Theoretical and Applied Genetics https://doi.org/10.1007/s00122-020-03602-3

ORIGINAL ARTICLE

Identifcation of two novel Hessian fy resistance genes H35 and H36 in a hard winter wheat line SD06165

Lanfei Zhao1,2 · Nader Ragab Abdelsalam2,3 · Yunfeng Xu2 · Ming‑Shun Chen4 · Yi Feng2,5 · Lingrang Kong1 · Guihua Bai4,2

Received: 31 January 2020 / Accepted: 1 May 2020 © This is a U.S. government work and its text is not subject to copyright protection in the United States; however, its text may be subject to foreign copyright protection 2020

Abstract Key message Two new Hessian fy resistance QTLs (H35 and H36) and tightly linked SNP markers were identifed in a US hard winter wheat SD06165. Abstract Hessian fy (HF), Mayetiola destructor (Say), is one of the most destructive pests in wheat (Triticum aestivum L.) worldwide. Growing resistant cultivars is the most efective approach to minimize Hessian fy damage. To identify new quantitative trait loci (QTLs) for HF resistance, a recombinant inbred line population was developed by crossing HF resistant wheat line SD06165 to a susceptible line OK05312. The population was genotyped with 1709 single-nucleotide polymorphisms (SNPs) generated from genotyping-by-sequencing and phenotyped for HF resistance in greenhouses. Two novel QTLs for HF resistance were identifed from SD06165. The major QTL, designated as H35, was closely linked to SNP marker SDOKSNP7679 on chromosome 3BS that explained 23.8% and 36.0% of the phenotypic variations; the minor QTL, designated as H36, was fanked by SNP markers SDOKSNP1618 and SDOKSNP8089 on chromosome 7AS and explained 8.5% and 13.1% of the phenotypic variation in the two experiments. Signifcant interaction was detected between the two QTLs. Seventeen SNPs that tightly link to H35 and eight SNPs that tightly link to H36 were converted to kompetitive allele specifc polymerase chain reaction markers for selecting these QTLs in breeding programs.

Introduction

Hessian fy (HF), Mayetiola destructor (Say), is a destructive Communicated by Aimin Zhang. pest of wheat (Triticumestivum) worldwide. HF originated from the Fertile Crescent in West Asia along with wheat, its Electronic supplementary material The online version of this primary host (Harris et al. 2003). HF can be transported with article (https​://doi.org/10.1007/s0012​2-020-03602​-3) contains supplementary material, which is available to authorized users. wheat across regions (Barnes 1956; Fitch 1846). Today, HF can be found in North Africa, South Europe, North America, * Guihua Bai Southwest Asia and other wheat growing regions worldwide [email protected] (Ando et al. 2018; Berzonsky et al. 2003; El Bouhssini et al. 1 State Key Laboratory of Crop Biology, Shandong Key 2013). In the USA, HF was the frst invasive insect to cause Laboratory of Crop Biology, College of Agronomy, economic havoc, especially in the Great Plains and southeast Shandong Agriculture University, Taian 271018, Shandong, region of the USA. (Hao et al. 2013; Morton et al. 2011; China Pauly 2002). HF can cause signifcant reduction in spike 2 Department of Agronomy, Kansas State University, 2004 number per plant, grain number and weight, eventually grain Throckmorton Hall, Manhattan, KS 66506, USA yield of wheat (Smiley et al. 2004; Schwarting et al. 2016). 3 Agricultural Botany Department, Faculty of Agriculture, Hessian fy is a member in the Cecidomyiidae family Saba Basha, Alexandria University, Alexandria 21531, Egypt (Mamaev 1975; Gagné 1994) and is sexually reproduced 4 Hard Winter Wheat Genetics Research Unit, USDA, 4008 with a short life cycle of 30 days. A female insect can lay Throckmorton Hall, Manhattan, KS 66506, USA 100–400 eggs on the adaxial surfaces of wheat tender leaves 5 College of Agronomy, Northwest A&F University, in a short time (approximately 3 h). Eggs will hatch within Yangling 712100, China

