Mapping an APR gene on 2NvS segment in wheat line K13-868
K13-868 seedlings were susceptible (IT 8) to race CYR34 in the greenhouse (Fig. 1a). However, in field environments, this line exhibited a high level of adult plant resistance (IT 3) to stripe rust when exposed to mixed races (Fig. 1b, c). IT and FDS for each line in the RIL population derived from the cross K13-868 × SY95-71, along with parental lines, were assessed across four different environments in 2020 and 2021 (Table S1). The frequency distribution for IT and FDS displayed characteristics typical of a quantitative trait (Fig. 1d, e). Subsequently, using closely linked molecular markers or functional markers for stripe rust resistance genes, commonly used Yr genes in Chinese wheat varieties were tested in this line (Table S2). The Yr17 linked marker SC_372 was detected in K13-868, suggesting it carried the 2NvS segment. To validate the genetic effect of the 2NvS segment on the adult plant stripe rust resistance of K13-868, SC_372 and six newly developed linked markers (Table S3) covering physical regions from 2.87 to 43.76 Mb on chromosome 2AS, were used for genotyping 214 F5:6 plants from K13-868 × SY95-71 to construct a genetic map spanning 25.60 cM (Table S4). Using the IT and FDS values of the four sets from the F5 and F6 populations (CZ2020, PX2020, WJ2020, and CZ2021) and their BLUP values (Table S1), a major QTL was identified consistently across all tests. This QTL was linked with markers InDel_2.87 and KP2A_34.29, explaining 18.55 to 33.16% of the phenotypic variation with LOD scores ranging from 9.69 to 18.73 (Fig. 1f; Table S5). Evidently, an APR locus exists in the 2NvS segment of the wheat line K13-868, conferring effective resistance against the current predominant Pst races of China.
Development and evaluation of NILs for stripe rust response
A pair of NILs for the 2NvS segment were selected from the progeny of a heterozygous F5 line, which was from the RIL population created by crossing K13-868 with SY95-71. Seedling tests showed that both NIL-R and NIL-S plants were susceptible to CYR34 (Fig. 2a). However, at the adult plant stage, the NIL-R plants (IT 5) exhibited greater resistance to mixed Pst races compared to the NIL-S plants (IT 8) (Fig. 2b). To better understand the dynamics of resistance conferred by the APR gene, we evaluated the response at four growth stages (ranging from the 2nd to 5th leaf stages) to race CYR34 under two temperature regimes: Low temperature (LT, 10–15°C) and High temperature (HT, 10–30°C) in the greenhouse (Fig. 2a). In the LT test, both K13-868 and NIL-R were highly susceptible to CYR34 until the 5th leaf stage when resistance was seen. In contrast, SY95-71 and NIL-S were susceptible at all four growth stages. Under the HT test, both K13-868 and NIL-R began to display resistance to CYR34 by the 4th leaf stage. These results further confirmed that the stripe rust resistance originating from the 2NvS segment in K13-868 was more effective under high temperature condition.
Fine mapping of the APR gene on the 2NvS segment using a large NIL-derived population
To narrow down the candidate gene region for the APR gene in the 2NvS segment, a large NIL-derived population of 6,389 plants was used for screening recombinants within the 2NvS segment region. In total, 22 molecular markers, located between 0.16 Mb and 31.05 Mb on the 2NvS segment (Table S3), were developed to precisely identify recombinant events. Although the 2NvS segment rarely recombines with the 2AS chromosome on wheat, we fortuitously identified 22 recombinants within this 33 Mb physical interval. Using these markers, we genotyped the 22 recombinants on the 2NvS segment (Table S6) and refined the location of the APR gene.46 families of 5 recombinants with critical recombination events were planted again in 2021–2022 (Table S7), providing additional support to the proposed location of the APR gene. We found that the recombination events were not randomly distributed across the 2NvS segment, but enriched in certain regions, e.g., the crossovers in the 14 lines were between 12.38 and 14.91 Mb. Based on the phenotypic identification of critical recombinants and families, we narrowed down the candidate gene region for the APR gene to 19.36-33 Mb in the Jagger v1.1 genome (Fig. 3).
RNA-seq analysis of NILs for stripe rust response during Pst infection
We conducted transcriptome sequencing of NILs inoculated with the fresh rust of Pst race CYR34 at both the 2nd and 5th leaf stages to investigate the key gene responses and pathways involved in the APR on the 2NvS segment. Leaves of NIL-R2, NIL-R5, and NIL-S5 for Illumina RNA sequencing were harvested at 3 dpi from Pst-inoculated and mock-inoculated plants. A total of 76.65 million 150-bp paired-end reads were obtained for 18 samples. After adapter trimming and filtering low-quality reads, a total of 74.26 million clean reads remained with an average Q20 of 96.96% and Q30 of 92.33% (Table S8). The number of reads from each sample ranged from 9.98 to 15.88 GB. The filtered clean reads were mapped to the Jagger Reference genome, and the mapping ratio of clean reads reached 96.29–97.47% (Table S8). Most reads for all samples were successfully mapped, and sequences sufficiently captured most of the expressed genes. Using RT-qPCR analysis on eight randomly selected genes, we verified that the results were consistent with the transcriptome data (Fig. S1; Table S9).
