Mapping stripe rust resistance genes by BSR-Seq:YrMM58 and YrHY1 on chromosome 2AS in Chinese wheat lines Mengmai 58 and Huaiyang 1 are Yr17

*,Zhiyong Liu,*

aCollege of Agronomy and Biotechnology,China Agricultural University,Beijing 100193,China

bState Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China

cCollege of Life Science,Sichuan Agricultural University,Ya'an 625014,Sichuan,China

dCollege of Horticulture,China Agricultural University,Beijing 100193,China

eWheat Research Institute,Henan Academy of Agriculture Sciences,Zhengzhou 450002,Henan,China

1.Introduction

Common wheat is a major food crop and wheat production is continually challenged by several diseases,including stripe rust,stem rust,leaf rust,powdery mildew,and Fusarium head blight.Stripe rust,caused by Puccinia striiformis f.sp.tritici(PST),is one of the most severe of those diseases worldwide[1].Breeding stripe rust resistant cultivars by deployment of effective stripe rust resistance genes is the main strategy for control[2,3].More than 70 stripe rust resistance genes have been formally named and some of them have been widely used in breeding[4–6].However,new virulent pathogen races are a constant threat once resistance genes are deployed[7].For example,Yr9 on the 1RS.1BL translocation became ineffective following emergence of the virulent race CYR29 in the late 1990s[8],and the Yr24/Yr26/YrCH42 was recently overcome by race CYR34(V26)[9].Therefore discovery of effective stripe rust resistance genes and identification of closely linked markers to enable marker assisted selection are essential for continued resistance.

Bulked segregant analysis (BSA) can rapidly identify molecular markers linked to traits of interest[10].Many genetic markers such as restriction fragment length polymorphisms(RFLPs),amplified fragment length polymorphisms(AFLPs),simple sequence repeats(SSRs),and sequence-tagged sites(STSs)linked to disease resistance genes have been used in BSA.However,all of these types of markers have limitations when working with large and highly repetitive plant genomes such as hexaploid wheat due to inadequate density and high levels of duplication.RNA-Seq is an extensively used next-generation sequencing(NGS)technology that not only detects differentially expressed transcripts but also numerous genomic variations in expressed exons,and is relatively inexpensive[11].With development of high-throughput sequencing technologies and protocols,BSR-Seq,an approach combining BSA and RNA-Seq has emerged as a new mapping strategy that offers the promise of rapid discovery of novel genes and genetic markers linked to target genes[12–14].This approach combines the advantages of high-throughput and cost-effectiveness in analysing large genomes.It can also be used to acquire gene expression data.Examples demonstrating the power of BSR-Seq include cloning of the maize glossy13 gene[12,15],mapping of stripe rust resistance gene Yr15 in hexaploid wheat[14],and mapping of high grain protein content gene GPC-B1[13]in tetraploid wheat.

Comparative genomics analyses using genome sequences of Brachypodium,rice and sorghum and the physical map of Aegilops tauschii provide an effective method for gene mapping in wheat.The stripe rust resistance gene Yr18 was mapped in a＜0.50 cM genetic interval using rice and Brachypodium genome sequences[16]and Yr36 was located to a 0.14 cM genetic interval between markers Xucw113 and Xucw111 using the rice collinear region sequences[17].These results thus facilitated map-based cloning and identification of the protein product of an ABC transporter(Yr18/Lr34/Pm38)[18]and a kinase-START(Yr36)protein[17].Using a similar approach,the stripe rust resistance gene Yr26 was located in a genomic interval orthologous to genomic regions Bradi3g28410 to Bradi3g29600 in Brachypodium and Os10g0470700 to Os10g0489800 in rice[19].Since the Ae.tauschii genome has a higher level of micro-collinearity with the wheat A and B subgenomes than any of Brachypodium,rice,and sorghum,Lu et al.[20]mapped the spot blotch resistance gene Sb3 on wheat chromosome 3BS to a 2.15 cM genetic interval using Ae.tauschii chromosome 3DS SNP marker sequences[21].

Mengmai 58 and Huangyang 1 are recently bred wheat lines from the Huang-Huai River Valleys Winter Wheat Zone that are highly resistant to prevailing PST races CYR32,CYR33,and CYR34(V26).In this paper,we report the rapid mapping of stripe rust resistance genes YrMM58 and YrHY1 in bread wheat lines Mengmai 58 and Huaiyang 1 using BSR-Seq and comparative genomic analysis.

