Mapping of a Major QTL, qBK1Z, for Bakanae Disease Resistance in Rice

Bakanae disease is a fungal disease of rice (Oryza sativa L.) caused by the pathogen Gibberella fujikuroi (also known as Fusarium fujikuroi). This study was carried out to identify novel quantitative trait loci (QTLs) from an indica variety Zenith. We performed a QTL mapping using 180 F2:9 recombinant inbred lines (RILs) derived from a cross between the resistant variety, Zenith, and the susceptible variety, Ilpum. A primary QTL study using the genotypes and phenotypes of the RILs indicated that the locus qBK1z conferring bakanae disease resistance from the Zenith was located in a 2.8 Mb region bordered by the two RM (Rice Microsatellite) markers, RM1331 and RM3530 on chromosome 1. The log of odds (LOD) score of qBK1z was 13.43, accounting for 30.9% of the total phenotypic variation. A finer localization of qBK1z was delimited at an approximate 730 kb interval in the physical map between Chr01_1435908 (1.43 Mbp) and RM10116 (2.16 Mbp). Introducing qBK1z or pyramiding with other previously identified QTLs could provide effective genetic control of bakanae disease in rice.


Introduction
Bakanae disease, which means foolish seedling in Japanese, was firstly identified in 1828 in Japan [1], and is widely distributed in temperate zones as well as tropical environments and occurs throughout rice growing regions of the world [2].
Four Fusarium species including F. andiyazi, F. fujikuroi, F. proliferatum and F. verticillioides in the G. fujikuroi species complex have been associated with bakanae disease in rice [3]. This disease is typically a seed-borne fungus, but may occur when the pathogen is present in plant material or soil. Infected seeds/plants result in secondary infections [4], which spread through wind or water. Bakanae disease has different symptoms such as tall, lanky tillers with pale green flag leaves. Infected plants also have fewer tillers, and plants surviving till maturity bear only empty panicles [5], resulting in yield loss [6,7]. Low plant survival and high spikelet sterility [5] may account for yield losses of up to 50% in Japan [7], 3.0-95% in India [8][9][10], 3.7-14.7% in Thailand, 5-23% in Spain, 40% in Nepal [10], 6.7-58.0% in Pakistan [11], 75% in Iran [12] and to 28.8% in Korea [13]. Germinating rice seeds in seed boxes for mechanical transplantation has caused many problems associated with diseases [14] including bakanae disease, which are not considered serious in direct seeding. Hot water immersion and fungicide treatment are the most common ways of seed disinfection [10,15,16]. However, both the hot water treatment and application of fungicide are insufficient to control bakanae disease. Thermal effect does not reach the pericarp of the severely infected rice seeds. The application of fungicides is not functioning well for destroying the spores of this fungal pathogen, and some pathogen showed resistance to the fungicides [13,[17][18][19]. Therefore, the genetic improvement of rice using the quantitative trait loci (QTLs)/genes providing bakanae disease resistance would be a more effective way to control bakanae disease.
Several QTLs associated with bakanae disease resistance have been identified and those can be used for marker-assisted selection in rice breeding as well as for understanding the mechanisms of resistance. Yang et al. [20] identified two QTLs located on chromosome 1 and chromosome 10 by in vitro evaluation of the Chunjiang 06/TN1 doubled haploid population. Hur et al. [21] identified a major QTL, qBK1, on chromosome 1 from 168 BC 6 F 4 near isogenic lines (NILS) generated by crossing the resistant indica variety Shingwang with susceptible japonica variety Ilpum. Lee et al. [22] delimited the location of qBK1 to 35 kb interval between two InDel (Insertion-deletion) markers, InDel 18 (23. [25] identified qBK1_628091 (0.6-1.0 Mbp on chromosome 1) and qBK4_31750955 (31.1-31.7 Mbp on chromosome 4) by GWAS (genome-wide association study) approach using 138 japonica rice germplasms. Kang et al. [26] discovered the QTL qFfR9 at 30.1 centimorgan (cM) on chromosome 9 from a japonica variety Samgwang. Lee et al. [16] found the QTL qBK1 WD located between markers chr01_13542347 (13.54 Mb) and chr01_15132528 (15.13 Mb) from the japonica variety Wonseadaesoo. They also found that resistance of gene pyramided lines harboring two QTLs, qBK1 WD and qBK1, was significantly higher than those with only qBK1 WD or qBK1. Identifying new resistance genes from diverse sources is important for rice breeding programs to acquire durable resistance against bakanae disease by either enhancing the resistance level, helping to overcome the breakdown of resistance genes, or both. In this study, we aimed to provide a new genetic source, qBK1 z with detailed gene locus information for developing resistant rice lines which contains single or multiple major QTLs to enhance bakanae disease resistance.

