Xingyng Xu,Dolores Mornhinweg,Amy Bernro,Genqio Li,Ruolin Bin,Brin J.Steffenson,Guihu Bi

a USDA-ARS Wheat,Peanut,and Other Field Crop Research Unit,Stillwater,OK 74075,USA

b USDA-ARS Hard Winter Wheat Genetics Research Unit,Manhattan,KS 66506,USA

c Department of Plant Pathology,Kansas State University,Manhattan,KS 66506,USA

d Department of Plant Pathology,University of Minnesota,St.Paul,MN 55108,USA

Keywords:

ABSTRACT 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_1318811 and 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.

1.Introduction

Barley(Hordeum vulgare L.)is one of the most widely grown crops in the world and is ranked fourth in world production among food crops in 2017[1].The crop has a number of important uses,including malt production for beer and spirits,feed for animals and also food for human consumption[2].Barley is often cultivated in marginal areas with few additional inputs.Thus,it is important to protect the crop from biotic constraints such as insects to secure sustainable and economic production.

Greenbug(Schizaphis graminum Rondani)is one of the most important insect pests in US barley production,with large scale outbreaks occurring every 5-10 years in the southern Great Plains[3-5].Greenbug feeding causes chlorosis,necrosis and even death of plants when they are heavily infested.Although outbreaks occur only occasionally,yield losses can be huge in an outbreak year without expensive pesticide applications.

Growing greenbug resistant barley is the preferred approach for insect control because it reduces production costs and eliminates contamination from pesticide applications.Although considerable efforts have been made to identify sources of resistance,only two barley greenbug resistance genes,Rsg1 and Rsg2,have been identified.Rsg1 was found in barley cultivar‘Post 90’,a reselection of barley cultivar‘Post’derived from the cross Harrison×Will with Will as the donor of the resistance gene[6,7].Rsg2 was first identified in Jao(PI 426756),a spring barley landrace collected from Sindh,Pakistan in 1976.Two breeding lines carrying Rsg2,STARS1501B and STARS1502B,were released in the USA in 2018[8].Previous studies indicated that Rsg2 was non-allelic to Rsg1[9],and that the two genes responded differently to greenbug biotypes TX1,WY10MC,WY81,and WY10B[10,11].Rsg1 was mapped to an 8.4-cM interval in the terminal region of chromosome arm 3HL using SSR and SNP markers[12]and has been used in barley production.However,the genomic location of Rsg2 remains unknown.

Rsg1 and Rsg2 confer resistance to some economically important greenbug biotypes.However,new virulent greenbug biotypes continuously occur.For example,Armstrong et al.[11]identified six new greenbug biotypes in Wyoming alone,including WY81,WY10MC,WY10B,WY12 MC,WY86 and WY4.Among them,biotype WY81 is virulent to Rsg1,while WY10MC and WY10B are virulent to Rsg2.Therefore,new greenbug resistance genes are urgently needed in barley breeding pipelines to manage greenbug.

Wild barley(Hordeum vulgare ssp.spontaneum)is the progenitor of cultivated barley and originated in the Fertile Crescent region.Today,its range extends from North Africa in the west,across Central Asia and into Southern Asia.Wild barley possesses abundant genetic variation and is considered an important source of biotic and abiotic stress resistance.To identify new genes for disease resistance in barley breeding,Steffenson et al.[13]assembled the Wild Barley Diversity Collection(WBDC),comprised of 318 accessions from the Fertile Crescent,Central Asia,North Africa,and the Caucasus regions.The WBDC accessions were screened for resistance to a set of greenbug biotypes by the USDA-ARS,Stillwater,OK,and only a few accessions were found resistant.One of these accessions(WBDC053)exhibited a unique response profile to a suite of greenbug biotypes.This accession is resistant to greenbug biotypes B,C,E,I,TX1,WY4A,WY4B,WY81,WY12MC,and WY86 and susceptible to F,H,WY10MC,and WY10B[11].Therefore,WBDC053 may carry new greenbug resistance gene(s).The objectives of this study were to characterize the greenbug resistance gene in WBDC053 and develop genomic tools for its introgression into elite barley cultivars.

2.Materials and methods

2.1.Mapping population

A set of 154 F5:6recombinant inbred lines(RILs)were developed from the cross Weskan×WBDC053 using single seed descent and used in this study.WBDC053(PI 681777)is a wild barley accession originally collected in the Baluchistan region of Pakistan.It was derived from a single plant selection and selfed five times at University of Minnesota,St Paul,MN,USA[14].Weskan,a winter barley cultivar with a pedigree of Purdue 6515A2/KY 66-7-63-1294,was released by the Kansas State University Experiment Station in 1990 and is susceptible to greenbug.

