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Development and Validation of a Perfect KASP Marker for Fusarium Head Blight Resistance Gene Fhb1 in Wheat
Plant Pathol. J. 2019;35:200-207
Published online June 1, 2019
© 2019 The Korean Society of Plant Pathology.

Lovepreet Singh1, James A Anderson2, Jianli Chen3, Bikram S Gill4, Vijay K Tiwari1, and Nidhi Rawat1,*

1Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA
2Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA
3Department of Plant, Soil, and Entomological Sciences, University of Idaho, Aberdeen, ID 83210, USA
4Wheat Genetics Resource Center, Department of Plant Pathology, Kansas State University, Manhattan, Kansas, KS 66506, USA
Correspondence to: *Phone) +1-301-405-9744, FAX) +1-301-314-9308
E-mail) nidhirwt@umd.edu
ORCID
Nidhi Rawat
https://orcid.org/0000-0003-4820-0020

Handling Editor: Park, Chang-Jin
Received January 27, 2019; Revised March 16, 2019; Accepted March 31, 2019.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Fusarium head blight (FHB) is a devastating wheat disease with a significant economic impact. Fhb1 is the most important large effect and stable QTL for FHB resistance. A pore-forming toxin-like (PFT) gene was recently identified as an underlying gene for Fhb1 resistance. In this study, we developed and validated a PFT-based Kompetitive allele specific PCR (KASP) marker for Fhb1. The KASP marker, PFT_KASP, was used to screen 298 diverse wheat breeding lines and cultivars. The KASP clustering results were compared with gel-based gene specific markers and the widely used linked STS marker, UMN10. Eight disagreements were found between PFT_KASP and UMN10 assays among the tested lines. Based on the genotyping and sequencing of genes in the Fhb1 region, these genotypes were found to be common with a previously characterized susceptible haplotype. Therefore, our results indicate that PFT_KASP is a perfect diagnostic marker for Fhb1 and would be a valuable tool for introgression and pyramiding of FHB resistance in wheat cultivars.

Keywords : Fhb1, Fusarium head blight, KASP assay, pore-forming toxin-like (PFT), Wheat
Body

Fusarium head blight (FHB), also known as head scab, is a devastating wheat disease with a significant economic impact. FHB incidences have been reported at epidemic levels across the USA, several parts of Canada, Europe, China and Korea since the 1990s (Goswami and Kistler, 2004; McMullen et al., 2012; Shin et al., 2018; Windels, 2000). The total direct and indirect losses due to FHB in wheat and barley were estimated to be $ 7.67 billion from 1993 to 2001 across nine states in the northern Great Plains and the Central USA (Nganje et al., 2004). In addition to lower yields, FHB infected grains accumulate trichothecenes and estrogenic mycotoxins that make grain unfit for animal and human consumption (Goswami and Kistler, 2004). Deoxynivalenol (DON) is the major toxin associated with FHB (McMullen et al., 1997) and its consumption causes vomiting, feed refusal, and diarrhea in animals (Bennett and Klich, 2003). In North America, Fusarium graminearum Schwabe is reported as a major causal agent of FHB. Besides wheat, the pathogen also infects other important cereal crops including barley, corn, oat, and rye (Goswami and Kistler, 2004).

Among various management practices, growing disease resistant cultivars is the most effective and economical disease management strategy against FHB (McMullen et al., 1997). Although quantitative trait loci (QTLs) associated with FHB have been found on all the wheat chromosomes, Fhb1 is the most important large effect and stable QTL for FHB resistance derived from Chinese cultivar ‘Sumai 3’ (Buerstmayr et al., 2009). Sumai 3 was developed by crossing two moderately susceptible parents ‘Funo’ and ‘Taiwanxiaomai’ and it has been widely used in wheat breeding programs as a source for FHB resistance (Bai and Shaner, 1994). Fhb1 confers resistance against spread of the pathogen on the spike after initial infection, also known as type II resistance (Schroeder and Christensen, 1963). Fhb1 is located on the short arm of chromosome 3B of Sumai 3 (Anderson et al., 2001; Xiao et al., 2011). A pore-forming toxin-like (PFT) gene was identified as an underlying gene for Fhb1 resistance. PFT is a 3,472 bp gene with two exons which is predicted to encode two agglutinin domains and one pore forming ETX/MTX2 domain (Rawat et al., 2016). Five different haplotypes for the Fhb1 region, including one resistant (R) and four susceptible (S1, S2, S3, and S4) haplotypes were reported by Rawat et al. (2016) in a panel of 40 landraces and cultivars with a well-characterized FHB phenotype. PFT was present only in R and S3 haplotypes, and both haplotypes differ by two SNPs (Rawat et al., 2016).