Bakanae Disease Bioassay in Parents and F 2:9 RILs
The proportion of healthy Zenith (resistant) and Ilpum (susceptible) plants was evaluated with 10 biological replicates after inoculation of the virulent F. fujikuroi isolate CF283 [27]. Most Zenith plants did not exhibit a thin and yellowish-green phenotype, which is a typical symptom of bakanae disease, unlike Ilpum ( Figure 1A).
The proportion of healthy Zenith plants was 63.2 ± 11.8% (ranging from 42.7% to 79.3%), which was significantly different from that of Ilpum (14.3 ± 11.4%, ranging from 3.3% to 37.0%) ( Figure 1B). Zenith and Ilpum were further inoculated with green fluorescent protein (GFP)-tagged F. fujikuroi isolate CF283. Ten days after inoculation, plants with typical disease symptom of each variety were subjected to a confocal microscopy analysis. Confocal imaging of radial and longitudinal sections of the basal stem showed that the fungus penetrated and was localized easily and abundantly at vascular bundle, mesophyll tissue and hypodermis in the susceptible Ilpum variety, while only a background level of GPF signal was detected in the resistant Zenith ( Figure 2).

QTL Analysis and Mapping of qBK1 z Using 180 F 2:9 RILs
Based on the bakanae disease bioassay (proportion of healthy plants), the 180 F 2:9 RIL population exhibited continuous distribution (ranged from 0% to 98.0%; Figure 3), which quantitatively confirmed the inheritance of bakanae disease resistance. We selected 164 markers showing polymorphism between Ilpum and Zenith from 1150 RM markers (http://gramene.org, accessed on 12 August 2018) tested. which covering the whole rice chromosome ( Figure A1). The genetic linkage map of Ilpum and Zenith for primary mapping was constructed with 164 polymorphic markers covering a total length of 3140 cM with average interval of 19.14 cM as described by Lee et al. [28]. Primary QTL mapping using the 180 F 2:9 populations showed that a significant QTL associated with bakanae disease resistance at the seedling stage was located between the SSR markers, RM1331 and RM3530 on chromosome 1, and it was designated qBK1 z . The LOD score of qBK1 z was 13.43, which accounted for 30.9% of the total phenotypic variation (Table 1). A finer localization of qBK1 z was determined by analyzing the chromosome segment introgression lines in the region detected from primary mapping. The qBK1 z region between RM1331 and RM3530 from primary mapping was narrowed downed with an additional 55 SSR markers and 12 InDel markers designed for the insertion/deletion sites based on the differences between the japonica (http://www.gramene.org, accessed on 12 August 2018) and indica (http://rice.genomics.org.cn, accessed on 12 August 2018) sequences. Four SSR markers and six InDel markers were selected as polymorphic markers between the parents to narrow down the position of the qBK1 z region (Table A1). Finally, seven homozygous recombinants were selected from the F 2:9 lines using 14 markers in the 2.8 Mb region around the SSR markers RM1331 and RM3530 (Figures 4 and 5). The proportion of healthy plants of the seven homozygous recombinants was evaluated with three biological replicates according to Duncan's new multiple range test. Based on this bioassay, lines classified to Group "a" were regarded as resistant, and Group "b" as susceptible (Figure 4). Considering the genotype and the phenotype of the recombinants, it is clear that qBK1 Z conferring resistance to bakanae was an approximate 730 kb interval delimited by the physical map between Chr01_1435908 (1.43 Mbp) and RM10116 (2.16 Mbp).

