Quantitative trait loci responsible for sharp eyespot resistance in common wheat CI12633

Sharp eyespot is a major fungal disease of wheat caused by Rhizoctonia cerealis in cool and humid environments worldwide. In this study, 224 single seed descent derived F13, F14 and F15 recombinant inbred lines (RILs) from the cross between CI12633 (a resistant cultivar) and Yangmai 9 (a susceptible cultivar) were assessed for sharp eyespot resistance (R.cerealis isolate R0301) in field and greenhouse conditions in three growing seasons. Different agronomic characteristics were also evaluated in the field with no disease infection. All the lines were genotyped with the Illumina iSelect 90 K SNP wheat chip and 101 SSR markers. Sharp eyespot resistance was significantly negatively correlated with heading date and tiller angle, and significantly positively correlated with the diameter of the basal first internode and second internode. Five QTL with a likelihood of odds ratio score of higher than 3.0 were detected on chromosomes 2BS, 4BS, 5AL and 5BS, respectively. These identified QTL may be used in future wheat breeding programs through marker assisted selection for developing sharp eyespot resistant cultivars.

CI12633 is an R. cerealis-resistant winter wheat line. Our study aimed to clarify the relationship between sharp eyespot resistance and the five independent agronomic factors and to identify sharp eyespot resistance QTL and tightly linked SNP markers for use in future marker-assisted selection programs or positional cloning.

Results
Performance of the measured traits in three years. The distribution pattern of the disease index for sharp eyespot in the 224 RILs indicated that resistance to sharp eyespot is controlled by multiple genes and not by a single gene in the CI12633/Yangmai 9 population (Fig. 1). The skewness and kurtosis were all near zero, which indicated that resistance trait in CI12633 exhibited a normal distribution at all the environments.  (Table 1).
Some disease index detected from RILs which influenced by others pathogen must be abandoned in the data analysis. In the investigation, we found that disease index of sharp eyespot can be infected by wheat powdery mildew and yellow mosaic virus. Usually, disease index of RILs infected by these pathogen was very high.  The analysis of variance (ANOVA) for disease index showed a significant variation for the genotypes and five environments ( Table 2). The disease was serious in all environments except F2016 for the small amount of rain in this year which led to air humidity was insufficient for sharp eyespot being hardly occurred even though sprinkled with water every day. Additionally, we found that the correlations for disease index between different trials were all positively significant at the P = 0.01 level ( Table 3), indicating that the expression of the disease resistance was consistent across the environments thus the data were further used for QTL analysis.
Correlations between disease index with other agronomic traits. The disease index were negatively correlated with heading data (HD) ( Table 3). Early heading lines tended to have greater disease index (more susceptible to sharp eyespot) in comparison to lines with late heading dates. The disease index also showed significant positive correlation with first internode diameter (FID) and second internode diameter (SID), indicating that plants with more slender stem are less prone to disease infection. Plant height (PH) is an important trait of wheat, which had no significantly negatively correlated with disease index in each environment (Table 3). A significant negative correlation between disease index and tiller angle (TA) was found in both F2014 and F2015 trials. Lines with greater TA showed less sharp eyespot symptom. SNP polymorphism and genetic mapping. The CI12633/Yangmai 9 RIL population was genotyped with the wheat 90 K SNP array which resulted in 2829 polymorphic markers. In addition to the SNP markers, 205 SSR markers were also used to genotype the population. After removing unlinked markers, the resulting map consisted of 2012 markers (1911 SNP markers and 101 SSR markers) spanning 12434.23 cM in length with an average locus density of 6.18 cM/locus.

Discussion
Phenotyping is a critical step for gene mapping. Limited research has been conducted to date on this disease because of the difficulties associated in accurately phenotyping the plant's response to sharp eyespot 3 . One of the methods is the use of wheat kernels inoculated with the pathogen and subsequent quantitative categorization using a scale of 0 to 5 based on disease severity 15 . This method is dependent on the placement of the wheat kernel, i.e. the infected kernels should be placed close to but not touching the roots of the plants. The direct contact of plant roots with the inoculated pathogen will allow the pathogen to attack the plant persistently, leading to a higher disease index even in resistant plants, particularly when the environmental conditions are suitable for pathogen growth 16 . In this study, the inoculation was carefully conducted to prevent the direct contact between plant roots and the pathogen and all inoculated plants were dug up for the evaluation of disease index. This is a time-consuming and labor-intensive phenotyping method but the reliability of the results was improved. Two other methods of inoculation, toothpick and seedbed inoculation, are generally used by researchers, with many studies finding that toothpick inoculation was more effective 2,6-8 . In this research, however, the toothpick inoculation method could not be used because the sheath of CI12633 and its descendants was too thin and narrow and would break with the insertion of the toothpick.  4 identified several QTL for sharp eyespot resistance. These QTL located on chromosomes 1A, 2B, 3B, 4A, 5D, 6B, and 7B, respectively. Among these, the QTL detected on chromosome 2BS was also identified in our study, though their precise location was unclear. BLAST analysis of the GrainGenes and WHEAT URGI websites found that the markers RAC875_c730_234 and RAC875_c16697_1502 were similar to the SSR markers Xbarc101-2 and barc200, respectively, suggesting that QSe.jaas-2BS and QSe.cau-2BS may be the same 5,7 . The QTL on Chromosomes 4BS, 5AL (2) and 5BS identified in the current study were nor reported before. Interestingly, the resistance QTL on 2B (QSe.jaas-2BS) with larger effect came from the susceptible wheat Yangmai 9. Some resistance gene have been identified previously in susceptible parents. For instance, a rice sheath blight (SB) resistance QTL allele came from the susceptible cultivar Lemont has been successfully fine mapped by Zuo et al. 16 . It is more likely that the wheat Yangmai 9 is not highly susceptible to sharp eyespot disease. In Yangmai 9, the resistance QTL QSe.jaas-2BS may play a decisive role. It's not uncommon that a lot of disease resistance QTL mapped so far do not behave consistently in difference environments. In rice, a similar phenomenon was observed by Channamallikarjuna et al. 17 and Zuo et al. 16 in maping rice SB resistance QTLs. In this study, none of the QTL could be detected in all environments, most likely due to that the outbreak of this disease is easily influenced by environments. Thus, the identification of QTL for sharp eyespot resistance should be based on multi-experiments/environments.

