg2.a3 in the RIL population were converted to Kompetitive allele specific PCR(KASP)markers KASP-Rsg2.a3-1 and KASP-Rsg2.a3-2,respectively.The two KASP markers can be used to select Rsg2.a3 and have the potential to tag Rsg2 in barley improvement programs.

Establishment and application of an SNP molecular identification system for grape cultivars

We aimed to develop a set of single nucleotide polymorphism(SNP) markers that can be used to distinguish the main cultivated grape(Vitis L.) cultivars in China and provide technical support for domestic grape cultivar protection, cultivar registration, and market rights protection. A total of 517 high-quality loci were screened from 4 241 729 SNPs obtained by sequencing 304 grape accessions using specific locus amplified fragment sequencing, of which 442 were successfully designed as Kompetitive Allele Specific PCR(KASP) markers. A set of 27 markers that completely distinguishes 304 sequenced grape accessions was determined by using the program, and 26 effective markers were screened based on 23 representative grape cultivars. Finally, a total of 46 out of 48 KASP markers, including 22 markers selected by the research group in the early stage, were re-screened based on 348 grape accessions. Population structure, principal component, and cluster analyses all showed that the 348 grape accessions were best divided into two populations. In addition, cluster analysis subdivided them into six subpopulations. According to genetic distance, V. labrusca, V. davidii, V. heyneana, and V. amurensis were far from V. vinifera, while V. vinifera×V. labrusca and V. amurensis×V. vinifera were somewhere in between these two groups. Furthermore, a core set of 25 KASP markers could distinguish 95.69% of the 348 grape accessions, and the other 21 markers were used as extended markers. Therefore, SNP molecular markers based on KASP typing technology provide a new way for mapping DNA fingerprints in grape cultivars. With high efficiency and accuracy and low cost, this technology is more competitive than other current identification methods. It also has excellent application prospects in the grape distinctness, uniformity, and stability(DUS) test, as well as in promoting market rights protection in the near future.

A lignified-layer bridge controlled by a single recessive gene is associated with high pod-shatter resistance in Brassica napus L.

Pod shattering causes severe yield loss in rapeseed(Brassica napus L.)under modern agricultural practice.Identification of highly shatter-resistant germplasm is desirable for the development of rapeseed cultivars for mechanical harvesting.In the present study,an elite line OR88 with strong shatter resistance and a lignified-layer bridge(LLB)structure was identified.The LLB structure was unique to OR88 and co-segregated with high pod-shatter resistance.The LLB structure is differentiated at stage 12 of gynoecium development without any gynoecium defects.Genetic analysis showed that LLB is controlled by a single recessive gene.By BSA-Seq and map-based cloning,the resistance gene location was delimited to a0.688 Mb region on chromosome C09.Transcriptome analysis suggested Bn TCP8.C09 as the gene responsible for LLB.The expression of Bn TCP.C09 was strongly downregulated in OR88,suppressing cell proliferation in the pod valve margin.KASP markers linked to the candidate gene were developed.This pod shatter-resistant line could be used in rapeseed breeding programs by direct transfer of the gene with the assistance of the DNA markers.

QTL mapping of seedling biomass and root traits under different nitrogen conditions in bread wheat(Triticum aestivum L.)

