*2.5. Assessment of the pi21-2428 Haplotype in 325 Rice Accessions*

Multiple sequence alignment of the *Pi21* alleles in 325 rice accessions revealed a total of 11 haplotypes among the tested accessions (Figure 5A, Supplementary Table S2). However, none of the accessions carried the resistant *pi21(-21*/*-48)* allele. The haplotypes were named based on insertion/deletion patterns. Among the 11 haplotypes, *Pi21-NPB* (the *Pi21* allele in Nipponbare), *Pi21(-9)*, and *Pi21(-24*/*-15)* were identical to the previously identified haplotypes B, C, and H, respectively (Figure 5A, Supplementary Table S2) [16], and the rest eight were novel types.

**Figure 5.** Assessment of haplotypes of *Pi21* in 325 rice accessions and blast inoculation of the *pi21-2428*-containing varieties. (**A**) A total of 11 haplotypes were identified among 325 rice accessions. The haplotypes were named based on insertion/deletion patterns. *Pi21-NPB*, *Pi21(-9)*, and *Pi21(-24*/*-15)* were identical to the previously identified haplotypes B, C, and H, respectively [16]. Open boxes represent exons, lines represent introns, and dotted lines represent deletions. Numbers above the diagrams indicate positions of deletions corresponding to the *Pi21-NPB* allele, and numbers below the diagrams represent the start and end nucleotide positions of exons of each allele. (**B**) Phenotypes of the *pi21-2428*-containing varieties 02428, ShuHui881 (SH881), Yong4 (Y4), and ZhengDa4Hao (ZD4) inoculated with *M. oryzae* isolates 2Y838-1, 501-3, CHNOS, IR16-1, KJ201, RB6, and RB22, respectively. Rice varieties LXG and MengGuDao (MGD) were used as highly susceptible controls.

Among the 325 rice accessions, in addition to 02428, three Chinese *indica* varieties ShuHui881, Yong4, and ZhengDa4Hao were detected carrying the resistant *pi21-2428* allele. The *pi21-2428*-containing varieties were inoculated with seven *M. oryzae* isolates. As shown in Figure 5B, the two susceptible varieties LXG and MengGuDao (MGD) were highly susceptible to most of the *M. oryzae* isolates. The four *pi21-2428*-containing varieties showed complete resistance, moderate resistance, or moderate susceptibility to the isolates. For example, the *indica* variety ZhengDa4Hao was highly resistant to isolates KJ201 and RB22, and moderately resistant to isolates CHNOS and IR16-1, suggesting that there might be some *R* genes conferring the high resistance in ZhengDa4Hao against the four isolates. ZhengDa4Hao also showed susceptibility to isolates 2Y838-1, 501-3, and RB6, but the lesions on leaves were limited in size and much less than those on leaves of LXG or MGD. Overall, none of the *pi21-2428*-containing varieties showed highly susceptible to the seven *M. oryzae* isolates, suggesting that the four varieties possessed high basal resistance with significantly delayed and reduced development of disease lesions.

#### **3. Discussion**

When compared with*R*gene-mediated, race-specific resistance, basal resistance has been presumed to be more durable [4,10]. Therefore, the identification of genes or QTLs conferring basal resistance is of significance in breeding crops with long-lasting resistance to plant diseases. In the present study, we employed BSA-Seq to identify four candidate QTLs *qBBR-4*, *qBBR-7*, *qBBR-8,* and *qBBR-11,* conferring basal resistance to rice blast disease on rice chromosomes 4, 7, 8, and 11, respectively. Building on advances in next-generation sequencing, the BSA-Seq method took advantage of pooled sequencing, which does not require the laborious process of genotyping of each individual from a large mapping population, allowing for rapid identification of candidate genes or QTLs controlling important agronomic traits [24,31].