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3–4 days at 20 °C. Newly hatched larvae migrate along the chromosome 2B (Amri et al. 1990), H31 on the terminus leaf-blade, enter wheat stem via the whorl, and continue to of chromosome arm 5BS (Williams et al. 2003), H34 and move to the base of the nearest node where their feeding QHara.icd-6B on chromosome 6B (Bassi et al. 2019; Li causes abnormal sheath and leaf growth, stunt plant, and et al. 2013). eventual seedling death (Anderson and Harris 2006). A sin- To date, 16 biotypes of HF have been reported, includ- gle wheat seedling can have up to 50 larvae, and infestation ing biotypes A to O and GP (the Great Plains biotype) in wheat can cause signifcant reduction in wheat grain yield based on their responses to the wheat resistant cultivars (Smiley et al. 2004; Schwarting et al. 2016). carrying genes H3, H5, H6 and a combination of H7H8 Currently, the most efective approach to HF control is (Gallun 1977; Ratclife and Hatchett 1997). The biotype integrated pest management (IPM) that mainly consists of GP is still the prevalent biotype of HF in the US Great cultural, chemical, biological and genetic controls (Buntin Plains, although it is the least virulent biotype among et al. 1992). The genetic control to use host-plant resistance these biotypes reported in the USA. (Chen et al. 2009; to the insect is the most important foundation of a successful Garcés-Carrera et al. 2014; Sardesai et al. 2005). IPM strategy. Growing resistant wheat cultivars is widely The relationship between wheat and HF fts a classical accepted as the most efective and environmentally friendly gene-for-gene genetic model, so deploying a single resist- approach to reduce HF damage worldwide (Berzonsky et al. ance gene in wheat cultivar may quickly lose its efective- 2003). ness due to the selection pressure that leads HF to adapt Wheat resistance to HF was frst reported in 1785 (Painter to the corresponding resistance gene (Foster et al. 1991; 1951). To date, a total of 35 HF resistance genes (H1-H34 Ratclife et al. 1994, 1996). Therefore, improving the and Hdic) from wheat and its wild relatives have been of- durability of HF resistance is an ultimate goal of wheat cially named (Bassi et al. 2019; Hao et al. 2013; Li et al. breeding programs worldwide. One approach is to pyramid 2013). All the HF resistance genes are dominant or partially multiple resistance genes in wheat cultivars to extend the dominant except that h4 was reported as a recessive gene duration of resistance; another way is to search for novel (Bassi et al. 2019). Among these resistance genes, nine were resistance genes to be used in wheat breeding programs. identifed from common wheat including H1, H2, H3, h4, Molecular markers closely linked to these resistance genes H5, H7, H8, H12 and H34; six were from Aegilops tauschii are essential for gene pyramiding. In addition, previously including H13, H22, H23, H24, H26 and H32; 15 were some genes have been localized to chromosomes using from durum wheat including H6, H9, H10, H11, H14, H15, monosomic analysis, which is difcult to locate a gene H16, H17, H18, H19, H20, H28, H29, H31 and H33; two to a short chromosome interval, sometimes results in an (H21 and H25) were from rye (Secale cereale); and three incorrect chromosomal location (Gallun and Patterson were from Ae. ventricosa (H27), Ae. triuncialis (H30) and 1977). Due to limited markers available and lack of refer- a cultivated primitive emmer wheat (Triticum turgidum ssp. ence genome sequence in early studies, some genes were dicoccum) (Hdic) (Bassi et al. 2019; Kong et al. 2005, 2008; mapped incorrectly to chromosomes even using DNA McIntosh et al. 1973; Sardesai et al. 2005). More recently, markers. For example, H26 was previously localized on two new major QTLs for HF resistance were identifed by chromosome 4D, but later it was remapped to chromosome association mapping, including QH.icd-2A transferred from 3D using a high-density map (Wang et al. 2006). Using a T. dicoccum and QHara.icd-6B transferred from T. ararati- high-density single-nucleotide polymorphism (SNP) map, cum (Bassi et al. 2019). H34 was identifed on chromosome 6B (Li et al. 2013) Many of these resistance genes have been mapped to and another HF resistance gene was mapped on the short various wheat chromosomes. Gallun and Patterson (1977) arm of chromosome 1A (Li et al. 2015). More recently, was the frst to locate H6 on chromosome 5A using mono- H7 and H8 have been reassigned to chromosomes 6A and somic analysis (Gallun and Patterson 1977). Later, Steb- 2B, respectively, using a SNP map (Liu et al. 2020). Fur- bins et al. (Stebbins et al. 1982) found that H6 closely thermore, those SNPs closely linked to a gene can be con- linked to H3, H9 and H10. Using molecular markers, H3, verted into kompetitive allele specifc PCR (KASP) assays H5, H6, H9, H10, H11, H12, H14, H15, H16, H17, H19, for marker-assisted selection (MAS) in wheat breeding H28, H29 and Hdic were further mapped to a HF resist- programs (Tan et al. 2017). ance gene cluster in the distal gene-rich region of short ‘SD06165’ is a hard-red winter (HRW) wheat line arm of wheat chromosome 1A (Kong et al. 2005, 2008; from South Dakota that showed stable resistance to Hes- Liu et al. 2005). H13, H22, H23, H24, H26 and H32 from sian fy biotype GP. In this study, a RIL population from Aegilops tauschii were located on the wheat chromosomes SD06165 × OK05312 was genotyped using SNP markers 1D, 3D, 4D and 6D (Cox and Hatchett 1994; Gill et al. generated by genotyping-by-sequencing (GBS) and phe- 1987; Raupp et al. 1993; Sardesai et al. 2005). Only four notyped for HF resistance in greenhouse experiments to genes were mapped in the B genome of wheat with H20 on identify HF resistance QTL in SD06165.