2.2.Evaluation of RILs for responses to greenbug biotypes E and TX1

WBDC053,Weskan and all RILs were evaluated for responses to greenbug biotype E at the USDA-ARS Wheat,Peanut,and Other Field Crop Research Unit,Stillwater,OK in 2020 and 2021.Greenbug biotype E,collected on wheat in Bushland,TX,USA,in 1982[15],is a dominant biotype that can be increased rapidly in greenhouses in the Great Plains of the USA.The experiment was conducted in a greenhouse with supplemental light of 14 h per day at 22±5°C.Greenbug biotype E was increased on susceptible barley cultivar Eight-Twelve(PI 537437).A randomized complete block design with two replicates was used.Screening flats were 152 cm long and 104 cm wide with 8 cm-diameter hills spaced 8 cm apart in the formation of 60 hills(6×10)per flat.For each RIL,six seeds were planted in each of two consecutive hills in each replicate,and the two parents were planted in each flat as resistant(WBDC053)and susceptible(Weskan)controls.Flats were infested as soon as seedlings emerged by laying heavily infested leaves from culture pots of greenbug biotype E between the rows.Plants were rated approximately two weeks after infestation when the susceptible checks had died.The dead plants were classified as susceptible,and the normal plants were classified as resistant.

WBDC053 also provides resistance to a few other biotypes[11].To confirm the assumption that a single gene in WBDC053 conditions resistance to different biotypes,we evaluated the responses of the RIL population to biotype TX1 recently collected in Texas of the USA using the same protocol described above in 2021.

2.3.Genotype the RIL population using the genotyping-by-sequencing

Leaf tissue was collected from each RIL and the two parents at the two-leaf stage,dried at-80 °C in a Freezemobile 35EL Sentry 2.0 Lyophilizer(Sp Scientific,Warminster,PA,USA),and grounded at 1500 rpm for one min in a MiniG Automated Tissue Homogenizer(Metuchen,NJ,USA).Genomic DNA was extracted using a method described by Dubcovsky et al.[16]and quantified using the Quant-iT PicoGreen dsDNA assay kits(Thermo Fisher Scientific,Waltham,MA,USA).A protocol described by Mascher[17]was used to construct GBS libraries.In brief,DNA normalized to 20 ngμL-1was digested with PstI and MspI,barcoded and ligated to a common Y-adaptor using T4 DNA ligase.Then,all DNA samples were pooled,purified and PCR-amplified.DNA fragments of 200-300 bp were size-selected for sequencing on an Ion Proton sequencer(Thermo Fisher Scientific,Waltham,MA,USA).SNPs were called using a universal network-enabled analysis kit(UNEAK)and the reference-based TASSEL GBS pipeline[18,19].SNP positions were determined by mapping sequence reads to the barley reference genome Hordeum vulgare r1[20],and SNPs with minor allele frequency greater than 20%,heterozygote frequency less than 5% and missing datapoints less than 11 were selected to map the target gene.

2.4.Allelism test

The greenbug resistance gene Rsg2 has not been mapped.Therefore,an F2population was developed from the cross STARS1501B×WBDC053 to determine whether the greenbug resistance gene in WBDC053 is allelic to Rsg2.The F2population was evaluated for response to greenbug biotype E in two replicates using the protocols described above for the RIL population.

2.5.Mapping the greenbug resistance gene in WBDC053

The ICI QTL Mapping V4.1 software program[21]was employed to map the greenbug resistance gene in WBDC053 using the Kosambi mapping function to convert recombination frequency into genetic distance[22].The GBS-SNPs were grouped using a logarithm of the odds(LOD)threshold of 4.5,and ordered in each linkage group using the Recombination Counting and ORDering(RECORD)algorithm.The linkage map was fine-tuned using the sum of adjacent recombination fractions(SARF)at a window size of 7 as rippling criteria.MapChart 2.2[23]was used to draw the linkage map.In addition,χ2tests were performed to determine the greenbug resistance gene number and gene action in WBDC053.