The association between sharp eyespot resistance and agronomic traits. The association between
QTL for sheath blight resistance and agronomic traits in rice has been reported 18 . Little research is conducted on the association between agronomic traits and wheat sharp eyespot 19 . Wheat sharp eyespot pathogens favor cool, humid and shady environments thus is generally more predominant in the lower part of the plant than the upper part. Small internode diameter is conducive to field ventilation and light crossing, thereby changing the growth condition of R. cerealis. Wheat varieties such as Shanhongmai, Xifeng, and Limai 16 with small internode diameter are generally more resistance to sharp eyespot 5 . Our experiment confirmed that disease index showed significant positive correlation with FID and SID and the resistant QTL on 2B (QSe.jaas-2BS) was associated with the internode diameter. Although significant correlations were observed between sharp eyespot resistance and HD, FID, SID, or TA, more detailed analysis are required to establish any definitive link between these phenological traits and plant's response to this disease.
In summary, the markers closely linked to sharp eyespot resistance genes identified in present investigation can be used for marker assisted selection in wheat improvement.

Materials and Methods
Plant materials. The 224 F 13-15 RILs used for QTL mapping were developed from a cross between CI12633 and Yangmai 9. Yangmai 9, a high-yielding wheat cultivar grown in the wheat region along the middle and lower   Table 3. Correlation coefficients among the four trial environments for the sharp eyespot disease index and between disease index and important agronomic traits in the RIL population of the CI12633/Yangmai 9 cross (**Significant at P < 0.01 and *significant at P < 0.05).  As humid and wet weather conditions prevail in Yangzhou during spring this area is very conducive for sharp eyespot outbreak. All research materials were sown on October 20 th for each of the three years, in a randomized complete block design with three replicates. CI12633 and Yangmai 9 were included every 50 lines in each replicate. Each plot was 0.8 m long and rows were spaced 0.4 m apart; in each row, approximately 50 seeds were sown. To achieve a plant density of 40 plants per row some seedlings were removed. High plant densities provided favorable conditions for the spread of sharp eyespot. In early March, when the weather became warm and wet, the plants were inoculated by placing R0301-colonized wheat kernels on the soil surface close to the plants, but the kernels should not be touched with the plants. The kernels were then covered with earth and water was sprinkled three times a day for the first month in non-raining days. Overall, the weather was very suitable for sharp eyespot outbreak two of three seasons with consistent rainfall occurring throughout the experimental period until the final disease recording.

QTL a Marker interval
In early May, when the susceptible parent Yangmai 9 began to succumb to R. cerealis infection, all sample plants except the edge plants were dug up and individually assessed for disease (approximately 200-250 wheat stems of each line). The infection types were categorized qualitatively from 0 to 5: 0, no lesion; 1, the lesion appeared on the sheaths rather than stems; 2, the width of the lesion was <50% of the infected stem perimeter; 3, the width of the lesion is >50 and <75% of the infected stem perimeter; 4, the width of the disease lesion is >75% of the infected stem perimeter; 5, white spike or dead plant. Disease index = ((0 × X 0  All materials were sown in early November in a randomized complete block design with three replicates. The planting and management patterns were similar to those in the field. A water sprinkler system was constructed over the plants that was used to keep the experimental environment moist. In mid-February, when wheat plants were at the tillering growth stage, R0301-colonized wheat kernels were added to the soil surface and covered with soil. The water sprinkler system and temperature control system were then used to facilitate R. cerealis infection and development. When Yangmai 9 began to succumb to sharp eyespot, all the plants were dug up for disease assessment. The disease score was calculated as described above. Agronomic trait data. All the RILs and both parents were planted in field conditions in the same rand- QTL analyses. QTL analyses were conducted by composite interval mapping (CIM) with the software WinQTLCart 2.5. To declare QTL significant, the threshold logarithm of the odds score was arbitrarily set at 3.0. Adjusted means for percent disease index values each year were calculated before pooling the results from all the years for statistical analyses. The QTL were named according to the International rules of Genetic Nomenclature (http://wheat.pw.usda.gov/ggpages/wgc/98/Intro.htm). If one QTL only detected one time, it will be discarded.