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Materials and methods genotypes into four classes based on the ratings of two par- ents. These RILs with 0–10% resistant plants were rated Plant materials highly susceptible as for OK05312; RILs with 11–50% resistant plants per RIL were rated moderately suscepti- SD06165 is an HF resistant HRW wheat breeding line ble; RILs with 51–70% resistant plants per RIL were rated developed by South Dakota State University with the moderately resistant; and RILs with 71–100% resistant pedigree of Wesley/SD97049, and OK05312 is an HF sus- plants per RIL were rated highly resistant as for SD06165. ceptible HRW wheat breeding line with the pedigree of A Chi-square test was conducted to ft the segregation TX93V5919/KS96WGRC40//OK94P549/KS96WGRC34. data to either a single gene (1R:1S) or two additive-gene A population of 154 F­ 5:6 recombinant inbred lines (RILs) (1R:1MR:1MS:1S) segregation model (Liu et al. 2020). from SD06165/OK05312 was evaluated for HF resistance using wheat accessions ‘Carol’ (H3), ‘Cardwell’ (H6) and DNA extraction ‘Molly’ (H13) as the HF resistant checks, and ‘Danby’ as the susceptible check. A set of Chinese Spring (CS) group Equal amount of leaf tissues was collected from all ­F5:6 3 nulli-tetrasomic lines were used to validate chromosome RILs and parents at two-leaf stage in 1.3 ml 96 deep-well location of the major HF resistance gene. plates with a 3.2-mm stainless steel bead in each well. The plates with leaf tissues were dried for 48 h in a freeze-dryer (Thermo Fisher, Waltham, MA, USA), and ground for 3 min HF resistance evaluation at 20 cycles per sec into fne powder in a Mixer Mill (Retsch GmbH, Germany). Plant genomic DNA was extracted using Biotype GP of HF was used to evaluate HF resistance of cetyltrimethyl ammonium bromide (CTAB) method (Bai the RIL population as described previously (Li et al. 2013; et al. 1999). Liu et al. 2020). HF biotype GP was maintained in the USDA-ARS Hard Winter Wheat Genetics Research Unit, Genotyping‑by‑sequencing (GBS) and SNP calling Manhattan, Kansas. Plants of the SD06165/OK05312 RIL population, two parents and four checks were infested DNA concentration was estimated in the FLUOstar Omega with the HF biotype GP in the spring (Rep. I) and fall of microplate reader (BMG LABTECH, Germany) using a ® 2018 (Rep. II). In each experiment, compacted growing Quant-iT™ ­PicoGreen dsDNA Assay Kit (Life Technolo- mix (2: 1: 1 of soil, sand and vermiculite) in each plas- gies, Grand Island, NY), and normalized to 20 ng/µl for GBS tic fat (56 × 36 × 10 cm) was uniformly marked with 12 library construction. The GBS library of 154 RILs and two rows, then divided to 24 half-rows with 15 cm long per parents (eight replications per parent) was constructed fol- row. Twenty lines and four controls with 20 seeds per line lowing Poland et al. (2012) and sequenced for three runs in were planted in each fat. The planted fats were placed on an Ion Proton Sequencer (Life Technologies). greenhouse benches at 18 ± 3 °C with a 14/10 h (day/night) GBS-SNPs were called using the reference-free Uni- photoperiod. When the frst true leaf fully expanded and versal Network-Enabled Analysis Kit (UNEAK) pipeline the second leaf emerging from all the seedlings (Zadoks implemented in the TASSEL (Bradbury et al. 2007). Raw scale 12), the fats on each bench were covered with a sequence reads were trimmed to 64 bps, which include cheesecloth tent (540 × 120 × 40 cm) and about 200 newly SNPs, for SNP calling. The SNPs showing polymorphisms mated female fies were released to each fat. Three weeks between parents and segregating in more than 80% lines after infestation, resistant and total seedlings were counted of the RIL population (< 20% missing data) were used for for each testing line. All the stunted plants with a dark further linkage map construction. To reduce false positive green color and some bloated live larvae at the base of SNPs, SNPs with more than 10% heterozygotes in the RIL stem were rated as susceptible (S), and the normally grown population were removed before further analysis. plants with light green and some small reddish dead larvae between leaf sheaths were rated as resistant (R). Those Linkage map construction and QTL analysis plants without distinct R or S characters, including these with dark green frst leaf but new healthy tillers and some An initial linkage map was constructed using the ‘Regres- ® smaller live larvae than in the susceptible plants, were also sion mapping’ algorithm in JoinMap­ v4.0 (Van Ooijen rated as R. The percentage of resistant plants per line was 2006). Recombination fractions were converted to genetic calculated for QTL analysis. distances in centiMorgans (cM) using the Kosambi func- Percentage of resistant plants was calculated for each tion (Kosambi 1944). Two adjacent markers with genetic RIL, parent and control were used to classify these distance < 30 cM and a logarithm of odds (LOD) score of 5.0 or above were considered closely linked markers. Marker