DNA marker-assisted selection (MAS) is highly desirable for selecting FHB resistance due to its quantitative nature, high Genotype x Environment interaction (GxE), and requirement of adult plant screening (Buerstmayr et al., 2009). Traditionally, MAS for Fhb1 has been reported using linked simple sequence repeat (SSR) markers (Anderson et al., 2007; Miedaner et al., 2006; Wilde et al., 2007; Xie et al., 2007; Zhou et al., 2003). However, linked SSR markers have limitations in their utility. First, recombination between the marker and underlying gene might occur after multiple selection cycles. Second, lack of polymorphism might become an issue with introgression into diverse genetic backgrounds. Third, haplotype variation of markers not associated with the causal gene can result in false positives or false negatives. Finally, SSR markers are gel-based markers and require a fragment analysis step in addition to polymerase chain reaction (PCR). Single Nucleotide Polymorphism (SNP) markers are the markers of choice among contemporary breeding programs due to high throughput, gel-free detection, and low cost (Gupta et al., 2001; Singh and Singh, 2015). Kompetitive allele specific PCR (KASP) is a high-throughput and breeder friendly fluorescence-based genotyping platform for SNP markers (Semagn et al., 2014). In wheat, KASP markers have been developed and validated for leaf rust resistance (Neelam et al., 2013), stem rust resistance (Babiker et al., 2015), wheat streak mosaic virus resistance (Tan et al., 2017) and pre-harvest sprouting resistance (Cabral et al., 2014). Bernardo et al. (2012) developed SNP markers flanking the Fhb1 locus using a Ning 7840/Clark population. KASP markers linked with Fhb1 have been developed (Rasheed et al., 2016; Steiner et al., 2017; Su et al., 2018), but still there is a need to develop a gene-specific perfect KASP marker for Fhb1. The objectives of this study were to develop and validate a gene specific perfect KASP marker for Fhb1. PFT_KASP will enhance breeding efficiency for FHB resistance.

Materials and Methods

Plant materials

A panel of 40 landraces was used to test diagnostic ability of the KASP marker (Supplementary Table 1). For marker validation, 75 breeding lines from University of Idaho’s spring wheat breeding program were used (Table 2). Additionally, 223 diverse wheat entries including cultivars and breeding lines maintained at the University of Minnesota and Pacific Northwest wheat breeding programs were used for genotyping (Supplementary Table 2).

Primer design

The PFT sequences were amplified using PFT_TILLING primer used for performing Targeting Induced Local Lesions in Genomes (TILLING) in Rawat et al. (2016) from the accessions representing R and S3 haplotypes. For developing a PFT specific KASP marker, we targeted the G/A polymorphism between R and S3 haplotype at the 2,181 nucleotide position where the G nucleotide was specific to R haplotype (Fig. 1). The KASP marker, PFT_KASP, was designed at the target SNP from LGC genomics (LGC Ltd, Teddington, UK). The sequences of two allele specific reverse primers and one common forward primer for PFT_KASP are given in Table 1. For validating the PFT_KASP marker, the 298 wheat breeding lines and cultivars were also screened for the STS marker UMN10 (Liu et al., 2008), gel-based gene specific markers for PFT (PFT_GSP), and a neighboring gene SGNH (SGNH_GSP) belonging to GDSL lipase superfamily in the Fhb1 region (Table 1). PFT_GSP was designed to target the G/A polymorphism between R and S3 haplotypes. SGNH_GSP was also designed to specifically amplify R haplotype. Table 1 enlists all the primer sequences used in this study.

