QTL mapping of adult-plant resistance to leaf and stripe rust in wheat cross L224-3/Zhengzhou5389

Wheat leaf rust and stripe rust are important diseases worldwide. Breeding resistant cultivars is an effective means to control wheat leaf and stripe rust. Wheat line L224-3 currently has high resistance to wheat leaf and stripe rust at the field. In this study, 166 recombinant inbred lines (RILs) derived from the L224-3 × Zhengzhou 5389 cross were used to map quantitative trait locus (QTL) for leaf and stripe rust resistance. The RILs and two parents were phenotyped for leaf rust severity at Baoding in Hebei province and Zhoukou in Henan province in the 2015/2016 and 2016/2017 cropping seasons, and for stripe rust severity at Baoding in Hebei Province and Mianyang in Sichuan Province in the 2015/2016 and 2016/2017 growth seasons. All the RILs and parents were also genotyped with the 660 K SNP array and simple sequence repeat (SSR) markers to screen for potential polymorphic markers associated with rust resistance. Four QTLs on chromosomes 1A, 2A, 4B and 7B, were detected using inclusive composite interval mapping (IciMapping). QLr.hbau-1A/QYr.hbau-1A, derived from susceptible parent Zhengzhou 5389, was pleiotropic for both leaf rust and stripe rust resistance and maybe a novel QTL. The second QTL, QLr.hbau-2A/QYr.hbau-2A derived from L224-3 for leaf rust and stripe rust resistance is possibly Lr37/Yr17. QLr.hbau-4B/QYr.hbau-4B might be a new locus for leaf rust and stripe rust resistance. The fourth QTL, QYr.hbau-7B is possibly a new QTL. The QTL identified in the present study with their flanking markers might be used for candidate gene mining and marker-assisted selection (MAS) in wheat breeding programs for rust resistance.


Introduction
Puccinia triticina-induced leaf rust (LR), and Puccinia striiformis-caused stripe rust (YR) are important wheat diseases worldwide.Typically, leaf rust epidemics cause losses of up to 40% (Khan et al. 2013(Khan et al. ), and which was recorded in 1969(Khan et al. , 1973(Khan et al. , 1975(Khan et al. , 1979(Khan et al. , 2012 and 2015 caused great yield losses in China (Peng et al. 2016;Zhou et al. 2013).Wheat stripe rust epidemic occurred in 1950, 1964, 1990, 2002 and 2017, with wheat annual yield losses up to 13.8 billion kg (Ma 2018).Breeding and rational utilization of resistant cultivars is the most economical, effective and safe measures to control these diseases (Ge et al. 2011).
L224-3, an advanced wheat line introduced by the Institute of Agricultural Sciences of Huangfanqu Farm in Henan Province, showed good APR to leaf rust and stripe rust in the field.Zhou et al. (2018) mapped a dominant gene LrL224 derived from L224-3 on 1B.Therefore, it is of great significance to identify the APR genes in the line for enriching wheat resistance genes in China.In this study, the RIL population from L224-3/Zhengzhou 5389 was used for mapping QTL for APR to leaf and stripe rust, providing a high-quality resistance source for wheat breeding program.

Plant materials and pathotypes
QTLs for leaf rust and stripe rust resistance were mapped using 166 RILs from L224-3/Zhengzhou 5389.Zhengzhou 5389, which is highly susceptible to leaf rust and stripe rust, was used as a susceptible check.Four Pt pathotypes (THTT, THTQ, THTS and PHPS) and three Pst pathotypes s (CYR32, CYR33 and CYR34) were adopted for phenotyping the RILs in the field.The population and four Pt pathotypes were preserved and propagated in our laboratory, and three Pst pathotypes were kindly provided by Dr. Taiguo Liu from the Institute of plant protection, Chinese Academy of Agricultural Sciences.

Field trial
The resistant parent L224-3, the susceptible parent Zhengzhou 5389 and all the 166 RILs were planted at Baoding, Hebei Province and Zhoukou, Henan Province in the 2015/2016 and 2016/2017 cropping seasons for the evaluation of LR severities.Both places are hotspots for leaf rust infection and spread.Similarly, the YR field experiments were conducted in Baoding, Hebei Province and Mianyang, Sichuan Province during the 2015/2016 and 2016/2017 growing seasons.Field trials were conducted in randomized complete blocks with two replicates.Each plot consisted of a single 1.5 m row with 0.25 m between rows with approximately 60 seeds were sown in each row.The susceptible line Zhengzhou 5389 was planted in each of ten rows to encourage the spread of ureadiaspores.Spreader rows of Zhengzhou 5389 were also planted perpendicular and adjacent to the test rows.
Inoculation for stripe rust was carried in beginning of January in Mianyang and mid of April in Baoding, and for leaf rust was conducted in April at both Zhoukou and Baoding.Stripe rust epidemics was initiated by spraying aqueous suspensions of urediniospores of an equal mixture of Pst pathotypes, CYR32, CYR33 and CYR34 to which a few drops of Tween 20 (0.03%) were added onto the spreader rows Zhengzhou 5389 at tillering stage.These Pst pathotypes were virulent to most Chinese wheat cultivars.Similarly, equal mixtures of Pt pathotypes (THTT, THTQ, THTS and PHPS) were added to Zhengzhou 5389 at tillering stage.Disease severities (percentage of leaf area covered with uredinia) were scored twice in each environment according to the modified Cobb scale by Peterson et al. (1948).Final disease severity (FDS) data was used for QTL analysis.

