2.3 Location of complementary genes on chromosomes 3BS and 4BL
In order to map two complementary genes separately, we chose 114 single-gene segregating families showing a ratio 3R : 1S from 441 4N0461×Nongda4503 F2:3 families (Table S2). We made pairs of resistance and susceptibility pools from these single-gene segregating F2:3 families to screen polymorphic SSR markers. Markers gwm251 on chromosome arm 4BL and gwm533 on 3BS were polymorphic among these families, respectively, suggesting that the complementary genes were located on these two chromosomes. Accordingly, these single-gene segregating F2:3 families were divided into gwm251-segregating families and gwm533-segregating families. Then the DNA pools of gwm251 (4BL)-segregating families and gwm533 (3BS)-segregating families were used for 90 K iSelect Chip analysis, respectively.
The results of 90 K iSelect Chip analysis showed that SNPs between the resistant and susceptible pools were mainly on chromosome 3B and 4B, respectively (Fig. 2A-D, S3). Therefore, we speculated that the two resistance genes of 4N0461 may be on chromosomes 4B and 3B and temporarily named them LrN3B and LrN4B, respectively. We found that the SNPs were enriched in 0–30 Mb on chromosome arm 3BS and in 500–650 Mb on chromosome arm 4BL (Fig. 2B, 2D, S3). Accordingly, markers 4B116, 3B298, and 3B384 were developed based on the reference sequences of these two SNPs-rich regions. Markers gwm251, wmc692, and 4B116 on chromosome arm 4BL and gwm533, 3B298, and 3B384 on chromosome arm 3BS were tested on the aforementioned 441 F2 plants of cross 4N0461×Nongda4503 to genotype locus of LrN3B and LrN4B, respectively. The associations of marker genotypes and phenotypes of 441 plants were consistent with the expectations of complementary dominant genes (Table S3).
Based on their marker genotypes, we selected two susceptible F2:3 lines 9T264 and 9T265 separately carrying LrN3B or LrN4B and crossed them. The F1 plants of 9T264×9T265 showed adult plant resistance to leaf rust. When crossed with 4N0461×Nnongda4503 F1 plants, the progenies of both tested crosses showed 1R: 1S (χ2 = 0.29 and 0.22, respectively) (Fig. 1C, Table S4). These results further confirmed the inheritance and locations of the complementary resistance genes of 4N0461.
From the single gene-segregating families, we chose 301 F3 plants segregating at the LrN3B locus and 312 plants segregating at the LrN4B locus according to their marker genotypes. Both populations segregated 3R:1S (χ2 = 1.55, χ2 = 0.155, p > 0.05) (Table S5). These two populations were used for mapping LrN3B and LrN4B separately. In addition to markers barc163 and gpw7390 known to be located on the target region of chromosome arm 4BL, more markers were developed based on the resequencing data of two parents and the IWGSC RefSeq v1.1 genome sequence. Finally, four new markers 3B288, 3B95, 3B330, 3B345 (near 3B298, 3B384 and gwm533) and six markers 4B96, 4B298, 4B301, 4B342, barc163 and gpw7390 (adjacent to gwm251, wmc692 and 4B116) were used to build the genetic maps of LrN3B and LrN4B, respectively. LrN3B was mapped within a 1.66-cM interval between 3B330 and gwm533, and LrN4B with its co-segregating marker wmc692 was flanked by 4B96 and gwm251 within a 2.08-cM interval (Fig. 3).
2.4 Fine Mapping of LrN3B
To map LrN3B more finely, a bigger population of 1328 F4 plants by selfing the F3 heterozygous plants from LrN3B-segregating population were used to test more markers (Table S5).
According to the IWGSC Refseq v1.1 reference genome, there were 1.08 Mb between 3B330 and gwm533. Three Indel markers 3 − 1, 3–9, and 3–79 and one STARP marker 3G152 were developed from the interval (Fig. 4A, Table S6). Seven markers (3B330, 3B384, 3 − 1, 3–9, 3–79, 3G152 and gwm533) were used to scan 1328 plants. LrN3B was located between markers 3 − 1 and 3G152 and co-separated with 3–9. There were 13 recombinant plants between the flanking markers. Using additional 1445 F5 plants derived from the heterozygous recombinants, we delineated LrN3B within a 0.06-cM interval between 3–62 and 3–70 (corresponding to 106 kb on IWGSC RefSeq v1.1) and found that markers 3–19, 3–33 and 3–35 co-segregated with LrN3B (Fig. 4).