1 3 Theoretical and Applied Genetics sequences in each linkage group were aligned to the cor- scored using GeneMarker v.1.97 (Soft Genetics LLC, State responding chromosome of the Chinese Spring reference College, PA, USA). genome RefSeq v1.0 (IWGSC 2018) using a Basic Local In addition, selected SSR and KASP markers that tightly Alignment Search Tool (BLAST) to determine their physical linked to the resistance QTL were used to genotype a set positions in the chromosome. of Chinese Spring genetic stock to validate QTL mapping HF resistance ratings of RILs from the two independ- result. The set of Chinese Spring genetic stock includes ent greenhouse experiments (Rep. I and Rep. II) and the a Chinese Spring positive control, a line missing chromo- mean over the two experiments were used separately for some arm 3BS (20 + 3BL), a line missing chromosome 3A QTL identifcation using composite interval mapping (CIM) but carrying two pairs of chromosome 3B (N3AT3B), a module in WinQTL cartographer v.2.5 (Wang 2007). LOD line missing 3B but carrying two pairs of chromosome 3A thresholds to claim signifcant QTLs were generated by 1000 (N3BT3A) and a line missing 3D but carrying two pairs of permutations (Doerge and Churchill 1996). Peak LOD val- chromosome 3B (N3DT3B). ues in QTL regions were used to estimate the QTL posi- The putatively annotated high confdence (HC) genes tions. MapChart v.2.32 was used to draw the linkage maps located between the fanking markers of the major HF resist- (Voorrips 2002). ance QTL were identifed based on the Chinese Spring ref- erence genome (IWGSC 2018). The fanking markers were Conversion of GBS‑SNPs into KASP markers identifed by the QTL analysis, and were aligned to the Chi- nese Spring reference genome using BLAST to determine their physical positions. To fll up the missing data of GBS-SNPs, the sequences of GBS-SNPs under QTL regions were used to design KASP Efects and interaction of QTLs in SD06165 primers using Primer3web version 4.1.0 (http://bioin​fo.ut. on Hessian fy resistance ee/prime​r3/). The KASP primers were assayed for poly- morphisms between the parents and the polymorphic mark- To analyze the allelic efects of the HF resistance genes on ers were used to genotype the mapping population. KASP 3B and 7A in the RIL population, two KASP markers closely assays were performed in a 4 μl polymerase chain reaction linked to the two resistance QTLs, SDOKSNP7679 linked to (PCR) mix including 1.94 μl 2X PACE™ Genotyping Mas- 3BS QTL and SDOKSNP1618 linked to 7A QTL, were used ter Mix (3CR Bioscience, Harlow, England, UK), 0.06 μl to represent the two resistance QTLs. The two KASP mark- primer assay mix and 2 μl genomic DNA. PCR started with ers in the RIL population segregated into four genotypes, an initial denaturation at 94 °C for 15 min, followed by 10 aabb (no resistance alleles), AAbb (resistance allele of 3B touch-down PCR cycles at 94 °C for 20 s, and 60 °C for QTL), aaBB (resistance allele of 7A QTL) and AABB (both 1 min with − 0.5 °C/cycle, and then by 30 cycles of 94 °C resistance alleles for 3B and 7A QTLs). The mean resistance for 20 s, 55 °C for 1 min. The genotypic data was col- ratings of the four genotypes calculated from each green- lected using the FLUOstar Omega microplate reader (BMG house experiments and the mean over the two experiments Labtech, Germany) and analyzed using Klustercaller soft- were used to analyze the interaction between the two QTLs ware (LGC group, Teddington, UK). The GBS-SNP data from SD06165, respectively. of RILs were replaced by the corresponding KASP data to re-construct a new linkage map. The QTL regions in the new map were reported as the fnal QTLs for HF resistance. Results