Genotyping assays

The KASP assay was performed in 10 μl reaction volume consisting of 5 μl KASP master mix, 0.14 μl KASP primer assay mix and 5 μl of 10 ng/μl DNA template. The amplification and Fluorescent end-point reading were performed on a BIO-RAD CFX 96 (Bio-Rad Laboratories Inc., Hercules, CA, USA). The following cycling conditions were used: 94°C for 15 min, ten touchdown cycles of 94°C for 20 s and 61–55°C (dropping 0.6°C per cycle) for 1 min; followed by 26 cycles of 94°C for 20s and 55°C for 1 min. The Fluorescent end-point reading was done at 30°C for 1 min. The allele discrimination mode of BIO-RAD CFX manager 3.1 software was used for genotype calling. It is important to note that the distinction of S3 haplotype from R haplotype in this KASP assay is based on just one SNP, we recommend the strict use of the above thermocycler profile. The PCR for PFT_GSP and SGNH_GSP was performed in 25 μl reaction volume including 5 μl of 5× MyTaqTM Buffer (Bioline), 2 μl of each forward and reverse primer (4 μM/μl), 0.1 μl of 5 U/ μl MyTaqTM DNA Polymerase (Bioline) and 75 ng of DNA template. The PCR amplifications were run on T100TM thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) by using cycling profile as follows: 95°C for 5 min, five touchdown cycles of 95°C for 45 s, 65–60°C (dropping 1°C per cycle) for 45 s and 72°C for 1 min; followed by 27 cycles of 95°C for 45 s, 60°C for 45 s and 72°C for 1 min; with final extension for 7 min at 72°C. The PCR products were visualized with ethidium bromide after running on 1.8% agarose gels. The methods for UMN10 genotyping were as described previously (Liu et al., 2008).

Results and Discussion

The KASP assay developed for PFT targeted the SNP between R and S3 haplotype (Fig. 1), and all the other susceptible haplotypes lack PFT. PFT_KASP therefore behaves as a dominant marker. The PFT_KASP assay accurately clustered genotypes into three clusters of R haplotype, S3 haplotype and other susceptible haplotypes in the panel of 40 landraces and controls for all the haplotypes (Fig. 2). As the rate of recombination is very low in the Fhb1 region (Rawat et al., 2016; Shweiger et al., 2016), other co-dominant markers known from the Fhb1 region such as widely used UMN10 (Liu et al., 2008) or recently reported HRC-GSM (Su et al., 2018) may be used to discern heterozygosity or homozygosity in breeding programs.

To validate the specificity of PFT_KASP, it was used in conjunction with SGNH_GSP, PFT_GSP and UMN10 marker to genotype a set of 75 spring wheat breeding lines from Idaho’s wheat breeding program (Table 2). Thirteen lines were found to be resistant with PFT_KASP, SGNH_GSP and PFT_GSP, whereas three additional lines gave ‘R’ genotype with UMN10. Out of 13 lines, seven lines were scored as heterozygous (H) containing both R and S alleles for UMN10. The lines that showed the resistant haplotype with PFT_KASP had Sumai 3- derived or related Fhb1 gene in their pedigree. Similarly, in the panel of 223 cultivar and breeding lines from Minnesota, 24 were found to be resistant using PFT_KASP, SGNH_GSP and PFT_GSP markers, whereas, five additional lines were scored as ‘R’ using UMN10 marker (Supplementary Table 2). Out of 24 resistant lines, 4 lines scored as H containing both R and S alleles for UMN10. The genotyping data for all 298 lines was in complete agreement with each other for PFT_KASP, SGNH_GSP and PFT_GSP markers, whereas 8 disagreements were observed between them and the UMN10 marker.

The disagreement in genotypes of the eight lines for PFT_KASP and UMN10 among the total 298 lines screened could be due to any genetic recombination that may be present between PFT and UMN10 or a possibility of a different haplotype in these lines as compared to other susceptible haplotypes. It should be noted that rate of recombination is known to be very low for this region (Schweiger et al., 2016). To discern the origin of the disagreement in these lines, we sequenced two genes located upstream of UMN10 in the Fhb1 region: Histidine rich calcium-binding protein (His) and Terpene Synthase (TS). Fig. 3 shows the location and order of all the markers used for resolving the disagreements. The sequences of His and TS matched to the ‘R’ haplotype in all the eight entries with disagreements, whereas PFT and SGNH genotyping showed them to be susceptible haplotypes (Table 3), this was similar to a previously characterized susceptible ‘S3’ haplotype reported in Rawat et al. (2016). The ‘R’ score for these eight lines identified with UMN10 was not accurate and further validated the sensitivity of PFT_KASP assay and lack of polymorphism in UMN10 between R and S3 haplotypes. Previously, UMN10 has been used for conducting MAS for Fhb1 with an assumption of tight marker and trait linkage (Zhang et al., 2016). However, the UMN10 based KASP marker developed in a previous study detected some false positives (Rasheed et al., 2016). Thus, our results underscore the limitation of linked markers over perfect markers.

