Identification and fine mapping of PmNJ3946 for powdery mildew resistance in einkorn wheat

Peisi Wng, Jun Hung, N Li, Jie Zhng, Cimei Gu, Yng Yun, Ziruo Wen, Hiyn Ji,b,Zhongxin Kong,b, Zhengqing M,b,*

a Crop Genomics and Bioinformatics Center and National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095,Jiangsu, China

b Zhongshan Biological Breeding Laboratory, Nanjing 210014, Jiangsu, China

Keywords:Powdery mildew Disease resistance Genetic mapping Triticum monococcum PmNJ3946

ABSTRACT Powdery mildew caused by Blumeria graminis f.sp.tritici(Bgt)is a destructive wheat disease.Although it can be easily overcome by deployment of resistance genes, the resistance is often quickly compromised by pathogen virulence.Thus,exploration and characterization of new resistance genes is always ongoing.Line NJ3946 derived from a cross of einkorn wheat accessions TA2032 and M389 showed resistance to powdery mildew.Inheritance analysis of an F2 population derived from a cross of NJ3946 and M389 suggested that the resistance was conferred by a dominant allele.With polymorphic markers identified through bulked segregant analysis (BSA), this gene was mapped to a novel locus on chromosome 3A,and was designated as PmNJ3946.Bulked segregant RNA-seq analysis(BSR-seq)was conducted to obtain more closely linked markers,which allowed delimitation of the PMNJ3946 locus to a 0.9 cM interval covering a physical distance of less than 1 Mb.PMNJ3946 was flanked by Xwgrc5153 and SNP-derived marker CHS21_3A008915069, and co-segregated with SNP-derived markers CHS21_3A008939814 and CHS21_3A008943175.The PmNJ3946 discovery expands the diversity of powdery mildew resistance genes and is useful for wheat breeding.

1.Introduction

Powdery mildew caused by the biotrophic fungus Blumeria graminis f.sp.tritici(Bgt)is a severely damaging disease of wheat(Triticum aestivum L.).Deployment of resistant cultivars is the most economic and effective method of control.Wheat breeders often rely on resistance genes that confer qualitative resistance(R genes)in breeding.However, a disadvantage of extensive R-gene deployment is rapid loss of effectiveness due to the emergence and increase of virulent races [1], for example, the losses of resistance associated with Pm2 and Pm8 [2,3].Thus, unearthing and utilizing new resistance genes is a constant task to meet breeding requirements.

Powdery mildew resistance can be under the control of genes with qualitative or quantitative effects, or both.At present, more than 100 R genes derived from common wheat or its relatives,distributed at 62 designated loci (PM1-PM69, excluding PM18, PM22,PM23, PM31, PM44, PM48, and PM17 as duplicates of other named genes or lacking formal publication) and dozens of temporarily designated or non-designated genes have been reported [4–7].Two or more powdery mildew R genes have been identified on most wheat chromosomes except 6D with only one gene and 3A,3D and 4D with none.Particularly,at least six loci for powdery mildew resistance are probably present on chromosome 7AL [8–27].Moreover, some resistance genes contain different functional alleles, such as Pm1, Pm3, and Pm60 [9,26–29], which probably provides different resistance spectrum to Bgt isolates and expands the resistance gene diversity.Apart from the qualitative resistance genes, more than 100 quantitative loci conferring resistance to powdery mildew have been identified so far [30].

R genes have been mainly targeted in wheat breeding.Although a single R gene can provide a high degree of resistance, it usually lacks durability as virulent Bgt strains inevitably follow.However,a few researches have demonstrated that combinations of different R genes can enhance resistance and prolong durability.For example, wheat lines SN0293-2 and SN0293-7 carrying Pm2,Pm52 and PmSN0293 showed high resistance at all growth stages[31].Common wheat lines D57, Liangxing 99, and AL69, emmer wheat Khapli (T.dicoccum), and T.boeoticum accession pau5088,which are highly resistant to powdery mildew, all possess two or more resistance genes [22,32–35].With the help of transgenic technology,pyramiding of four Pm3 alleles enhanced powdery mildew field resistance as well [36].Therefore, expanding the R gene repertory is desirable for wheat improvement.

Wheat relatives are valuable gene resources for wheat breeding.As the major contributors, the closely related wheat species from diploid to hexaploid, including Aegilops speltoides, are donors of 65 reported powdery mildew resistance genes [4,7,37], of which 12 are from the diploid A genome species T.monococcum L.and T.urartu L.The close relatives are preferred in gene exploitation because the genes can be readily transferred to wheat through sexual cross and homologous recombination.In this study, we found that NJ3946,a recombinant inbred line derived from a cross of cultivated einkorn wheat (Triticum monococcum L.subsp.monococcum) accession TA2032 with T.monococcum accession M389,showed a high level of resistance to powdery mildew in fields.Here, we report the identification and fine mapping of a novel dominant resistance gene in this line.

