Allele-specific marker-based assessment revealed that the rice blast resistance genes Pi2 and Pi9 have not been widely deployed in Chinese indica rice cultivars

Abstract

Background: The most sustainable approach to control rice blast disease is to develop durably resistant cultivars. In molecular breeding for rice blast resistance, markers developed based on polymorphisms between functional and non-functional alleles of resistance genes, can provide precise and accurate selection of resistant genotypes without the need for difficult, laborious and time-consuming phenotyping. The Pi2 and Pi9 genes confer broad-spectrum resistance against diverse blast isolates. Development of allele-specific markers for Pi2 and Pi9 would facilitate breeding of blast resistant rice by using the two blast resistance genes.

Result: In this work, we developed two new markers, named Pi9-Pro and Pi2-LRR respectively, targeting the unique polymorphisms of the resistant and susceptible alleles of Pi2 and of Pi9. The InDel marker Pi9-Pro differentiates three different genotypes corresponding to the Pi2/Piz-t, Pi9 and non-Pi2/Piz-t/Pi9 alleles, and the CAPS marker Pi2-LRR differentiates the Pi2 allele from the non-Pi2 allele. Based on the two newly developed markers and two available markers Pi2SNP and Pi9SNP, the presence of Pi2 and Pi9 was assessed in a set of 434 rice accessions consisting of 377 Chinese indica cultivars/breeding materials and 57 Chinese japonica cultivars/breeding materials. Of the 434 accessions tested, while one indica restorer line Huazhan was identified harboring the Pi2 resistance allele, no other rice line was identified harboring the Pi2 or Pi9 resistance alleles.

Conclusions: Allele-specific marker-based assessment revealed that Pi2 and Pi9 have not been widely incorporated into diverse Chinese indica rice cultivars. Thus, the two blast resistance genes can be new gene sources for developing blast resistant rice, especially indica rice, in China. The two newly developed markers should be highly useful for using Pi2 and Pi9 in marker-assisted selection (MAS) breeding programs.

Keywords: Blast disease; Molecular marker; Pi2; Pi9; Resistance gene; Rice.

Fig. 1

An InDel marker targeting the promoter region of Pi9. a Physical location of the identified InDel region in the Pi2/9 locus. b Sequence alignment showing the 27-bp InDel between the Pi2/Piz-t and non-Pi2/Piz-t alleles and the 10-bp InDel between the Pi9 and non-Pi9 alleles in diverse cultivars. c PCR amplification patterns of the InDel marker Pi9-Pro that differentiates the Pi2/Piz-t, Pi9 and non-Pi2/Piz-t/Pi9 alleles. M: DNA ladder; 1: C101A51 (Pi2 donor line); 2: Toride-1 (Piz-t donor line); 3: 75-1-127 (Pi9 donor line); 4: Nipponbare; 5: 93–11; 6: CO39; 7: D62B; 8: SE21S; 9: Katy; 10: 02428; 11: Longtepu B; 12: Lijiangxintuanheigu; 13: Shuhui 527; 14: Minhui 3301; 15: Ce 64; 16: Fu 838; 17: Guanghui 998; 18: Miyang 46; 19: Minghui63; 20: Gang 46B; 21: Zaogang B; 22: Gufeng B; 23: Jin 23B; 24: Zhenshan 97B; 25: Guangzhan 63S; 26: Peiai 64S. Asterisk indicates the InDel region; Grey arrows indicate the promoter regions of Pi2, Piz-t or Pi9

Fig. 2

A CAPS marker targeting the LRR domain of Pi2. a DNA polymorphisms corresponding to the eight amino-acid differences in between Pi2 and Piz-t. b Sequence alignment showing that the polymorphic variations corresponding to the first six amino-acid differences in between Pi2 and Piz-t were conserved between the Pi2 and non-Pi2 alleles in diverse cultivars. c Schematic diagrams indicating sizes and recognition sites for HinfI and PstI in the PCR fragment of the Pi2 and Piz-t alleles amplified with primers 2-LRR-F/2-LRR-R. d Electrophoresis profiles of the CAPS marker Pi2-LRR that differentiates the Pi2 and non-Pi2 alleles. Upper panel showing the PCR products without digestion with restriction enzymes, middle panel showing the PCR products digested with PstI, and lower panel showing the PCR products digested with HinfI. M: DNA ladder; 1: C101A51 (Pi2 donor line); 2: Toride-1 (Piz-t donor line); 3: 75-1-127 (Pi9 donor line); 4: Nipponbare; 5: 93–11; 6: CO39; 7: D62B; 8: Xianghui 68; 9: Shuhui 527; 10: Zhenshan 97B; 11: Zaogang B; 12: Minghui 63; 13: Guangzhan 63S; 14: Gang 46B; 15: Miyang 46; 16: Fu 838; 17: SE21S; 18: Peiai 64S; 19: Lijiangxintuanheigu; 20: Minhui 3301; 21: Longtepu B; 22: 02428; 23: Guanghui 998; 24: Jin 23B. PstI recognition sequence (CTGCAG) is underlined; HinfI recognition sequence (GAATC) is marked by dotted underling

Fig. 3

Pi9-Pro and Pi2-LRR analyses of 15 cultivars displaying a Pi2/Piz-t-type genotype for the Pi9-Pro marker. Upper panel showing the PCR amplification patterns of the InDel marker Pi9-Pro; Middle panel showing the patterns of Pi2-LRR products digested with PstI; and lower panel showing the patterns of Pi2-LRR products digested with HinfI. M: DNA ladder; 1: C101A51 (Pi2 donor line); 2: Toride-1 (Piz-t donor line); 3: 75-1-127 (Pi9 donor line); 4: Duohui 43; 5: Minghui 86; 6: Huazhan; 7: Minghui 1259; 8: Xianghui 68; 9: Fengxinzhan; 10: GIZA 176; 11: Jiabala; 12: Jiazao No.1; 13: Jiazao 935; 14: Haomake (K); 15: Lemont; 16: Shuiyuan 290; 17: Shuiyuan 377; 18: Zhonghua No.8