2.Materials and methods

2.1.Plant materials

Stripe rust resistant wheat lines Mengmai 58 and Huaiyang 1 obtained from the Huang-Huai River Valleys Winter Wheat Zone were crossed with the highly susceptible wheat variety Nongda 399 and F2:3populations were used for genetic analysis.Mengmai 58 is a selection from Zhoumai 22 and Huaiyang 1 is a derivative of cross 951-99/Aikang 58.Wheat cultivar Jagger was used as a control possessing Yr17[5].

Jagger,Mengmai 58,Huaiyang 1,Nongda 399,and 30 F1plants and more than 200 F2:3progenies from each cross,were evaluated for adult plant stage stripe rust resistance at Sichuan Agricultural University,Ya'an,Sichuan during the 2014–2015 growing season.PST race CYR34(V26)was used for inoculation.To ensure optimal disease responses the susceptible cultivars Nongda 399 and Taichung 29 were planted after every 20 rows as spreaders.About 30 seeds were planted in 1.5 m rows spaced 20 cm apart.

Disease severities(DS)estimating the areas of sporulation on upper leaves were scored across the range of 0–100%[22].Infection types(ITs)were also recorded using a 0–9 scale previously described[23],where ITs 0–3,4–6,and 7–9 were considered to be resistant,intermediate resistant and susceptible,respectively.IT and DS were scored twice at 4-to 5-day intervals when DS on Nongda 399 and Taichung 29 reached approximately 90%–100%.

2.2.BSR-Seq

Leaf tissues from 20 homozygous resistant and 20 homozygous susceptible F2:3families from Nongda 399/Mengmai 58,and 40 homozygous resistant and 40 homozygous susceptible F2:3families from Nongda 399/Huaiyang 1,respectively,were collected from seedlings for preparation of resistant and susceptible RNA bulks.The RNA samples were sequenced on anIllumina HiSeq4000platform.Sequence quality was controlled using software Trimmomatic v0.32[24].RNA reads of the resistant and susceptible bulks were aligned to the draft assembly(IWGSC)of the wheat genome survey sequence(http://www.wheatgenome.org/)usingsoftware STAR v2.4.0j[25].SNP and Indels were called using software GATK v3.2-2 with its module “Haplotype Caller”applied[26].

2.3.DNA isolation

Genomic DNA was extracted from seedling leaf tissue from each F2:3family of the two crosses using the CTAB method[27].Based on infection type and disease severity data,20 homozygous resistant and 20 homozygous susceptible F2:3lines in each cross were used to construct resistant and susceptible DNA bulks for SNP marker validation.

2.4.PCR amplification and electrophoresis

PCR was performed in a Thermal Cycler(Bio-Rad)in 20 μL total volumes as follows:denaturation for 5 minat 94°C,followed by 35 cycles of 40 s at 94 °C,40 s at 60 °C,1 min at 72 °C and a final extension for 7 min at 72°C.The PCR products were sequenced either by the Sanger method to validate true polymorphisms or separated by electrophoresis on 8%non-denaturing polyacrylamide gels(39 acrylamide/1 bisacrylamide,w/w)to detect differences in band size using silver staining.

2.5.SNP and STS markers development

Single nucleotide polymorphisms(SNPs)associated with stripe rust resistance identified by BSR-Seq analysis were selected for marker development and validation.The flanking sequences of candidate SNPs were used as templates for PCR primer design using Primer3web(http://primer3.ut.ee/).

Ae.tauschii extended SNP marker sequences homologous to the stripe rust resistant genetic region were used as queries for BLAST search of Chinese Spring contigs generated by the International Wheat Genome Sequencing Consortium(IWGSC,http://www.wheatgenome.org/)and 454 shotgun sequences[28].The sequences were used as templates to design sequence-tagged site (STS)primer pairs with Primer3web.

The designed SNP and STS primers were screened for polymorphisms between the parental lines,as well as the resistant and susceptible DNA bulks.Polymorphic STS markers were used to genotype the segregating populations.

2.6.Statistical analysis and genetic linkage map construction

Chi-squared(χ2)tests were used to evaluate goodness-of-fit of observed and expected segregation ratios.The polymorphic SNP and STS markers were used to construct a genetic linkage map with the Map Draw 2.1 software[29].

3.Results

3.1.Genetic analyses of stripe rust resistances in Mengmai 58 and Huaiyang 1

Jagger(Yr17),Mengmai 58,Huaiyang 1,and the F1plants were highly resistant(IT 0),whereas Nongda 399 and Taichung 29 were highly susceptible(IT 9)(Table 1).The data indicated that resistance in both crosses was conferred by single dominant genes,which were designated YrMM58 and YrHY1.