2.6.Development of KASP markers

Primer 3(v.4.0)(http://bioinfo.ut.ee/primer3-0.4.0/)was used to design primers.KASP assays were performed in an ABI ViiA 7 real-time PCR system(ThermoFisher Scientific,MA,USA),with 10μL reaction mixtures containing 5μL KASP master mix(2X),0.12μL of each allele-specific primer(100μmolμL-1),0.3μL of common primer(100μmolμL-1)and 1μL genomic DNA(20 ng μL-1).The PCR began at 30 °C for 1 min and 94 °C for 15 min,and then continued for 40 cycles of 94 °C for 20 s and 60 °C for 1 min,with a final step of 30°C for 1 min.KASP marker data were analyzed using the ABI ViiA 7 software.

3.Results

3.1.Responses of Weskan×WBDC053 RILs to greenbug biotype E and TX1

WBDC053 exhibited high resistance to a set of 10 greenbug biotypes[11].We first selected greenbug biotype E,the dominant biotype in the Great Plains of the USA,to test the two parents and the RIL population.Typical damage symptoms,chlorotic spots with necrotic lesions,were observed on Weskan and susceptible RILs at 3-4 days after infestation,and the damaged plants gradually turned yellow,withered and died at about two weeks after infestation.WBDC053 and the resistant RILs generally grew normally without observed damage.Of the 154 RILs,84 were resistant and 64 were susceptible,which fit a 1:1 segregation ratio for a single gene(χ2=2.7,df=1,P greater than 0.1).The remaining six lines were still segregating,from each of which 23 or 24 plants were tested with a total of 99 plants rated as‘‘resistant”and 42‘‘susceptible”(χ2=1.7,df=1,P greater than 0.18 for a 3:1 segregation ratio).Therefore,the greenbug resistance gene in WBDC053 is dominant.The experiment was repeated in 2021,leading to highly consistent results.

In addition,the RIL population was evaluated for responses to greenbug biotype TX1,a new biotype collected from Texas,and the responses of these RILs to biotypes E and TX1 were consistent,confirming the assumption that a single gene in WBDC053 conditions resistance to multiple greenbug biotypes.

3.2.Linkage analysis

A total of 181.7 million high quality reads were generated from sequencing of the RIL population,with each RIL having 422,433-4,871,356 reads and 100.54 Mb-1.16 Gb sequence.These sequences were mapped to the barley reference sequence Hordeum vulgare r1,which led to the identification of 69,506 SNPs with a minor allele frequency greater than 0.2.We further selected 3347 SNPs which have no more than 10 missing datapoints in the RIL population and mapped 466,521,506,238,617,402,and 540 GBS-SNPs on chromosomes H1 to H7,respectively.The remaining 57 SNPs were not located to a specific chromosome.On average,each SNP had 2.5 missing datapoints in the RIL population.

Linkage analysis positioned the greenbug resistance gene in WBDC053 to the distal end(0-2.35 Mb)on the short arm of chromosome 2H.The gene co-segregated with GBS-SNPs 2H_1318811,2H_1368709 and 2H_1839499,with all 84 resistant,64 susceptible,and 6 heterozygous RILs having the WBDC053,Weskan,and heterozygous alleles at each locus,respectively.The greenbug resistance gene in WBDC053 was 0.68 cM distal to SNP 2H_2354645 in the map(Fig.1).

3.3.Allelism test

Given that Rsg1 resides on chromosome 3H and the genomic location of Rsg2 is still unknown,we performed an allelism test to determine whether the gene in WBDC053 is allelic to Rsg2 by evaluating an F2population derived from STARS1501B×WBDC053.A total of 1226 F2plants,with 596 in replicate 1 and 630 in replicate 2,were evaluated for response to greenbug biotype E.All 1226 plants exhibited high resistance,indicating the gene in WBDC053 is likely allelic to Rsg2 and therefore officially designated Rsg2.a3.The former Rsg2 gene is officially re-designated as Rsg2.a1.

Fig.1.A linkage map containing Rsg2.a3 in the terminal region of chromosome 2H.The GBS-SNPs and genetic distances(in cM)are shown on the right and left sides of the linkage map,respectively.Each SNP was named using a combination of its chromosome and physical location in the Hordeum vulgare r1 reference sequence(in bp).Only a portion of the chromosome 2H linkage map is shown.