Chromosomal localization and putative gene HF resistance in the SD06165/OK05312 RIL annotation of the major Hessian fy resistance QTL population

To confrm the location of the major HF resistance QTL The biotype GP of HF was used to infest seedlings of par- on chromosome 3BS, 12 SSR markers (Barc68, Barc75, ents, resistant and susceptible controls and RILs in the Barc102, Barc133, Barc147, Barc156, Barc259, Cfa2191, spring (Rep. I) and fall (Rep. II) of 2018. All plants of Cfd79, gwm389, gwm493 and gwm533) from the distal ‘OK05312’ and susceptible control ‘Danby’ showed suscep- region of chromosome arm 3BS (Blake et al. 2019) were tible symptoms and resistant controls ‘Carol’ (H3), ‘Card- selected to screen the RIL population. PCR was performed well’ (H6) and ‘Molly’ (H13) were resistant to the biotype following Li et al. (2013) with minor modifcations. The GP. ‘SD06165’ shown partial resistance to biotype GP with PCR products were separated using ABI PRISM 3730 DNA 73.7% and 78.5% resistant plants in the spring (Rep. I) and Analyzer (Applied Biosystems, Foster City, CA, USA) and fall 2018 (Rep. II) greenhouse experiments, respectively. In the RIL population, 106 (68.8%) and 99 (64.3%) lines

1 3 Theoretical and Applied Genetics showed high susceptibility with < 10% resistant plants to SNPs with 6 and 4 SNPs in each chromosome, respectively, biotype GP as the susceptible parent in both experiments, and 14 SNPs were mapped to the unknown chromosome. and only 25 (16.2%) and 27 (17.5%) RILs showed high Using the genetic map, two QTLs were identifed for HF resistance with more than 75% resistant plants to biotype GP resistance, which agrees with the frequency distribution of as ‘SD06165’ (Fig. 1). The segregation ratio of the percent- percentage of resistant plants per line in the RIL population. age of resistant lines in the mapping population suggested One of the QTLs with a higher LOD value was located on that the HF resistance in SD06165 might be conditioned by the distal region of chromosome arm 3BS, and the other was more than one gene. Furthermore, the χ2-test of the segrega- located on the chromosome arm of 7AS in all experiments. tion ratios in the RIL population did not ft either one or two SD06165 contributed resistance alleles at both QTLs. gene model (Supplemental Table 1). In addition, the trans- gressive segregation was observed for resistant genotypes Conversion of GBS‑SNPs to KASP markers in the RIL population, more than 16% lines showed higher resistance than SD06165 in the two greenhouse experiments. To verify the GBS-SNP data of the RIL population and fll in missing data generated from GBS, the GBS-SNPs Linkage map construction and QTL analysis in the QTL regions were converted to KASP markers. For the 3BS QTL, 37 GBS-SNPs were used for KASP marker A total of 10,045 SNPs was generated and 1709 SNPs with conversion. Parental screening identifed 17 polymorphic < 20% missing data were used for linkage map construction. KASP markers that were used to genotype the RIL popula- Among these SNPs, 1671 SNPs were mapped to 44 linkage tion. For the 7AS QTL, 15 GBS-SNPs in the QTL region groups (Supplemental Table 2). Using Chinese Spring ref- were used for KASP conversion and 8 of them segregated erence genome (IWGSC 2018), the 44 linkage groups were in the population (Supplemental Table 3). Genotypic data assigned to 21 chromosomes with one group to unknown of all the 25 polymorphic KASP markers had similar seg- chromosome. The linkage map was 2080 cM long with a regation patterns to their corresponding GBS-SNPs. For 25 mean marker density of 1.24 cM per marker. Among all the KASP markers, only 27 KASP data points disagreed with chromosomes, chromosome 6D had the most SNPs with a corresponding GBS-SNPs with 0–3 mismatches per marker total of 204 SNPs, chromosomes 5D and 6A had the fewest and 537 missing SNP data points from GBS were flled in the RIL population with 11–30 missing data points per

Fig. 1 Frequency distribution of the percentage of Hessian fy resistant lines in the population of SD06165/OK05312 evaluated in 2018 spring (Rep. I) and fall (Rep. II) and the mean over the two experiments