PFT_KASP is a perfect diagnostic marker for Fhb1 and will be a valuable resource for wheat breeders to develop FHB resistant cultivars. It will enhance the efficiency of introgression and pyramiding of FHB resistance genes in wheat varieties.

Supplemental Materials
Acknowledgements

This work was supported by the USDA National Institute of Food and Agriculture, Hatch project 1016879 and Maryland Agricultural Experiment Station via MAES Grant No. 2956952.

Figures
Fig. 1. Multiple sequence alignment of wheat accessions representing R and S3 haplotypes for PFT. WZHHS, Shanasui, Wannin2, Sumai-3, Huoshawmai, Shuilizhan, and 701 Chokwang contain R haplotype, whereas, Jingzhou1, Nanda2419, and Emai6 possess S3 haplotype. Asterisks designate the consensus sequence among all the genotypes. The position of allele specific and common primer is highlighted as bold underlined. The SNP targeted for developing PFT_KASP is highlighted as bold italics.
Fig. 2. Genotyping results of PFT_KASP marker on a panel of different genotypes and controls. Genotypes clustered as blue squares in the upper left corner are homozygous resistant (R) haplotype. Genotypes clustered in the lower right corner as orange circles are homozygous susceptible (S3) haplotype. Genotypes which do not possess either allele are clustered with NTCs (No template controls) as black diamonds in the lower left corner.
Fig. 3. Order and location of genes and markers on Fhb1 genomic assembly () used to resolve the haplotypes of the eight breeding lines/cultivars with disagreement between UMN10 and PFT.
Tables

List of primer sequences used in this study

Marker Primer Sequence (5′-3′)
PFT_KASP Reverse_G AGCTGGGAATGAAGGAACCTGC
Reverse_A AGCTGGGAATGAAGGAACCTGT
Common Forward GTGATTATCTCTCCCATCTTATGTTTGCAA
PFT_TILLING Forward TGGCACACGCTACATTGCT
(Rawat et al., 2016) Reverse CAACTTCGCCGTCAACTACA
PFT_GSP Forward ATAATCTCCTTGATGCTTTTACT
Reverse ACTATCAAGGCCCTCAATACCA
SGNH_GSP Forward CAATTCCAGCAGTTCATCAAC
Reverse CATACCCGCAACACACAT
His Forward AAGGAGAAGAAGCTCAAGTCG
Reverse CTGGGTTCAGCAGAGTTCGCAC
TS Forward GTACACTCGGGACCGTATGGTC
Reverse GGAGATGTCGTTGAGGAAGCGG
UMN10 Forward CGTGGTTCCACGTCTTCTTA
(Liu et al., 2008) Reverse TGAAGTTCATGCCACGCATA

Genotyping results of PFT_KASP, UMN10, SGNH_GSP, and PFT_GSP markers on the set of University of Idaho wheat breeding lines used for validation of PFT_KASP marker