3.4.Development of KASP mar kers

Two of the three GBS-SNPs co-segregating with Rsg2.a3,2H_1318811 and 2H_1839499,were converted to KASP markers KASP-Rsg2.a3-1 and KASP-Rsg2.a3-2,respectively.KASP-Rsg2.a3-1 from 2H_1318811 was located at 1,318,811 bp in the Morex reference genome(Hordeum vulgare r1).WDBC053 carries the‘‘G”allele,while Weskan carries the‘‘A”allele at this locus.The allele-specific primers locate from 1,318,791 to 1,318,811 bp and the common primer extends from 1,318,878 to 1,318,899 bp in the Hordeum vulgare r1 reference genome(Table 1).KASP-Rsg2.a3-2 was converted from the GBS-SNP 2H_1839499 at 1,839,499 bp in the reference genome,where Weskan and WDBC053 carry the‘‘C”and‘‘T”alleles,respectively.We used the genomic sequence from 1,839,479 to 1,839,499 bp to design the allele-specific primers and 1,839,544 to 1,939,563 bp to design the common primer(Table 1).

Table 1Alleles,allele-specific primers,and Common primers of KASP-Rsg2.a3-1 and KASP-Rsg2.a3-2.

KASP-Rsg2.a3-1 and KASP-Rsg2.a3-2 were used to genotype the RIL population(Fig.2),and the KASP genotyping results were in complete agreement with the GBS-SNP data,with all 84 resistant RILs carrying the WBDC053 alleles and 64 susceptible RILs possessing the Weskan alleles at these loci.RILs that were still segregating at the Rsg2.a3 locus exhibited heterozygous genotype.Therefore KASP-Rsg2.a3-1 and KASP-Rsg2.a3-2 can be reliably used to tag Rsg2.a3 in barley breeding.

3.5.Candidate genes in the target region

Rsg2.a3 resides in the interval of 0-2,354,645 bp(SNP 2H_2354645)in the terminal region of chromosome arm 2HS.There are 59 annotated genes in this region with 46 genes encoding proteins with known functions in the Hordeum vulgare r1 reference sequence(Table 2).Among the 46 genes,15 are R genes that encode proteins containing leucine-rich-repeat (LRR), including HORVU2Hr1G000150,HORVU2Hr1G000160,HORVU2Hr1G000170,HORVU2Hr1G000180,HORVU2Hr1G000190,HORVU2Hr1G000200,HORVU2Hr1G000290,HORVU2Hr1G000510,HORVU2Hr1G000560,HORVU2Hr1G000630,HORVU2Hr1G000640,HORVU2Hr1G000700,HORVU2Hr1G000770, HORVU2Hr1G000780, and HORVU2Hr1G000900.Genes encoding cytochrome P450-related proteins,aldo/keto reductase,protein phosphatase 2,L-3-cyanoalanine synthase/cysteine synthase,transferase,tetrahydroberberine oxidase/THB oxidase,and hexokinase were also identified in this region(Table 2).

4.Discussion

4.1.Rsg2.a3 is a new allele at the Rsg2 locus

Wild species has proven to be an important source for greenbug resistance in small grain cereals.In wheat,seven of the eight greenbug resistance genes originated from wild species or wheat relatives,including Gb5 from Ae.speltoides,Gb2 and Gb6 from rye,and Gb3,Gb4,Gb7,and Gb8 from Ae.tauschii[24].In this study,we identified a greenbug resistance gene in wild barley for the first time.This finding highlights the value of this subspecies for identifying new resistance genes of value for barley.

Of the known greenbug resistance genes,Rsg1 was previously mapped to chromosome 3H and Rsg2 has not been mapped.We mapped Rsg2.a3 to the terminal region of the short arm of chromosome 2H,and allelism test indicated that it is likely allelic to Rsg2(officially Rsg2.a1),indicating that Rsg2 also resides in this region.A previous study indicated that STARS 1051B(the source of Rsg2)and WDBC053 were resistant and susceptible to greenbug biotype F,respectively[11],indicating that Rsg2.a1 and Rsg2.a3 are indeed different from each other.

Fig.2.Segregation of the WBDC053(red dots),Weskan(blue dots)and heterozygous(green dots)alleles at the KASP-Rsg2.a3-1(left)and KASP-Rsg2.a3-2(right)loci in a RIL population from WBDC053×Weskan.The‘‘×”represents negative control.