3.2.BSR-Seq analysis

The BSR-Seq approach was applied to map the stripe rust resistance genes YrMM58 and YrHY1.One hundred and fifty bp length mode RNA-Seq bulks of MM58R,MM58S,HY1R,and HY1S produced 40,488,082,40,562,867,52,941,318,and 40,869,720 raw reads pairs,respectively.Less than 1%of the raw reads pairs were filtered for each bulk after quality control.During read mapping,we found that 81%,72%,84%,and 85%of the filtered read pairs were uniquely mapped for bulks MM58R,MM58S,HY1R,and HY1S,respectively.Subsequent SNP calling identified 128,502 high-quality variants(SNPs and Indels)between bulks MM58R and MM58S,and 131,825 between bulks HY1R and HY1S.Finally,at the cut off of allele frequency difference(AFD)＞0.8 and Fisher's Exact Test P-value＜1e?10,19 SNPs were found to be associated with the YrMM58 resistance,and 24 SNPs were associated with YrHY1 resistance (Fig. 1). Twelve of the YrMM58-associated SNPs and 14 of the YrHY1-associated SNPs were located in 14.0 Mb and 15.9 Mb distal regions of chromosome 2AS,suggesting that YrMM58 and YrHY1 were located in that region.

3.3.Candidate SNP validation and genetic mapping

The YrMM58-and YrHY1-associated SNPs on 2AS were validated for polymorphisms between the parental lines as well as contrasting resistant and susceptible DNA bulks.Two SNP markers,WGGB191 and WGGB196 showed clear polymorphisms between the parental lines as well as the resistant and susceptible DNA bulks(Fig.2).SNP markers showing linkage with the rust resistance in 20 resistant and 20 susceptible F2:3families were then genotyped in the entire Nongda 399/Mengmai 58 and Nongda 399/Huangyang 1 F2:3families usingSanger sequencing to construct genetic linkage maps of stripe rust genes YrMM58 and YrHY1(Fig.3).Both YrMM58 and YrHY1 were located on the distal end of chromosome arm 2AS.

Table 1–Stripe rust responses to PST race CYR34 of parents,F1and F2:3lines from crosses Nongda 399/Mengmai 58 and Nongda 399/Huaiyang 1.

Fig.1–Distribution of candidate SNPs within wheat chromosomes.

3.4.Comparative genomics analysis with Ae.tauschii and STS marker development

The contig sequences of SNP markers WGGB191 and WGGB196 were used to search the Ae.tauschii SNP marker extended sequences database.WGGB191 and WGGB196 are orthologous to Ae.tauschii SNP markers AT2D1146 and AT2D1108,respectively.From the distal end of the chromosome to AT2D1108,there are 36 SNP markers(AT2D974 to AT2D1108)in the terminal region of Ae.tauschii chromosome arm 2DS.Low level synteny was observed for the corresponding genomic regions between Ae.tauschii and Brachypodium,rice and sorghum(Table 2).Therefore,only the Ae.tauschii sequence information was used for further comparative genomic analysis and marker development.

The Ae.tauschii SNP marker(AT2D974 to AT2D1108)extended sequences were used to search the Chinese Spring IWGSC chromosome 2AS sequences (https://www.wheatgenome.org/)and Chinese Spring CS42 TGAC v1 WGS sequence assembly (https://wheatis.tgac.ac.uk/grassrootsportal/blast)to develop STS markers linked to the stripe rust resistance genes.Ten new polymorphic STS markers were developed and genotyped in the two F2:3populations to construct genetic linkage maps of YrMM58 and YrHY1(Table 3).Finally,YrMM58 and YrHY1 were located to the terminal region of wheat chromosome arm 2AS and were respectively 7.7 cM and 3.8 cM distal to STS marker WGGB148(Fig.3).

Fig.2–Sanger sequencing profiles of SNP markers WGGB191 and WGGB196 in homozygous resistant(R),homozygous susceptible(S),and heterozygous F2:3families(H).

Fig.3–Comparative genetic linkage maps of YrMM58,YrHY1,and other stripe rust genes on chromosome 2AS.(a)Genetic linkage map of YrMM58;(b)genetic linkage map of YrHY1;(c)Ae.tauschii 2DS SNP linkage map corresponding to the YrMM58 and YrHY1 genetic region;(d)integrated genetic linkage map of stripe rust resistance genes Yr17,Yr69,YrR61,Yr56,and YrSph on wheat chromosome 2AS.