2.5 Candidate genes of LrN3B
According to the IWGSC RefSeq v1.1 genome, there are four annotated genes in the 106 kb interval (from 3–62 to 3–70): TraesCS3B02G014700, TraesCS3B02G014800, TraesCS3B02G014900, and TraesCS3B02G015000. The four genes encode glycerol-3-phosphate acyltransferase (G3PA), transducin family protein/WD-40 repeat family protein (TFP), tetratricopeptide repeat protein (TPR), and coatomer subunit beta'-1 (CSB), respectively (Table S7, Fig. 4C).
Our Sanger sequencing results showed that only TraesCS3B02G014800 and TraesCS3B02G014900 were different between resistant and susceptible genotypes (Table S8). We found two sequences (PP274033 and PP274034) of TraesCS3B02G014800 in 4N0461 with only one SNP (T1754C) between them and one TraesCS3B02G014800 sequence in Nongda 4503 identical to PP274034 (Fig. S3). A STARP marker TFPa was designed based on this SNP (Table S9). Using this marker, we could amplify two bands in 4N0461 and only one band in Nongda4503 and found that the marker co-segregated with LrN3B (Fig. 5A). We named the two sequences of TraesCS3B02G014800 in 4N0461 as 148A and 148B, respectively. TraesCS3B02G014800 sequence in Nongda4503 was the same as 148B (Fig. S3). There were 88 mismatches and 55 insertions/deletions between the sequence of TraesCS3B02G014800 in CS and 148B in 4N0461 and Nongda4503 and we got no amplification in CS with marker TFPa (Fig. 5A). We named this sequence variant in CS as 148C.
We found 156 mismatches and 28 insertions/deletions between the sequences of TraesCS3B02G014900 in 4N0461 (PP274036) and Nongda4503 (PP274037) (Fig. S5). Since the TraesCS3B02G014900 in Nongda4503 was the same as in CS, we named the TraesCS3B02G014900 in 4N0461 (PP274036) as 149R and that in Nongda4503 and CS as 149S. In fact, the abovementioned LrN3B-co-segregating marker 3–35 was by the sequence variation in TraesCS3B02G014900 (Fig. 5B).
Domain analysis showed that proteins encoded by TraesCS3B02G014800 and TraesCS3B02G014900 in 4N0461 had typical transmembrane structures at the N-terminal (Fig. S6). Compared with 148A, the SNP (T1754C) in 148B caused the change of an amino acid (V→A), but there is no fundamental change in the domains. The protein encoded by 149R in 4N0461 had three repetitive TPR domains at the C-terminal, a typical interaction motif. Both TraesCS3B02G014800 and TraesCS3B02G014900 could probably be the candidate genes for LrN3B.
2.6 Functional validation of LrN3B candidate genes by BSMV-VIGS
BSMV-VIGS was adopted to verify the function of candidate genes of LrN3B in 4N0461. The phytoene desaturase (PDS) gene-silencing construct was used to prove the efficacy of the VIGS system in 4N0461. After treated with BSMV virus carrying LrN3B silencing constructs targeting TraesCS3B02G014800 (TFP) and TraesCS3B02G014900 (TPR) genes, 4N0461 exhibited susceptibility to Pt PHT at the 7th-leaf stage with obvious pustules containing LR urediospores, while the negative controls (BMSV: 00) showed no visible Pt symptoms (Fig. 6A).
RT-PCR showed that the expression levels of two genes in the leaves inoculated with BSMV: TFP and BSMV: TPR virus were significantly lower than those in the leaves inoculated with the no-load virus (p < 0.05) (Fig. 6B). These results indicated that both TFP and TPR genes were necessary for the resistance of LrN3B to LR.
2.7 Sequence variations at LrN3B locus in common wheat
The diagnostic co-segregating markers of LrN3B TFPa and 3–35 were used to test the polymorphisms in 211 wheat lines. Four haplotypes of LrN3B were identified: HⅠ (148A + 148B + 149R); HⅡ (148B + 149R); HⅢ (148B + 149S); HⅣ (148C + 149S). HIV was predominant in 71.6% of all lines (151 lines). HII was the least, only 3 cultivars (Nongda189, Bainong201, and Nongda413, 1.4%) had this haplotype. HⅠ and HⅢ accounted for 5.2% and 21.8%, respectively (Fig. S7, Table S11). Next, we used co-segregating marker wmc692 of LrN4B to screen the 211 lines and found only 6 materials containing LrN4B + LrN3B: 4N0461, Nongda189, Zhoumai22, Zhou8425B, Zhoumai30, and Youbaomai. All these materials were highly resistant to LR (Table S12).