Plant nitrogen assimilation and use efficiency in the seedling's root system are beneficial for adult plants in field condition for yield enhancement.Identification of the genetic basis between root traits and N uptake plays a crucial role in wheat breeding.In the present study,198 doubled haploid lines from the cross of Yangmai 16/Zhongmai 895 were used to identify quantitative trait loci(QTLs)underpinning four seedling biomass traits and five root system architecture(RSA)related traits.The plants were grown under hydroponic conditions with control,low and high N treatments(Ca(NO_(3))_(2)·4H_(2)O at 0,0.05 and 2.0 mmol L^(-1),respectively).Significant variations among the treatments and genotypes,and positive correlations between seedling biomass and RSA traits(r=0.20 to 0.98)were observed.Inclusive composite interval mapping based on a high-density map from the Wheat 660 K single nucleotide polymorphisms(SNP)array identified 51 QTLs from the three N treatments.Twelve new QTLs detected on chromosomes 1 AL(1)in the control,1 DS(2)in high N treatment,4 BL(5)in low and high N treatments,and 7 DS(3)and 7 DL(1)in low N treatments,are first reported in influencing the root and biomass related traits for N uptake.The most stable QTLs(RRS.caas-4 DS)on chromosome 4 DS,which were related to ratio of root to shoot dry weight trait,was in close proximity of the Rht-D1 gene,and it showed high phenotypic effects,explaining 13.1%of the phenotypic variance.Twenty-eight QTLs were clustered in 12 genetic regions.SNP markers tightly linked to two important QTLs clusters C10 and C11 on chromosomes 6 BL and 7 BL were converted to kompetitive allele-specific PCR(KASP)assays that underpin important traits in root development,including root dry weight,root surface area and shoot dry weight.These QTLs,clusters and KASP assays can greatly improve the efficiency of selection for root traits in wheat breeding programmes.

A major and stable QTL for wheat spikelet number per spike validated in different genetic backgrounds

The spikelet number per spike(SNS)contributes greatly to grain yield in wheat.Identifying various genes that control wheat SNS is vital for yield improvement.This study used a recombinant inbred line population genotyped by the Wheat55K single-nucleotide polymorphism array to identify two major and stably expressed quantitative trait loci(QTLs)for SNS.One of them(QSns.sau-2SY-2D.1)was reported previously,while the other(QSns.sau-2SY-7A)was newly detected and further analyzed in this study.QSns.sau-2SY-7A had a high LOD value ranging from 4.46 to 16.00 and explained 10.21-40.78%of the phenotypic variances.QSns.sau-2SY-7A was flanked by the markers AX-110518554 and AX-110094527 in a 4.75-cM interval on chromosome arm 7AL.The contributions and interactions of both major QTLs were further analyzed and discussed.The effect of QSns.sau-2SY-7A was successfully validated by developing a tightly linked kompetitive allele specific PCR marker in an F_(2:3) population and a panel of 101 high-generation breeding wheat lines.Furthermore,several genes including the previously reported WHEAT ORTHOLOG OF APO1(WAPO1),an ortholog of the rice gene ABERRANT PANICLE ORGANIZATION 1(APO1)related to SNS,were predicted in the interval of QSns.sau-2SY-7A.In summary,these results revealed the genetic basis of the multi-spikelet genotype of wheat line 20828 and will facilitate subsequent fine mapping and breeding utilization of the major QTLs.

Cytogenetic identification and molecular marker development for the novel stripe rust-resistant wheat- Thinopyrum intermedium translocation line WTT11

Stripe rust,caused by Puccinia striformis f.sp.tritici(Pst),is one of the most destructive diseases of wheat(Triticum aestivum L)worldwide.Xiaoyan 78829,a partial amphidiploid developed by crossing common wheat with Thinopyrum intermedium,is immune to wheat stripe rust.To transfer the resis-tance gene of this excellent germplasm resource to wheat,the tr anslocation line WTT11 was produced by pollen irradiation and assessed for immunity to stripe rust races CYR32,CYR33 and CYR34.A novel stripe rust-resistance locus derived from Th.intermedium was confirmed by linkage and diagnostic marker analyses.Molecular cytogenetic analyses revealed that WTT11 carries a TTh 2DL translocation.The breakpoint of 1B was located at 95.5 MB,and the alien segments were found to be homoeologous to wheat-group chromosomes 6 and 7 according to a wheat660K single-nucleotide polymorphism(SNP)array analysis.Ten previously developed PCR-based markers were confirmed to rapidly trace the alien segments of WTT11,and 20 kompetitive allele-specific PCR(KASP)markers were developed to enable genotyping of Th.intermedium and common wheat.Evaluation of agronomic traits in two consecutive crop seasons uncovered some favorable agronomic traits in WTT11,such as lower plant height and longer main panicles,that may be applicable to wheat improvement.As a novel genetic resource,the new resistance locus may be useful for wheat disease-resistance breeding.