Among the four QTLs detected in this study, *qBBR-4* had the largest additive effect (Table 2). The *qBBR-4* locus is located in a region of chromosome 4 with a known recessive blast disease resistant QTL *pi21,* identified in rice variety Owarihatamochi [16]. *Pi21* encodes a proline-rich protein, consisting of a putative heavy metal-binding domain and protein-protein interaction motifs. While the dominant *Pi21* appears to slow the plant's defense responses, loss-of-function of *Pi21* (double deletions of 21 bp and 48 bp in the proline-rich region; haplotype L) confers durable resistance to blast disease in rice [16]. Sequence analysis revealed that the *pi21* allele in 02428, the parental line with high basal resistance, had double deletions of 30 bp and 33 bp, resulting in deletions of 10 aa and 11 aa in the proline-rich region, which house the core motif "PxxPxxP" (Figure 4A,B) for protein–protein interaction in multicellular organisms [16,32,33]. We further performed genotype-phenotype correlation analysis of 61 ER individuals and 59 ES individuals, and the results showed that all the homozygous *pi21-2428* individuals belonged to the ER bulk (Figure 4C). Taken together, these results suggest that double deletions of the 30 bp and 33 bp sequences of *Pi21* led to high basal resistance to blast disease, and that *pi21-2428* was the candidate gene of *qBBR-4*.

Previously, sequence analysis of the *Pi21* locus revealed 12 haplotypes (A to L) among 80 Asian cultivated rice varieties. Eleven of the haplotypes (A to K) carried an insertion or smaller deletions compared with the resistant *pi21(-21*/*-48)* allele, and did not confer resistance to blast disease [16]. In the present study, we detected a total of 11 haplotypes of *Pi21* among 325 rice accessions (Figure 5A, Supplementary Table S2), of which three were identical to the previously identified haplotypes B, C, and H [16], and eight were novel. Interestingly, the DNA variations of all the 20 detected haplotypes identified by Fukuoka et al. [16] and in this study were found to result in amino acid insertion/deletions, but not to cause premature termination of the predicted *Pi21* product, implying that the *Pi21* or *pi21* alleles maintain certain functions important for rice [34]. Besides 02428, we identified three more varieties, ShuHui881, Yong4, and ZhengDa4Hao, possessing the resistant *pi21-2428* allele. Inoculation testing with seven *M. oryzae* isolates indicated that, while the susceptible varieties LXG and MGD were highly susceptible to most of the *M. oryzae* isolates, the four *pi21-2428*-containing varieties showed

complete resistance moderate resistance or moderate susceptibility to the *M. oryzae* isolates (Figure 5B). The results suggest that there should be some *R* genes other than *pi21-2428* conferring the complete or high resistance in the four *pi21-2428*-containing varieties. On the other hand, the moderately susceptible reactions with limited lesion size and number, to virulent *M. oryzae* isolates indicated that ShuHui881, Yong4, ZhengDa4Hao, as well as 02428, possessed high basal resistance to blast disease (Figure 5B). It is worth noting that while the resistant *pi21(-21*/*-48)* allele was found only in *japonica* rice [16], the three *pi21-2428*-containing varieties ShuHui881, Yong4, and ZhengDa4Hao were *indica* rice (Supplementary Table S2). Therefore, the varieties identified in the present study provide valuable resources for breeding rice varieties, especially *indica* varieties, with durable resistance to blast disease.

Transgressive segregation was observed in the F2 population of 02428 × LXG, where some F2 segregants showed more resistance than both parents (Figure 1B). This phenomenon implies that favorable alleles from both resistant and susceptible parents could be combined in the progeny, leading to a higher resistance than in the parents. In the present study, in addition to *pi21-2428*, we detected three other QTLs, *qBBR-7*, *qBBR-8*, and *qBBR-11*, on chromosomes 7, 8, and 11, respectively. Further identification and functional characterization of the three QTLs should be helpful to better understand the mechanisms underlying rice basal resistance to blast disease. Furthermore, the finding of transgressive segregation for blast resistance in the F2 population of 02428 × LXG indicates that pyramiding more basal resistance genes or QTL alleles with *pi21-2428* would be an effective approach to enhance durable resistance to rice blast disease [10,35].