1 3 Theoretical and Applied Genetics marker (Supplemental Table 4). Thus all 25 KASP markers GWM493 and GWM0533) were polymorphic between the were used to replace corresponding GBS-SNPs in the RIL two parents and were screened in the RILs, only Cfd79 was population to refne the map. Using the new map, two HF mapped in the peak position of 56.6 cM on 3BS (Fig. 2). resistance QTLs were still mapped on the chromosome arms Cfd79 and three KASP markers (SDOKSNP6180, SDOK- 3BS and 7AS; however, the 3BS QTL was further delimited SNP9140 and SDOKSNP5826) were used to screen Chinese to a 3.0 cM region between GBS-SNP SDOK-M6771 and Spring, a ditelosomic line DT3BS (20 + 3BL), three Chi- KASP-SNP SDOKSNP2713, and explained 30.6% of the nese Spring nulli-tetrasomic lines (N3AT3B, N3BT3A and phenotypic variation when the mean phenotypic data from N3DT3B), and two parents (OK05312, SD06165). KASP two experiments were used (Table 1). Blasting the sequence data showed that all lines were amplifed and clustered with harboring each SNP in the 3BS QTL region against the SD06165 except lines DT3BS (20 + 3BL) and N3BT3A that Chinese Spring reference genome (IWGSC 2018) located were clustered with water controls (NTCs) (Supplemental the QTL in the physical interval between the 664,035 and Fig. 1). The SSR marker Cfd79 amplifed four diferent size 4,310,482 bps. The QTL peak was near KASP marker bands, 270 bp, 281 bp, 296 bp and 316 bp in CS, respec- SDOKSNP7679. When two greenhouse experiments were tively, but could not amplify the 296 bp band in lines DT3BS separately analyzed, similar result was obtained for the fall (20 + 3BL) and N3BT3A, which indicated that the 296 bp 2018 experiment, in which the QTL was mapped in a 2.7 cM band was 3BS specifc (Supplement Table 5). These results interval between KASP SDOKSNP2708 (1,227,497) and confrmed that the major QTL for HF resistance in SD06165 SDOKSNP3010 (4,805,823). However, in the spring 2018 linked to these markers was on 3BS. experiment, the QTL was mapped in a much shorter interval (0.9 cM) between SNPs SDOK-M6771 and SDOKSNP2708 and explained 23.82% of the phenotypic variation. The putative annotated genes within the fanking The QTL mapped on chromosome arm 7AS had a lower region of the major QTL LOD value than the 3BS QTL and was delimited to a 11.1 cM interval between KASP marker SDOKSNP8760 The QTL analysis identifed KASP markers SDOKSNP8067 (63,386,114) and GBS-SNP SDOK-M5043 (76,425,871) and SDOKSNP3010 as fanking markers for the 3B QTL and explained 13.07% of the phenotypic variation when and sequence alignment of their sequences to Chinese the mean phenotypic data from two experiments were used. Spring reference genome physically located the region Two individual experiments identifed the same right fank- between 274,576 and 4,805,886 bp on chromosome 3B. ing marker for the QTL, although spring 2018 experiment Based on Chinese Spring reference, 100 HC genes were supported a diferent left fanking marker (SDOKSNP1618) annotated in the fanking region (Supplemental Table 6). that made the QTL interval 4.3 cM shorter. Among these, genes encoding a defensin-like protein (TraesCS3B01G001800), a cellulose synthase family protein Confrmation of the chromosome location (TraesCS3B01G000500), a cellulose synthase-like protein of the major QTL using SSR and KASP markers (TraesCS3B01G000600), two isofavone reductase-like pro- teins (TraesCS3B01G003900 and TraesCS3B01G004500), To confrm the chromosome location of the 3BS QTL, 12 five pectinesterase inhibitors (TraesCS3B01G008200, SSR markers that were reported on the distal region of chro- TraesCS3B01G008300, TraesCS3B01G008400, mosome 3BS were screened for polymorphism between two TraesCS3B01G008800 and TraesCS3B01G008900) and a parents. Five SSR markers (Barc156, Cfd79, GWM389, proteinase inhibitor (TraesCS3B01G009700) may relate to