Rsg2.a3 was mapped to a 2.35 Mb interval in the terminal region of chromosome 2H.In this interval,59 annotated genes were identified with 15 genes carrying LRR protein domains.LRR-containing proteins are usually associated with plant innate immune responses against pathogens and pests.Several pest resistance genes encoding LRR-containing proteins have been cloned,including the potato aphid resistance gene Mi-1 that also conditions tomato root-knot nematode resistance[25],melon aphid resistance gene Vat[26],as well as rice brown planthopper resistance genes BHP14[27],BPH18[28]and BPH26[29].Notably,some of the 15 genes are either very close to or between GBS-SNPs markers that co-segregated with greenbug resistance in the RIL population.For example,HORVU2Hr1G000510 is 1579 bp distal to 2H_1318811,and HORVU2Hr1G000560, HORVU2Hr1G000630, HORVU2Hr1G000640,HORVU2Hr1G000700,HORVU2Hr1G000770,HORVU2Hr1G000780,and HORVU2Hr1G000900 reside between 2H_1318811 and 2H_1839499.To date,no cereal aphid resistance gene has been cloned and the mechanisms underlying cereal greenbug or any other cereal aphid resistance are still elusive.Given that LRR genes often provide plant innate immune responses to pest infestation[25-29],these LRR genes are good candidates for Rsg2.a3.Further fine-mapping of Rsg2.a3 is needed to pinpoint the candidate genes.This should be feasible given the availability of residual heterozygosity in the RIL population.Of the 154 F6RILs evaluated in this study,six lines were segregating for resistance to greenbug biotype E.The corresponding F5lines of these RILs can be advanced for fine mapping Rsg2.a3.Additionally,WBDC053 and other WBDC accessions are currently being sequenced by the International Wild Barley Sequencing Consortium(https://iwbsc.umn.edu/).This effort will facilitate the cloning of the greenbug resistance gene from this wild barley accession.

Table 2Genomic locations and function annotation of the candidate genes in the terminal region of chromosome 2H where Rsg2.a3 was located.

4.2.Use of Rsg2.a3 in barley breeding

Since the greenbug resistance gene identified was from wild barley,it is likely that some undesirable genes will be carried along when introgressing it into adapted germplasm.Thus,pre-breeding will be necessary to efficiently transfer Rsg2.a3 into modern barley cultivars.The KASP markers co-segregating with Rsg2.a3 developed in this study can be used for marker-assisted selection in barley breeding.WBDC053 is resistant to greenbug biotypes B,C,E,I,TX1,WY4,WY81,WY12 MC,and WY86[11].Given that Rsg1 is overcome by biotypes TX1 and WY81[11],Rsg2.a3 will be valuable for enhancing greenbug resistance in combination with Rsg1.Molecular markers flanking Rsg1 are available[12].Further conversion of those markers into KASP markers is still necessary to meet the requirements for high throughput screening of breeding materials.Moreover,Rsg2.a1 has been recently transferred to elite barley lines by recurrent backcrossing of STARS1501B,a selection from PI 426756,to Weskan,leading to the development of STARS1502B.STARS1502B showed similar grain yield,plant height,and test weight in fields as Weskan[8],suggesting that Rsg2.a1 does not negatively affect yield or quality traits.Given that Rsg2.a1 is allelic to Rsg2.a3,the KASP markers developed in this study have the potential to tag Rsg2.a1 in barley breeding,depending on whether they are polymorphic between the parents of breeding populations.

The only WBDC accessions resistant to greenbug biotypes found by Armstrong et al.[11]were WBDC053 from Pakistan,WBDC157 from Iraq,and WBDC177 and WBDC366 from Turkmenistan.This suggests that greenbug resistance may have originated in wild barley populations of South and Central Asia and Middle East.To identify additional sources of resistance,greater sampling and testing of wild barley accessions from this region should be conducted.In addition to greenbug resistance,WBDC053 possesses a high level of resistance to powdery mildew,an important foliar disease of barley caused by the fungus Blumeria graminis f.sp.hordei.The resistance spectrum of WBDC053 was also very wide as it exhibited low infection types to 38 of 40 B.graminis f.sp.hordei pathotypes tested[14].

CRediT authorship contribution statement

Xiangyang Xu:Conceptualization;Supervision;Formal analysis;Writing-original draft.Dolores Mornhinweg:Conceptualization;Investigation;Writing-review & editing.Guihua Bai:Conceptualization;Supervision;Writing-review & editing.Brian J.Steffenson:Conceptualization;Resources;Writing-review &editing.Ruolin Bian:Data curation;Software;Writing-review& editing.Genqiao Li:Formal analysis;Investigation;Writingreview & editing.Amy Bernardo:Investigation;Methodology;Writing-review & editing.

Acknowledgments

This study was supported by USDA-ARS CRIS project 3072-21000-009-00D.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.The USDA is an equal opportunity provider and employer.

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.