2.Materials and methods

2.1.Plant materials

Triticum monococcum L.subsp.monococcum accession TA2032(PI 191097) was obtained from Dr.B.S.Gill, Wheat Genetics Resource Center,Kansas State University.NJ3946 is an F8recombinant inbred line derived from the cross of TA2032 with susceptible einkorn wheat M389.A population of 497 F2plants, derived from NJ3946 × M389, was used in gene mapping.Susceptible bread wheat cultivar Sumai 3 was used for propagation of Bgt isolates.

2.2.Resistance evaluation

Seedlings were grown in rectangular trays in a growth chamber with 14 h light, 80% humidity, and a 22 °C/18 °C day/night cycle.NJ3946,M389 and the F2population were inoculated with Bgt isolate Bgt38 at the one-leaf stage.A few plants of Sumai 3 were randomly planted in each tray as the susceptible control.Resistance was investigated when all the control plants showed susceptibility,which usually takes about 5–7 d.Disease response was scored according to a scale of 0–5, with 0 for no visible symptom, or 1 and 2 for visible necrosis without sporulation or sparse sporulation(rated as resistant) and 3 to 5 for moderate to abundant sporulation rated as susceptible [29].F2:3progeny test was performed for some key F2recombinants for accurate mapping.

2.3.Bulk segregant analysis

Bulked segregant analysis (BSA) was used in initial marker screening [38].Separate DNA bulks were made separately with six resistant and six susceptible F2plants.Markers mapping to the diploid A genome(https://wheat.pw.usda.gov/GG3/),including those prefixed with BARC[39],GWM[40],WMC(wheat.pw.usda.-gov), CFA [41], CFD [42], and MAG [43], were tested for polymorphism.PCR product digestion and separation followed routine laboratory procedures [43,44].

2.4.RNA-seq and SNP calling

Seedling leaf samples 48 h post Bgt inoculation were bulked separately from 12 homozygous-resistant F2plants and 12 homozygous-susceptible F2plants, which were identified according to genotypes determined by the initially mapped molecular markers, and were used in RNA preparation for bulked segregant RNA-seq (BSR-seq) [45].Both resistant and susceptible bulks had two biological replicates.Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacture’s protocol.Sequencing was performed on an Illumina X-ten platform (Novogene Bioinformatics Technology Co., Ltd., Beijing).The 150-bp paired-end reads were mapped to the Chinese Spring(CS) genome v2.1 [46] with Hisat2 [47] for SNP calling with Genome Analysis Tool Kit 3.8.0 (software.broadinstitute.org/gatk/).The total effective counts per sample ranged from 13 to 17 million.SNPs with a sequencing depth of ≥5 were kept for ΔSNP-indexestimation, where ΔSNP-index=|SNP-index (susceptible)-SNP-index (resistance)|.To avoid SNP omission due to genome diversity between the hexaploid and diploid wheat, the reads were also mapped to the durum wheat cultivar Svevo genome[48].Mapping of the RNA-seq reads to the 10+genomes[49]were also performed but did not produce more information.

2.5.Marker design

SNPs in the target interval were used to design cleavage amplification polymorphism sequence (CAPS) or degenerate cleavage amplification polymorphism sequence (dCAPS) markers.Simple sequence repeats(SSR)in the corresponding intervals of the durum wheat genome, defined by positions of flanking markers, were identified in the MISA-web (webblast.ipk-gatersleben.de/misa/index.php?action=1) for additional marker development.Primers were designed using the primer design tool at NCBI.Primer information for critical markers is presented in Table 1.

2.6.Linkage analysis

Chi squared tests were used to determine the goodness-of-fit of segregation data to postulated genetic ratios.A genetic map was constructed using Mapmaker/EXP 3.0 with the Kosambi mapping function on [50].The map was drawn with MapDraw Version 2.1[51].New gene and locus nomenclature followed the updated guidelines [52].

2.7.Candidate gene identification and micro-collinearity analysis

Genes annotated in the corresponding intervals of hexaploid wheat CS (IWGSC RefSeq v2.1) and durum wheat cultivar Svevo(Svevo RefSeq Rel.1.0)were used for candidate gene identification based on expression changes and sequence variations revealed in the RNA-seq data.Micro-collinearity analysis was conducted using the annotated genes that are located in the target interval and were expressed in Bgt-induced seedling leaves.