Table 1 Chromosome locations, Experiments Chr. arm Position (cM) Left marker Right marker LOD PVE (%) Adda fanking markers, Log of odd (LOD) value, phenotypic Rep. I 3BS 0.91 SDOK-M6771b SDOKSNP2708 9.50 23.82 17.88 variation explained (PVE) and Rep. II 3BS 2.01 SDOKSNP2708 SDOKSNP3010 15.28 36.00 24.54 additive efect (Add) of the b wheat Hessian fy resistance Mean 3BS 1.61 SDOK-M6771 SDOKSNP2713 12.67 30.59 21.55 QTLs mapped in SD06165/ Rep. I 7AS 95.01 SDOKSNP1618 SDOK-M5043b 3.61 8.50 10.48 OK05312 RIL population Rep. II 7AS 94.01 SDOKSNP8760 SDOK-M5043b 6.03 11.80 15.40 Mean 7AS 94.01 SDOKSNP8760 SDOK-M5043b 5.96 13.07 14.91

a Additive efect where a positive value implies that the SD06165 allele contributed increased resistance to Hessian fy b Conversion of Kompetitive allele specifc polymerase chain reaction (KASP) was not successful for SDOK-M6771 and SDOK-M5043

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Fig. 2 Linkage groups corresponding to chromosome arms 3BS and using red, green and blue lines, respectively. Bars for the QTLs posi- 7AS that carry the two QTLs for Hessian fy resistance. Genetic dis- tions from left to right are calculated using data from Rep. I, Rep. tances in centiMorgans (cM) are shown on the left side of each link- II and Mean, respectively. The numbers on the top of QTL plots are age map and marker names are shown on the right side. QTL data LOD values. Cfd79 is an SSR marker mapped on chromosome 3BS from Rep. I, Rep. II and mean over the two experiments were plotted

Hessian fy resistance although typical disease resistance- showing signifcant interaction between the two QTLs on related gene was not identifed. the chromosomes 3BS and 7AS.

Efects of QTLs on HF resistance in SD06165 Discussion

KASP markers SDOKSNP7679 and SDOKSNP1618 are Hessian fy resistance genes in wheat the most closely linked markers to the QTLs 3BS and 7AS, respectively; thus they were used to analyze the QTL efects To date, 35 HF resistance genes have been formally named on HF resistance and the interaction between the QTLs in and some of them have been used in wheat breeding pro- the RIL population. The two QTLs have four allele combi- grams (Bassi et al. 2019; Li et al. 2013). In this study, a nations: AABB, AAbb, aaBB and aabb, where AA repre- RIL population of SD06165 × OK05312 was genotyped sents the resistance allele of 3BS QTL and BB represents and phenotyped in two experiments, and two QTLs for HF the resistance allele of 7AS QTL. Among the 154 RILs, resistance were identifed on chromosome arms 3BS and 36 lines with aabb genotype (susceptibility alleles at both 7AS in both experiments. As expected, SD06165 contrib- QTLs) had the average HF rating of 1.11–5.97% resistant uted both QTLs. The QTL on the distal end of chromosome plants per line; 38 RILs with aaBB (resistance allele at only 3BS showed a major efect on HF resistance, and the sec- 7AS QTL) had 1.45–13.16% HF resistant plants per line; ond QTL on 7AS had a much smaller efect than the one 40 RILs with AAbb genotype (resistance allele at only 3BS on 3BS. SD06165 is an elite hard-red winter wheat breed- QTL) had 26.38–29.63% HF resistant plants per line; and 40 ing line with the pedigree of Wesley/SD97049. Wesley (PI RILs with AABB genotype (resistance alleles at both QTLs) 605742) is a hard-red winter wheat cultivar with superior had 57.50–68.50% of HF resistant plants per line (Fig. 3), bread making quality and high yield potential. However, it