Discussion
Rice varieties with a single resistance gene are at an increased risk of being overcome by new pathological races [16,28]. The development of a rice variety with a higher level of resistance against bakanae disease is a major challenge in many countries [21,23,[29][30][31]. In this study, we identified qBK1 z locus related to bakanae disease resistance based on genotype and phenotype analyses of homozygous recombinants on the recombinant progeny of Ilpum and Zenith, using SSR and newly developed InDel markers.
It was reported that successful infection of Fusarium species is a complex process that includes adhesion, penetration (through wounds, seeds, stomatal pores) and subsequent colonization within and between cells [32,33]. Lee et al. [16] revealed that the fungus F. fujikuroi was more abundant in the stem of the susceptible variety than it was in the resistant one. Elshafey et al. [34] indicated that F. fujikuroi prefer to grow in aerenchyma, pith, cortex and vascular bundle of both sheath and stem of rice. In this study, we examined both the localization and abundance of F. fujikuroi isolate CF283 in the basal stem of rice using GFP-tagged F. fujikuroi isolate CF283 (Figure 2). Consistent with previous reports [16,34], F. fujikuroi isolate CF283 was extensively observed on the vascular bundle, mesophyll tissue and hypodermis of infected stems in susceptible the Ilpum variety, whereas this was rarely observed in resistant Zenith.
Many QTLs on bakanae disease resistance have been identified on chromosome 1. Three QTLs, qBK1 z , qBK1.2 and qBK1.3, were found in a similar region in spite of the different source of resistant varieties ( Figure 6). Fiyaz et al. [23] mapped qBK1.1 to a 20 kb region between markers RM9 and RM11232 from the Pusa 1121/Pusa1342 cross. These authors hypothesized that qBK1.1 and qBK1 [21] might be the same QTL as they had overlapping positions. Ji et al. [24] found that QTL qFfR1 was located in a 230 kb region of rice chromosome 1 in Korean japonica variety Nampyeong, and suggested that the three QTLs qBK1, qBK1.1 and qFfR1 might indicate the same gene. Lee et al. [22] narrowed down the position of the qBK1 locus to a 35 kb region between InDel 18 and InDel 19-14, and revealed that location of qBK1 is close to those of qBK1.1 and qFfR1, and do not overlap each other. Two additional QTLs including qB1 from Chunjiang 06 [20] and qBK1 WD from Wonseadaesoo [16] were also found on chromosome 1. Gene pyramiding via phenotypic screening assays for crop breeding is considered to be difficult and often impossible due to dominance and epistatic effects of genes governing disease resistance, and the limitation of screenings being all year-round [35]. Pyramiding of multiple resistant QTLs/genes by using marker-assisted breeding (MAB) in a single plant might confer either higher, durable, or both, resistances against bakanae disease. The effects of pyramiding resistance genes have been observed for several plant-microbe interactions. Pyramiding three bacterial blight resistance genes resulted in a high level of resistance and were expected to provide a durable pathogen resistance [36,37]. On the other hand, pyramiding of resistant genes resulted in a level of resistance that was comparable to or even lower to than that of the line with a single gene. For example, Yasuda et al. [38] reported rice lines with pairs of blast resistance genes to be only comparable to lines with a single gene which may have a stronger suppressive effect. Our previous study of bakanae disease resistance [16] revealed that the gene pyramided lines harboring qBK1 WD + qBK1 Plants 2021, 10, 434 8 of 12 had a much higher levels of resistance than those possessing either qBK1 WD or qBK1. The novel QTL, qBK1 Z , identified in this study can be utilize in MAB and gene pyramiding to achieve higher resistance in many bakanae disease prone rice growing areas.
In this study, we identified a new major QTL qBK1 z conferring bakanae disease resistance from a new genetic source of indica variety, Zenith. Through QTL analysis and fine mapping, we narrowed down the qBK1 z locus into 730 kb on the short arm of chromosome 1 which is a novel locus compared with all the previously identified bakanae disease resistance QTLs. Together with the previously identified QTLs of the bakanae disease resistance, the new qBK1 z can be introduced to the elite favorable background varieties by a marker-assisted backcrossed breeding. Furthermore, the information of the localization and different abundance on the vascular bundle, mesophyll tissue and hypodermis of infected stems of infected rice between resistant and susceptible varieties will be useful for further studying an interaction between the pathogen (F. fujikuroi) and rice host plants.

Plant Materials
Zenith, a medium grain type indica variety from USA released in 1936, was identified as resistant to bakanae disease in a preliminary screening of rice germplasm (data not shown). We generated 180 F 2:9 RILs from a cross between susceptible variety, Ilpum, and resistant variety, Zenith, for QTL analysis. The population was developed in the experimental fields of the National Institute of Crop Science in Miryang, Korea.