Breeding line ID Pedigree PFT_KASP UMN10 SGNH_GSP PFT_GSP
A12023S-1 IDO850/W14//IDO852 S S S S
A12023S-4 IDO850/W14//IDO852 S S S S
A12023S-5 IDO850/W14//IDO852 S S S S
A12025S-1 IDO850/W14//IDO854 S S S S
A12025S-2 IDO850/W14//IDO854 R R R R
A12025S-4 IDO850/W14//IDO854 S S S S
A12035S-1 IDO851/W14//IDO851 S S S S
A12037S-3 IDO851/W14//IDO854 S S S S
A12037S-5 IDO851/W14//IDO854 S S S S
A12037S-7 IDO851/W14//IDO854 S S S S
A12056S-10 IDO852//IDO852/W14 S S S S
A12056S-11 IDO852//IDO852/W14 S S S S
A12056S-12 IDO852//IDO852/W14 S S S S
A12056S-13 IDO852//IDO852/W14 S S S S
A12062S-6 IDO854/W14//IDO851 R R R R
A12062S-8 IDO854/W14//IDO851 R H R R
A12062S-9 IDO854/W14//IDO851 R H R R
IDO1405S 2016 F311 E border S S S S
A12063S-9 IDO854/W14//IDO854 S S S S
A12063S-10 IDO854/W14//IDO854 S S S S
A12063S-12 IDO854/W14//IDO854 S S S S
A12064S-1 IDO854/W14//IDO854 S S S S
A12064S-2 IDO854/W14//IDO854 S S S S
A12064S-8 IDO854/W14//IDO854 S S S S
A12064S-9 IDO854/W14//IDO854 S S S S
A12064S-11 IDO854/W14//IDO854 S S S S
A12077S-2 IDO696//IDO696/W14 S S S S
A12084S-3 IDO687//IDO687/W14 R H R R
A12084S-5 IDO687//IDO687/W14 R H R R
A12084S-6 IDO687//IDO687/W14 R H R R
A12084S-7 IDO687//IDO687/W14 S S S S
A12086S-3 IDO687/W14//IDO687 R R R R
A12086S-10 IDO687/W14//IDO687 R R R R
A12086S-12 IDO687/W14//IDO687 R R R R
A12086S-13 IDO687/W14//IDO687 S S S S
A12004S-1 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-3 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-10 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-14 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
IDO1603S 2016 Gp 100 S3 R S S
UI Stone 2016 Gp 100 S S S S
A12004S-15 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-16 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-17 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-18 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-19 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S3 R S S
A12004S-20 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-21 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-24 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S3 R S S
A12004S-27 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-28 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-29 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12004S-30 IDO694/N9016/3/JFSN*4/IDO584 (70-5)//Lassik S S S S
A12041S-1 IDO851/N9016//IDO851 S S S S
A12041S-3 IDO851/N9016//IDO851 S S S S
A12041S-4 IDO851/N9016//IDO851 S S S S
A12041S-5 IDO851/N9016//IDO851 S S S S
A12079S-1 IDO686/N9016//IDO686 S S S S
A12089S-3 IDO687/N9016//IDO687 S S S S
A12089S-6 IDO687/N9016//IDO687 R H R R
A12089S-7 IDO687/N9016//IDO687 S S S S
A12089S-8 IDO687/N9016//IDO687 S S S S
A12089S-9 IDO687/N9016//IDO687 S S S S
A12089S-10 IDO687/N9016//IDO687 S S S S
A12089S-11 IDO687/N9016//IDO687 S S S S
A12089S-12 IDO687/N9016//IDO687 R R R R
A12089S-13 IDO687/N9016//IDO687 S S S S
A12089S-14 IDO687/N9016//IDO687 S S S S
A12089S-16 IDO687/N9016//IDO687 S S S S
A12089S-18 IDO687/N9016//IDO687 S S S S
A12089S-19 IDO687/N9016//IDO687 S S S S
A12089S-20 IDO687/N9016//IDO687 S S S S
A12089S-21 IDO687/N9016//IDO687 S S S S
A12039S-2 IDO851/Futai 8944//IDO854 R H R R
A12039S-3 IDO851/Futai 8944//IDO854 S S S S
Disagreements between PFT_KASP and UMN10 are highlighted in red font.
R- presence of resistant allele; S- presence of susceptible allele in case of UMN10 and absence of allele for PFT_KASP, PFT_GSP and SGNH_GSP; H-presence of both resistant and susceptible alleles; S3- presence of non-functional PFT allele (S3 haplotype)

Haplotypes of genes spanning Fhb1 region in the eight lines showing disagreements between PFT_KASP and UMN10 genotyping

Entry Gene/markera Haplotypes

TSb Hisb UMN10c PFTc SGNHc
IDO1603S R R R S3 S
A12004S-19 R R R S3 S
A12004S-24 R R R S3 S
H0900081 R R R S3 S
Lassik R R R S3 S
UC1603 R R R S3 S
UC1618 R R R S3 S
9263 R R R S3 S
Genes/markers in Sumai 3 Fhb1 region sequence (KX907434.1) where TS: Terpene Synthase, His: Histidine rich calcium-binding protein, PFT: Pore-forming toxin-like gene, and SGNH: SGNH Plant lipase
Haplotypes identified based on sequencing
Haplotypes identified based on genotyping with markers
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