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Fig. 3 Comparison of the mean Hessian fy resistance ratings (%) of ferent letters (a, b and c) on the top of the fgure indicate these means diferent resistance gene combinations in the recombinant inbred pop- difered signifcantly at p = 0.05 (LSD). For the gene combinations, ulation evaluated in two experiments. The solid line inside the box ‘A’ and ‘a’ refer to the resistance and susceptibility alleles, respec- refers to the mean value, the bottom and top lines of each box refer tively, on 3BS; ‘B’ and ‘b’ refer to the resistance and susceptibility to the 2nd and 3rd quartiles; the bottom and top whiskers refer to the alleles on 7AS 1st and 4th quartiles; and the open circles represent outliner. The dif- was reported to be highly susceptible to the HF GP-biotype the QTL region are aligned to multiple physical locations (Peterson et al. 2001). SD97049 [ND8889 (Mvr/KS79397// in the Chinese Spring reference genome (IWGSC 2018). Nsr/3/Cody)/Karl] showed heterogeneous HF resistance in To further confrm its chromosome location, we analyzed the USDA 2003 Northern Regional Performance Nursery a SSR and three KASP markers that closely link to H35 (NRPN), as observed for SD06165 in this study, suggesting in a set of Chinese Spring group 3 nulli-tetrasomic lines SD06165 inherits HF resistance QTLs from SD97049. and a ditelosomic line (DT3BS) and the result indicated In previous studies, only four HF resistance genes (H20, that these markers are on the short arm of chromosome 3B H31, H34 and QHara.icd-6B) have been mapped on B (3BS), confrming H35 from SD06165 is on 3BS. genome of wheat, with H20 on 2B (Amri et al. 1990), In previous studies, multiple disease resistance loci were H31 on the 5BS (Williams et al. 2003), QHara.icd-6B detected on the distal end of 3BS in wheat, including Fhb1 and H34 on chromosome 6BS (Bassi et al. 2019; Li et al. for Fusarium head blight resistance (Anderson et al. 2001; 2013). To date, QTLs for HF resistance have not been Bai et al. 1999; Su et al. 2019), Yr57 for stripe rust resist- reported on 3BS; therefore, the QTL on 3BS identifed ance (Randhawa et al. 2015), Sr2 for stem rust resistance, in this study is a novel QTL for HF resistance and desig- Lr27 for leaf rust resistance and a powdery mildew resist- nated as H35. This QTL was mapped in the distal end of ance gene (Mago et al. 2011). In this study, H35 is mapped chromosome 3BS fanked by SDOK-M6771 and SDOK- in the similar region, suggesting that this short chromosome SNP2713 when the mean of two greenhouse phenotypic region (< 10 Mb) is gene rich with many genes for diseases data was used for QTL mapping. KASP marker SDOK- and insect resistance. These genes are present in diferent SNP9140 that closely links to H35 could not be aligned germplasm lines. Pyramiding some of these genes in one to the reference genome of wheat. Also, KASP markers line may provide a permanent source for multiple pest resist- SDOKSNP3010, SDOKSNP1690 and SDOKSNP5826 in ance. For an example, Fhb1 has been successfully stacked

1 3 Theoretical and Applied Genetics with Sr2 (Zhang et al. 2016) although low recombination H35, therefore, pyramiding diferent resistance genes in a was reported in this region (Schweiger et al. 2016). cultivar may signifcantly enhance the levels of HF resist- Several types of molecular markers have been used to tag ance. The KASP markers we developed for both genes can HF resistance, including restriction fragment length poly- be directly used for selection of the two QTLs in wheat morphism (RFLP), randomly amplifed polymorphic DNA breeding programs. (RAPD), simple sequence repeats (SSR), sequence-tagged sites (STS) and recently SNPs (Dweikat et al. 1997, 2002; Acknowledgements This is contribution number 20-077-J from the Kansas Agricultural Experiment Station. This project is partly funded Kong et al. 2005, 2008; Ma et al. 1993). With the rapid by the National Research Initiative Competitive Grants 2017-67007- development of next-generation-sequencing, SNP markers 25939 and 2017-67007-25929 from the US Department of Agriculture have been widely used in QTL mapping and marker-assisted National Institute of Food and Agriculture. Mention of trade names or breeding. Some SNPs linked to HF resistance genes have commercial products in this publication is solely for the purpose of providing specifc information and does not imply recommendation been used for MAS in wheat breeding. SNPs SynopGBS901 or endorsement by the USDA. USDA is an equal opportunity provider and IWB65911 linked to H32 were converted into KASP and employer. markers for MAS (Tan et al. 2017). In addition, two SNPs linked to the HF resistance gene HfrDrd have been identifed Author contribution statement GB designed the research; LZ, NRA, for deployment of HfrDrd in new wheat varieties (Tan et al. YX, MC, YF performed the research; LZ, YX and LK analyzed the H35 data; LZ and GB wrote the paper. All the authors read and approved 2015). In this study, is closely linked to SDOKSNP7679 the manuscript. and fanked by SDOKSNP2708 and SDOKSNP2713, and these KASP markers can be directly used to select H35 in Compliance with ethical standards breeding programs. Several QTLs on wheat chromosome 7AS were reported Conflict of interest The authors declare that they have no confict of for diferent traits, but not for HF resistance. Therefore, the interest. QTL on 7AS identifed in this study is also a novel QTL Ethical standards H36 The authors declare that the experiments comply for HF resistance, designated as . SDOKSNP8760, with the current laws of the country in which they were performed. SDOKSNP1618, SDOKSNP8089 and SDOKSNP2546 tightly linked to H36 are all KASP markers and can be use- ful for deployment of the resistance gene in wheat breeding programs. 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