Development of molecular marker and introgression of Bph3 into elite rice cultivars by marker-assisted selection

Abstract

The brown planthopper (BPH) is a serious insect pest of rice and a substantial threat to rice production. Identification of new BPH resistance genes and their transfer into modern rice cultivars are effective breeding approaches to reduce the damage caused by BPH. In this study, we mapped a BPH resistance gene to a 50-kb genomic interval between two InDel markers 4M03980 and 4M04041 on the short arm of chromosome 4 in indica rice cultivar BP60, where the BPH resistance gene was mapped in Rathu Heenati by Liu et al. (2015) and named "Bph3". This region contains two annotated genes Os04g0201900 and Os04g0202300, which encode lectin receptor kinases responsible for BPH resistance. We also developed a molecular marker "MM28T" for Bph3, and introgression Bph3 into susceptible rice restorer lines Guihui582 and Gui7571 by the marker-assisted selection (MAS) approach. The BPH resistance level is significantly enhanced in the Bph3-introgression lines, the resistance scores decrease from 8.2 to 3.6 for Guihui582 and decrease from 8.7 to around 3.8 for Gui7571. Therefore, developing molecular markers for the BPH resistance gene Bph3 and using them for molecular breeding will facilitate the creation of BPH-resistance rice cultivars to reduce damage caused by BPH.

Keywords: brown planthopper; fine mapping; molecular breeding; resistance gene.

Fig. 1

The evaluation result of BPH resistance of BP60 and other nine BPH susceptible lines ten days after infestation (A). The data are presented as mean ± SD (three biological repeats), and letters indicate a significant difference at p < 0.05 by Tukey’s HSD test (B).

Fig. 2

Fine mapping of Bph3 allele to a 50-kb genomic region. Four predicted ORFs are in the mapped interval. Numbers under the linkage map indicate the number of recombinants detected between the DNA markers closely linked to Bph3. Gene models annotated in The Rice Annotation Project Database (http://rapdb.dna.affrc.go.jp/) are shown as black arrows, and the actual Bph3 gene is shown with a red arrow.

Fig. 3

Molecular marker design based on alignment of the Bph3 sequence from different rice cultivars. According to Bph3 sequence difference between BP60 and other rice cultivars, an additional intermediate reverse primer (IR) was designed to amplify a 330 bp PCR product only from BP60, and a 809 bp PCR product from all rice cultivars. One nucleotide mutation (blue color) on the IR primer to get one nucleotide mismatch with BP60 and two nucleotides mismatch with other rice cultivars. DNA sequence alignment was performed on website: https://www.ebi.ac.uk/Tools/msa/mafft/.

Fig. 4

Validation of the Bph3 molecular marker using different rice cultivars. M: DNA marker 2000, 1: 9311, 2: Gui7571, 3: 75-1-27, 4: BL122, 5: 112B, 6: TeB, 7: LiangfengB, 8: IRBB7, 9: BP60, 10: IR1552. Using three primers in the PCR reactions, two different PCR products (809 bp and 330 bp) were obtained when the rice cultivars contained the Bph3 gene. As BP60 contains the Bph3 gene, both the 809 bp and 330 bp fragments were amplified in the PCR reaction, while the nine other cultivars only had the 809 bp PCR fragment after PCR amplification.

Fig. 5

BPH-resistance phenotype in Bph3-containing introgression lines and hybrid lines. (A and D) Seedling stage plants after 10 days of infestation by BPH; (B) 83-day-old plants after 18 days of infestation by BPH; (C and E) The data are presented as means ± SD (three biological repeats), and letters indicate a significant difference at p < 0.05 by Tukey’s HSD test.