4.Discussion

Genetic analyses indicated that a single dominant gene was responsible for the stripe rust resistances in wheat lines Mengmai 58 and Huaiyang 1.In combination with BSR-Seq and comparative genomics analysis,both stripe rust resistance genes YrMM58 and YrHY1 were mapped to the distal end of wheat chromosome 2AS.Five STS markers,WGGB148,WGGB152,WGGB159,WGB171,and WGGB176,closely linked to YrMM58 and YrHY1 generated the same amplification patterns in Mengmai 58,Huangyang 1,and Jagger(Fig.4).However,two STS markers,WGGB179 and WGGB186,further away from YrMM58 and YrHY1 than the above five markers showed different amplification patterns between Mengmai 58,Huangyang 1 and Jagger(Fig.4).We also tested Yr17-linked SSR markers Xgwm636 and Xgwm359[30],and SSR markersXbarc124,Xbarc212,Xgwm512,andXgwm372locatedon chromosome arm 2AS on Mengmai 58,Huangyang 1 and Jagger.The same amplification patterns were obtained for all SSRs in the three genotypes.These results suggested that Mengmai 58,Huangyang 1,and Jagger might contain the same sequences from WGGGB176 to the distal end of chromosome 2AS.Since VPM 1 and its derivatives have been widely used in wheat breeding programs in China in recent years,and VPM 1 carries Yr17 in a wheat-Aegilops ventricosa translocation[31],stripe rust resistance genes YrMM58 and YrHY1 are most likely Yr17.

Table 2–Comparative genomics analysis among Ae.tauschii extended SNP marker sequences,Brachypodium,rice,sorghum genome sequences and new STS markers.

Table 3–SNP and STS markers developed for mapping YrMM58 and YrHY1.

The 12 SNP and STS markers in the YrMM58 and YrHY1 genetic linkage maps on wheat chromosome 2AS showed the same order as their 11 corresponding orthologous SNP markers on Ae.tauschii chromosome 2DS,indicating a high level micro-collinearity between the two subgenomes(Fig.3).WGGB148,the STS marker most closely linked to YrMM58 and YrHY1,was developed from a Chinese Spring chromosome 2AS contig orthologous to a SNP marker AT2D974 extended sequence.However,the genetic distances between WGGB148 and stripe rust resistance genes YrMM58 and YrHY1,as well as the physical end position of AT2D974 on the Ae.tauschii chromosome 2DS physical map[21]reveals that the VPM 1 segment(Yr17)in wheat is probably an extension to the corresponding Ae.tauschii 2DS.This is in agreement with the VPM 1 chromosome segment translocation derived from Ae.ventricosa.

The efficiency and power of BSR-Seq was recently demonstrated in mapping plant genes.This technology provides not only the chromosome position of a target gene but also many associated SNPs for marker development[14].In the present study about 60%of the stripe rust resistance-associated SNPs were located in a 14.0–15.9 Mb distal region of chromosome 2AS(Fig.1).Two SNP markers WGGB191 and WGGB196 linked to YrMM58 and YrHY1 were then developed using the associated SNPs.

We used comparative genomics analysis of Ae.tauschii to develop 10 new STS markers to construct high-density genetic maps of YrMM58 and YrHY1.Our results also showed a consistent gene order between corresponding genetic regions of wheat chromosome 2AS and Ae.tauschii chromosome 2DS,suggesting a high level of micro-collinearity between the two subgenomes.Similar results were also reported when mapping powdery mildew resistance gene MlHLT[32]and spot blotch resistance gene Sb3[20].Thus,comparative genomics analysis using the high-density SNP Ae.tauschii linkage map and genomic sequences[21,33]may be more effective than using Brachypodium,rice and sorghum genomic resources to develop markers for high-density genetic linkage map construction in wheat.Prospectively,the most recently released IWGSC Chinese Spring WGS assemblies (http://www.wheatgenome.org/)will be increasingly informative for fine mapping and map-based cloning in wheat.

5.Conclusions

Combining BSR-Seq with comparative genomics analyses,stripe rust genes in wheat lines Mengmai 58 and Huaiyang 1 were precisely and rapidly mapped on the distal end of chromosome 2AS.The markers developed in the current research provide useful information for marker-assisted selection of stripe rust resistance genes YrMM58 and YrHY1 which proved to be Yr17.

Fig.4–PCR amplification patterns of STS markers in Mengmai 58,Huaiyang 1,Nongda 399,and Jagger(Yr17).

This work was financially supported by the National Key Research and Development Program of China(2016YFD0101802).

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