Evaluation of Bakanae Resistance and Statistical Analysis
The inoculation and evaluation of bakanae disease were conducted using a method described by Lee et al. [22]. The isolate CF283 of F. fujikuroi was obtained from the National Academy Agricultural Science in Korea. Isolate was inoculated in potato dextrose broth (PDB) and cultured at 26 • C under continuous light for one week. The F. fujikuroi culture was washed by centrifugation with distilled water, and the spore concentration was adjusted to 1 × 10 6 spores/mL. Forty seeds per each line were placed in a tissue-embedding cassette (M512, Simport, Beloeil, QC, Canada). Before inoculation, the seeds in the tissue-embedding cassette were surface sterilized with hot water (57 • C) for 13 min, then allowed to drain and cool. Subsequently, the seeds were soaked in the spore suspensions (1 × 10 6 spores/mL) for 3 d for inoculation with gentle shaking four times a day for equilibration. After inoculation, thirty seeds per line were sown in commercial seedling trays, and seedlings were grown in a greenhouse (28 ± 3 • C day/23 ± 3 • C night, 12 h light). Bakanae disease symptom on each line was evaluated by calculating the proportion of healthy plants at 1 month after sowing. The healthy and non-healthy plants are classified as described by Kim et al. [27]. The plants exhibiting elongation with thin and yellowish-green, stunted growth, and dead seedlings were classified as non-healthy plants.
The plants showing the same phenotype as the untreated plants, slight elongation then normal growth without thin and yellowish-green were regarded as healthy plants.
Statistical differences between means were analyzed using Duncan's multiple range test after one-way analysis of variance (ANOVA). The level of significance was designated as p < 0.05 and was determined using the SAS Enterprise Guide 4.3 program (SAS Institute Inc., Cary, NC, USA).

Localization of F. fujikuroi in Zenith and Ilpum Plants
Shoot bases of 10-day-old seedlings derived from Zenith and Ilpum seeds inoculated by GFP-tagged F. fujikuroi isolate CF283 [16] were observed under confocal laser-scanning microscopy (LSM-800, Zeiss, Germany) at GFP channel, and images were obtained using Zeiss LSM Image Browser. All experiments were conducted twice with at least three replicates.

DNA Extraction and Polymerase Chain Reaction
Genomic DNA from young leaf tissue was prepared according to the CTAB method [39] with minor modifications. Polymerase chain reaction (PCR) was performed in 25-µL reaction mixture containing 25 ng template DNA, 10 pmol of each primer, 10× e-Taq reaction buffer, 25 mM MgCl2, 10 mM dNTP mix and 0.02 U of SolGent e-Taq DNA polymerase (SolGent, Daejeon, South Korea). The reaction conditions were set as follows: initial denaturation at 94 • C for 2 min; 35 cycles of denaturation at 94 • C for 20 s, annealing at 57 • C for 40 s and extension at 72 • C for 40 s; and a final extension at 72 • C for 7 min. The amplification products were electrophoresed on a 3% (w/v) agarose gel and visualized by ethidium bromide staining.

QTL Analysis of the F 2:9 Population and Development of InDel Markers for Fine Mapping
Polymorphic SSR markers (n = 164) that were evenly distributed on rice chromosomes were selected from the Gramene database (http://www.gramene.org, accessed on 12 August 2018). These markers were used to construct a linkage map and for QTL analysis of the F 2:9 populations. The linkage map was constructed using Mapmaker/Exp v.3.0, and the genetic distance was obtained using the Kosambi map function [40]. Putative QTLs were detected using the composite interval mapping (CIM) function in WinQTLcart v.2.5 (WinQTL cartographer software [41]. A logarithm of the odds (LOD) ratio threshold of 3.0 was used to confirm the significance of a putative QTL. InDel markers were developed based on the fragment size differences in the sequence (in the range of 20 bp) between japonica (Gramene database http://www.gramene-.org, accessed on 12 August 2018) and indica (BGI-RIS; http://rice.genomics.org.cn, accessed on 12 August 2018) in the target region on chromosome 1. The primers were designed using Primer3 software (http://web. -bioneer.co.kr/cgi-bin/primer/primer3.cgi, accessed on 12 August 2018).

Conclusions
In this study, we identified a new major QTL qBK1 z conferring bakanae disease resistance from a new genetic source of indica variety, Zenith. Through QTL analysis and fine mapping, we narrowed down the qBK1 z locus to 730 kb on the short arm of chromosome 1 which is a novel locus compared with all the previously identified bakanae disease resistance QTLs. This new qBK1 z can be introduced to the elite favorable background varieties by a marker-assisted backcrossed breeding together with the previously identified QTLs of the bakanae disease resistance. Furthermore, the information of the localization and different abundance on the vascular bundle, mesophyll tissue and hypodermis of infected stems of infected rice between resistant and susceptible varieties will be useful for further studying the interaction between the pathogen (F. fujikuroi) and rice host plants.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.

Appendix A
Funding: This research was funded by the Rural Development Administration, Republic of Korea, grant number PJ01477401 (Project title: QTL mapping for development of functional rice with bakanae disease resistance).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest. Appendix A Figure A1. Linkage map constructed with 180 F2:9 recombinant inbred lines (RILs) derived from a cross between Zenith and Ilpum.