*2.1. Development of BC3F4 Pyramided Lines Using Marker-Assisted Breeding*

Tainung82 is one of the most widely cultured elite *japonica* varieties in Taiwan, but it exhibits a high susceptibility to bacterial blight disease. In order to develop a BB-resistant *japonica* cultivar, TNG82 was used as the recurrent parent to backcross with IRBB66 for three generations, and then self-crossed to produce a BC3F4 population. The polymorphism was detected between donor parent IRBB66 and recurrent parent TNG82 with the markers Xa4F/4R, RM604F/604R, Xa7F/7-1R/7-2R, Xa13F/13R and Xa21F/21R for *Xa4*, *xa5*, *Xa7*, *xa13* and *Xa21*, respectively. In addition, the parents were screened with 216 rice microsatellite markers, of which 143 were polymorphic and 117 were used for background selection (Figure S1). The breeding scheme using molecular markers for the selection of the five BB-resistant genes is shown in Figure 1. During the breeding procedure, functional marker selection was practiced from the F1 generation until the BC3F2 generation. The plants possessing all five resistance genes were selected in each stage, of which only two progenies were advanced to the next generation. A total of two plants having all five BB resistance genes (*Xa4*, *xa5*, *Xa7*, *xa13* and *Xa21*) were screened from 960 F2 plants and confirmed by lined molecular markers [10]. The two F2 plants were backcrossed to TNG82. A total of 53 of 147 BC1F1 plants containing different BB resistance genes were selected by MAS. The percentages of recurrent parent genome (%RPG) of BC1F1 ranged from 60% to 85%, with an average of 73.8% (Figure 2). Ten BC1F1 plants containing both the five BB resistance genes, as well as a high %RPG (average of 81.7%), were used for further backcrossing with TNG82.

**Figure 1.** Schematic diagram for pyramiding bacterial blight resistance genes into Taiwanese *japonica* rice cultivar, TNG82, using marker-assisted selection and number of plants selected in every generation.

**Figure 2.** The frequency distribution of recurrent parent genome (RPG) recovered rate using marker-assisted backcrossing in BC1F1, BC2F1 and BC2F2 populations derived from the backcross of IRBB66/TNG82. The numbers inside the right side of frame indicate the mean values (SD) of RPG recovered.

A total of 50 of 1228 BC2F1 plants containing different BB resistance genes possessed the recurrent genome content of TNG82, ranging from 72% to 94%, with an average of 83% (Figure 2). The 20 selected BC2F1 plants, heterozygous for all five BB resistance genes and possessing a high %RPG (average of 87.3%), were selfed to obtain the BC2F2 population. The plants homologous for all five target genes were segregated with a Mendelian pattern (homozygous preference genotype = 1/4n). The four BC2F2 plants carrying five positive homozygous alleles of the donor genes, including *Xa4*, *xa5*, *Xa7*, *xa13* and *Xa21*, were screened from 5012 BC2F2 plants. Four BC2F2 plants showed recurrent genome content of TNG82 with %RPG of 92.05% (29), 84.3% (18), 83.1% (5) and 79.4% (43), with an average of 84.71% (Table S1). In the BC2F3 generation, 17 plants containing different BB resistance genes were used to confirm resistance reaction by inoculation with *Xoo* isolate XF89-b and evaluated for agronomic performance. Four BC2F3 plants with the five BB resistance genes were backcrossed to TNG82. In the BC3F2 generation, 16 of 685 plants containing the five BB resistance genes were identified and grown as BC3F3. These 16 five-gene-pyramided genotypes were selfed and evaluated for agronomic performance. The nine BC3F4 lines containing five BB resistance genes, *Xa4*, *xa5*, *Xa7*, *xa13* and *Xa21* (Figure 3) were selected and evaluated for agronomic performance in the field, as well as analyzed for grain quality.

**Figure 3.** Multiplex PCR amplification of five bacterial blight resistance genes, *Xa4, xa5, Xa7, xa13* and *Xa21*. The five expected band sizes of 217, 106, 179, 381 and 595 bp, correlated with *Xa4, xa5, Xa7, xa13* and *Xa21*, respectively, were amplified in IRBB66 and nine five-gene-pyramided lines using multiplex PCR. P1:IRBB66, P2:TNG82. DNA products were separated by 6% polyacrylamide gel in 0.5 × TBE at 100 v for 60 min. M: DNA ladder marker.
