Genetic Diversity and Evolutionary Analyses Reveal the Powdery Mildew Resistance Gene Pm21 Undergoing Diversifying Selection

Abstract

Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a devastating disease that threatens wheat production and yield worldwide. The powdery mildew resistance gene Pm21, originating from wheat wild relative Dasypyrum villosum, encodes a coiled-coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) protein and confers broad-spectrum resistance to wheat powdery mildew. In the present study, we isolated 73 Pm21 alleles from different powdery mildew-resistant D. villosum accessions, among which, 38 alleles were non-redundant. Sequence analysis identified seven minor insertion-deletion (InDel) polymorphisms and 400 single nucleotide polymorphisms (SNPs) among the 38 non-redundant Pm21 alleles. The nucleotide diversity of the LRR domain was significantly higher than those of the CC and NB-ARC domains. Further evolutionary analysis indicated that the solvent-exposed LRR residues of Pm21 alleles had undergone diversifying selection (dN/dS = 3.19734). In addition, eight LRR motifs and four amino acid sites in the LRR domain were also experienced positive selection, indicating that these motifs and sites play critical roles in resistance specificity. The phylogenetic tree showed that 38 Pm21 alleles were divided into seven classes. Classes A (including original Pm21), B and C were the major classes, including 26 alleles (68.4%). We also identified three non-functional Pm21 alleles from four susceptible homozygous D. villosum lines (DvSus-1 to DvSus-4) and two susceptible wheat-D. villosum chromosome addition lines (DA6V#1 and DA6V#3). The genetic variations of non-functional Pm21 alleles involved point mutation, deletion and insertion, respectively. The results also showed that the non-functional Pm21 alleles in the two chromosome addition lines both came from the susceptible donors of D. villosum. This study gives a new insight into the evolutionary characteristics of Pm21 alleles and discusses how to sustainably utilize Pm21 in wheat production. This study also reveals the sequence variants and origins of non-functional Pm21 alleles in D. villosum populations.

Keywords: Dasypyrum villosum; Pm21 allele; diversifying selection; evolutionary analysis; genetic diversity; wheat powdery mildew resistance.

Figure 1

Geographic distribution of the D. villosum accessions used in this study. GRE, Greece. ITA, Italy. TUR, Turkey. BUL, Bulgaria. UKR, Ukraine. MKD, Macedonia. FSU, former Soviet Union. Unknown, the origins of the accessions are unclear.

Figure 2

Powdery mildew responses of different D. villosum lines, wheat genetic stocks, and F₁ plants of the crosses DvSus-1/DvSus-2, DvSus-1/DvSus-3, DvSus-1/DvSus-4, Dv6V#1/ Dv6V#3. The plants were inoculated with Bgt isolate YZ01 at one-leaf stage. The resistant wheat cv. Yangmai 18 and the susceptible cv. Yangmai 9 were used as the controls. Line DvRes-1 carries Pm21. Lines DvRes-2 and DvRes-3, carrying Pm21-C4 and Pm21-G2, were the resistant individuals of the accessions GRA961 and GRA1114, respectively. Lines DvSus-1 to DvSus-4, carrying non-functional Pm21 genes, were the susceptible individuals in the accessions GRA2738, GRA962, GRA1105, and PI598390, respectively.

Figure 3

Nucleotide diversity of 38 non-redundant Pm21 alleles isolated from the resistant D. villosum accessions. All the positions containing gaps were eliminated. Therefore, there were a total of 2,718 positions in the final dataset. The predicted protein structure is shown at the bottom.

Figure 4

Sequence logos of 16 LRR motifs encoded by Pm21 alleles. In the LxxLxLxx motifs, x represents the predicted solvent-exposed LRR residues, and L represents a leucine or another aliphatic amino acid residue. The sites at positions 628, 885, 903, and 905 pointed by arrows are predicted to be under positive selection.

Figure 5

Phylogenetic tree of Pm21 alleles constructed by the Neighbor-Joining method. The GenBank accession numbers are shown in brackets.

Figure 6

Origins of the non-functional alleles Pm21-NF1 to Pm21-NF3. Among them, the origin of Pm21-NF1 is unclear yet.