Genotyping and linkage map construction
Genomic DNA of RILs and parents was extracted from uninfected seedling leaves by the cetyltrimethylammonium bromide (CTAB) method (Sharp et al. 1988).DNA concentration was determined using Thermo Scientific NanoDrop 2000.Based on the Bulked Segregation Analysis (BSA) proposed by Michelmore et al. (1991), and with reference to the Prefer Screened Group (PSG) approach proposed by Hao et al. (2008), five lines extremely resistant to both rusts and five lines extremely susceptible to both rusts were selected.Ten lines and both parents were used as subpopulations and genotyped with the Affymetrix 660 K SNP Array for screening polymorphic SNP markers.Based on the chromosomal position of polymorphic SNP markers, SSR markers in this region will be selected for marker screening and genetic linkage map construction.

QTL analysis
QTL mapping was performed with the ICIM methods of IciMapping software (Meng et al. 2015).A logarithm of odds (LOD) threshold of 3.0 was adopted for considering a significant QTL.Stepwise regression was used to detect the percentages of phenotypic variance explained (PVE) by individual QTL and additive effects at the LOD peaks.QTL identified in individual environments with overlapping 20 cM intervals were considered to be identical.The identified flanking sequences of all SNP and SSR probes were subjected to BLAST against the Chinese Spring wheat reference sequence (IWGSC RefSeq v1.0, https:// urgi.versa illes.inra.fr/ blast_ iwgsc/ blast.php) to hit their physical positions.

Phenotypic evaluation to stripe rust and leaf rust
The FDS for leaf rust of L224-3 was less than 10% indicating good resistance to leaf and stripe rust.The FDS of Zhengzhou 5389 was 100% across four environments, while the mean stripe rust FDS of Zhengzhou 5389 ranged from 50 to 100% in all environments (Figs. 1, 2).The FDS in eight environments were significantly correlated with Pearson correlation coefficients (r) ranging from 0.215 to 0.910 (P < 0.01) (Table 1).Coefficients of correlation between leaf rust and stripe rust disease scores were 0.215 to 0.910 across trials, showing likelihood of pleotropic genes conferring resistance to both rusts.

Linkage map construction
After removing SNPs with monomorphism, markers with missing rate > 10%, and sites with distortion ratios (P < 1E −5 ), 2,941 polymorphic markers were retained, 96 for the 1A chromosome, 213 for the 2A chromosome, 177 for the 2B chromosome, 192 for the 3A chromosome, 1882 for the 4B chromosome, 172 for the 6D chromosome, and 209 for the 7B chromosome (Fig. 3).A total of 1142 pairs of molecular markers (SSR primers) were screened to detect polymorphism between resistant parent L224-3 and susceptible parent Zhengzhou 5389.The results showed that 356 pairs of SSR primers showed polymorphism in the two parents before being analyzed for polymorphism in five resistant and five susceptible lines.Finally, 19 pairs of molecular markers linked to potentially resistant genes were screened to detect the whole RIL population and construct a genetic linkage map for QTL analysis.

Discussion
The small group analysis In the present research, a small population was used for SNP array scanning to screen polymorphic markers that are potentially associated with APR genes.Using a small population with five resistant and five susceptible lines allows for more efficient and rapid detection of markers associated with resistance genes than traditional resistant and susceptible bulks, and then constructing a genetic linkage map including resistance QTL to obtain the genetic effects of the QTL.

Fig. 3
Fig.3 Frequency distributions of number of SNPs with 0-1 exchange in 5 resistant and 5 susceptible lines scanned with wheat 660 K SNP array

Fig
Fig.4 Likelihood plots of stripe rust and leaf rust adult-plant resistance quantitative trait loci (QTLs) on chromosomes 1A, 2A, 4B and 7B identified by ICIM in the RIL population from L224-3/Zhengzhou 5389.The LOD threshold is 2.5 for declaring significance.Positions (in cM) of the molecular markers along chromosomes are shown on the vertical axes; genetic distances between markers are shown