Totals of 8,513, 7,685, and 4,747 DEGs were identified in NIL-R5, NIL-S5, and NIL-R2, respectively (Fig. 4a, b). Among them, 6,570, 6,603, and 719 DEGs were up-regulated, whereas 1,943, 1,082, and 4,028 DEGs were down-regulated in NIL-R5, NIL-S5, and NIL-R2, respectively. Twenty-five up-regulated DEGs were detected only in NIL-R5. Most of them encoded proteins related to disease resistance, e.g., TraesJAG6B01G518500 encodes chitin elicitor receptor kinase 1, TraesJAG2D01G557400 encodes wall-associated receptor kinase-like 8, and TraesJAG2A01G490300 encoding probable L-type lectin-domain containing receptor kinase S.5 (Fig. 4c).
These DEGs in NIL-R5, NIL-S5, and NIL-R2 plants were further subjected to GO analysis. This analysis revealed that 248, 185, and 56 terms related to biological processes were enriched (FDR < 0.05) in up-regulated DEGs of NIL-R5, NIL-S5, and NIL-R2, respectively, whereas 163, 101, and 202 functional biological process categories were enriched in down-regulated DEGs. GO terms enriched in up-regulated DEGs of NIL-R5 were more abundant than in NIL-S5 and NIL-R2 (Fig. S2a-f). Specifically, plant immune-related pathways, including calmodulin binding (GO:0005516), protein kinase activity (GO:0004672), transmembrane receptor protein serine/threonine kinase activity (GO:0004675), and plant-type hypersensitive response (GO:0009626), were more enriched in DEGs of NIL-R5 compared to NIL-S5 and NIL-R2 (Fig. 4d, e).
Candidate genes for the APR gene in the 2NvS segment
The candidate region for the APR gene on the 2NvS segment, spanning from 19.36 Mb to 32.53 Mb on the 2AS chromosome of the Jagger reference genome, includes 266 high-confidence genes. Based on the RNA-seq data and applying the threshold (RPKM ≥ 1, observed at least once at either seedling or adult plant stages of NIL-R during Mock and Pst infection), 49 expressed genes were identified within the candidate gene region (Fig. 5a). Eleven DEGs were identified between mock and Pst infections in the adult plant stage of NIL-R. Among these, only two genes exhibited expression levels more than twice as high at the adult plant stage against Pst infection as the seedling stage (Fig. 5b). According to the gene annotations provided by the Triticeae-Gene Tribe (TGT; http://wheat.cau.edu.cn/TGT/), the two genes, TraesJAG2A01G041000 and TraesJAG2A01G046200, encode for L-type lectin-domain containing receptor kinase SIT2 and Disease resistance protein RPM1, respectively.
Marker-based detection of the 2NvS segment distribution in Sichuan wheat cultivars
Compared to the dominant marker SC_372 for detecting the 2NvS segment, the five co-dominant InDel markers developed in this study differentiate the 2NvS segment from its homoeologous wheat 2AS chromosome. This advances the marker-assisted selection (MAS) of the 2NvS segment, especially enhancing the efficiency of heterozygote genotyping screening MAS. Utilizing the newly developed marker InDel_31.05, we analyzed the 2NvS segment distribution in 259 wheat varieties from the Sichuan region released after 1996 (Fig. 6a; Table S10) and identified 106 cultivars carrying the 2NvS segment. Notably, of 81 varieties selected from years 2021 and 2022, 42 were detected with the segment, indicating a rise in proportion to 52% in the recent two years (Fig. 6b). Based on the BLUP values of FDS of the 142 varieties (Ye et al. 2019), clearly, the varieties that carried the 2NvS segment showed a significantly lower FDS than those without it, and the mean BLUP_FDS values of the varieties carrying it were reduced by 41% (Fig. 6c, d).
Agronomic trait evaluation of NILs for the 2NvS segment
During the 2021–2022 and 2022–2023 growing seasons in CZ and WJ, agronomic traits of NIL-R and NIL-S lines were compared under an equal mix of Pst inoculated conditions (Fig. 7). The presence of the 2NvS segment resulted in significantly higher (P < 0.05) values for grain number per spike (GNPS) in CZ2022, WJ2022 and CZ2023, thousand kernel weight (TKW) (P < 0.01) in WJ2022, kernel width (KW) (P < 0.01) in CZ2023. On the other hand, plant height (PH), spike length (SL), tiller number (TN), spike number (SN), and kernel length (KL) were not significantly different between the NIL-R and NIL-S plants. These findings demonstrated that the 2NvS segment offset the adverse impacts of stripe rust on yield, without negatively influencing the evaluated agronomic traits.