3.Results

3.1.Inheritance of the powdery mildew resistance in line NJ3946

Line NJ3946 and TA2032 showed immunity to powdery mildew in fields and was immune to isolate Bgt38, a locally predominant Bgt race, in evaluation in a growth chamber (Fig.1A).When challenged with Bgt38 at the one-leaf stage, the resistance response in the F2population derived from the cross of NJ3946 with susceptible M389 displayed a discrete distribution.Of 497 plants, 381 were resistant (with a response score of 0–2), and the remaining were susceptible (with a response score of 4–5), fitting a 3:1 ratio(χ2= 0.644, P1dfgreater than 0.25), indicating that resistance was conferred by a single dominant gene.

Table 1 Information of the newly developed key markers in this study.

Fig.1.Primary mapping of PmNJ3946.(A)Resistance of NJ3946,TA2032 and M389 to Bgt isolate Bgt38 in a growth chamber.Sumai 3 is the susceptible control.(B)Linkage map of PmNJ3946,numbers to the left indicate genetic distance in cM,the top points to the telomere.(C)Amplification patterns of flanking markers BARC294 and BARC012 in TA2032 (1); NJ3946 (2); M389 (3); resistant pool (4), and susceptible pool (5).Arrows indicate the polymorphic bands linked to PmNJ3946.Numbers to the left represent molecular sizes (bp); M, pUC19/MspI marker.

3.2.Primary mapping of PmNJ3946

In BSA,four of 183 surveyed SSR markers,GPW1106,BARC012,BARC294 and BARC310 (all distributed on chromosome 3AS),detected polymorphism between the parents and the bulks.Genotyping the F2population with these four markers showed that they were all associated with powdery mildew resistance in NJ3946.The resistance gene was located in a 13.6–cM interval flanked by Xbarc294 and Xbarc012(Fig.1B).Since no previously reported powdery mildew resistance gene was mapped to that region,the resistance locus was temporarily named PMNJ3946.The polymorphic patterns displayed by BARC294 and BARC012 among the parents and pools clearly showed that the resistance gene PmNJ3946 was derived from TA2032 (Fig.1C).

3.3.Fine mapping of PmNJ3946

Of a few SSR markers designed based on the durum wheat genome sequence(Svevo RefSeq Rel.1.0)covering the Xbarc310–Xbarc012 interval, WGRC5174, which was polymorphic between the resistant and susceptible pools, was placed between PMNJ3946 and Xbarc012.PMNJ3946 was consequently confined to a smaller region delimited by Xbarc294 and Xwgrc5174.

Fig.2.Genome distribution of SNPs mined from BSR-seq.Top,number of SNPs per chromosome; bottom, number of SNPs in different physical intervals of chromosome 3A, by reference to Chinese Spring RefSeq v2.1.

BSR-seq was then conducted to develop more closely linked markers.By mapping pool reads to the CS reference genome(v2.1)and SNP calling,5786 SNPs with a depth of ≥5 were identified.Most of the 1319 SNPs with a ΔSNP-index ≥0.8 were distributed on chromosome 3A, and mainly congregated in the terminal 20 Mb region of the short-arm(Fig.2),consistent with the physical locations of Xbarc294 and Xwgrc5174.Seven additional markers located in the Xbarc294-Xwgrc5174 interval were then designed based on four expressed genes with SNPs and three genes expressed only in the susceptible pool.They were all in close linkage with PMNJ3946 that was eventually confined to a 0.9 cM interval encompassed by CHS21_3A008915069 and Xwgrc5153 and co-segregated with markers CHS21_3A008939814 derived from the TraesCS3A03G0021500 homolog and CHS21_3A008943175 derived from the TraesCS3A03G0021700 homolog (Fig.3A).

3.4.Candidate gene analysis

The 0.9 cM PMNJ3946 interval in T.monococcum corresponds to a 702-kb sequence in durum wheat cultivar Svevo and a 915-kb sequence in the CS genome and shows perfect collinearity with both genomic blocks(Fig.3B,C).It possesses eight genes expressed in the Bgt-infected leaf tissues (Fig.3B).These genes were not expressed differentially between the resistant and susceptible pools and did not show changes in splicing pattern.Moreover,six of them were monomorphic in the CDS regions and were excluded as the PmNJ3946 candidates.SNPs were present only in homologs of TRITD3Av1G002600 (TraesCS3A03G0021500 in CS)and TRITD3Av1G002590 (TraesCS3A03G0021700 in CS) and cosegregated with powdery mildew response.Therefore, they were possible PmNJ3946 candidates.TRITD3Av1G002600 encodes a WVD2-like protein and TRITD3Av1G002590 encodes a dephospho-CoA kinase.

4.Discussion

In this study, we identified a novel powdery mildew resistance gene temporarily named PmNJ3946 in line NJ3946, a derivative of cultivated einkorn wheat accession TA2032.The PMNJ3946 locus was close to the telomere of chromosome arm 3AS and far from a minor QTL for powdery mildew resistance identified in a bread wheat Forno × spelt variety Oberkulmer population, and a QTL mapped in a RE714×Festin population[53,54],but could be close to a minor 3AS QTL mapped in a Saar×Avocet population[55].We concluded that PmNJ3946 is a previously unreported powdery mildew resistance gene.Chen et al.reported Pm44 on chromosome 3AS in a draft manuscript [56], but the paper was not formally published.

Einkorn wheats are valuable genetic resources for powdery mildew resistance.Up to now, 12 powdery mildew resistance genes have been identified in accessions of T.monococcum L.subsp.aegilopoides, T.monococcum L.subsp.monococcum, and T.urartu.Except for Pm25 and TmPm3 on chromosome 1A [57,58], Pm4d on 2A[59],and pm2026 on 5A[60],all others are in the PM1 cluster on 7A [9,13,14,22,25,29].The discovery of PmNJ3946 expands the diversity of powdery mildew resistance genes and is a valuable supplement to the powdery mildew resistance gene pool.

We narrowed PMNJ3946 down to a less than 1 Mb region by referring to the durum and common wheat genomes.With high collinearity between the PMNJ3946 interval and corresponding sequences in durum wheat cultivar Svevo and common wheat Chinese Spring,we conducted a candidate search taking advantage of the BSR-seq results.Among eight expressed genes, only TraesCS3A03G0021500 and TraesCS3A03G0021700 had SNPs between the resistant and susceptible DNA pools,and were identified as potential gene candidates.However, their known functions are not related to powdery mildew resistance.The WVD2-like protein encoded by TraesCS3A03G0021500 is currently only known for regulating plant and organ development [61,62] and the dephospho-CoA kinase encoded by TraesCS3A03G0021700 is a nuclear-localized protein involved in epigenetic regulation of gene expression in adaptation to environmental changes [63].We cannot exclude the possibility that a PmNJ3946 homolog was not present in the Svevo and CS genomes.Indeed, there are indels and sequence variations between the resistant and susceptible lines in the region surrounding PMNJ3946.For instance, homologous transcripts of TraesCS3A03G0019900, TraesCS3A03G0020000,TraesCS3A03G0029200, and TraesCS3A03G0030200 were present only in the susceptible pool and markers designed for the corresponding genes displayed presence-absence polymorphism.A higher-resolution genetic map and PMNJ3946 interval assembly are required to clarify this issue.

Fig.3.PmNJ3946 fine mapping.(A)Mapping with markers developed based on sequence variations in the expressed genes.(B,C)Alignment of the linked markers with genes annotated in chromosome 3A of durum wheat cv Svevo and chromosome 3A of CS.In(A),numbers to the left indicate genetic distance in cM,the top points to the telomere.In(B) and (C), numbers to the left indicate physical positions in Mb.All annotated genes expressed in Bgt-induced seedling leaves in the PMNJ3946 interval are shown.

Up to now, seven genes from T.monococcum and T.urartu,including Pm1b, Pm25, PmNCA4, PmNCA6, Pm4d, Pm60 and pm2026,have been transferred to tetraploid and/or common wheat and displayed the expected powdery mildew resistance[9,59,60,64,65].The discovery of PmNJ3946 enriches the powdery mildew resistance gene pools and its utilization is facilitated by the availability of closely linked markers.

CRediT authorship contribution statement

Peisi Wang:Investigation,Writing– original draft.Jun Huang:Data curation,Investigation.Na Li:Data curation,Investigation.Jie Zhang:Data curation, Investigation.Caimei Gu:Data curation,Investigation.Yang Yuan:Software, Funding acquisition.Ziruo Wen:Data curation, Investigation.Haiyan Jia:Supervision, Funding acquisition.Zhongxin Kong:Project administration.Zhengqiang Ma:Funding acquisition, Supervision, Writing – review &editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was partially supported by the‘JBGS’Project of Seed Industry Revitalization in Jiangsu Province (JBGS (2021) 013),National Natural Science Foundation of Jiangsu Province for Young Scholars (BK20170176), National Natural Science Foundation of China(30771165),the Ministry of Science and Technology of China(2016YFD0101004),and Jiangsu Collaborative Innovation Initiative for Modern Crop Production.We are grateful to Dr.R.A.McIntosh,University of Sydney, Australia for reviewing the article.