Most japonica rice cultivars exhibit high susceptibility to BB disease, except to race K1 in Korea, because of their narrow genetic diversity. It is imperative to develop new BB-resistant rice cultivars with high yield potential and grain quality using modern tools of biotechnology. However, it is often difficult to introduce the BB resistance genes from indica germplasm sources into a japonica genetic background by conventional breeding methods due to the unexpected linkage drag. Pyramiding resistance genes is difficult to accomplish using conventional breeding because of the dominance and epistasis effects of the genes controlling disease resistance. Nevertheless, using the tools of biotechnology, it is possible to transfer or pyramid valuable genes of BB resistance into rice without linkage drag (Rajpurohit et al.2011; Shanti et al.2010; Singh et al.2001; Sundaram et al.2008). Mangeumbyeo is a japonica cultivar with good grain and cooking quality and high yield potential but it is highly susceptible to BB races. An IRBB57 NIL carrying Xa4, xa5 and Xa21 genes in an IR24 genetic background conferred strong resistance to all Korean BB races, including K3a (Jeung et al.2006; Suh et al.2009a). We have introduced the three BB resistance genes (Xa4 + xa5 + Xa21) from IRBB57 into Mangeumbyeo through simultaneous foreground and phenotypic selection. Eventually, it was possible to introduce three BB resistance genes with desirable agronomic traits using marker-assisted backcrossing. All three co-dominant molecular markers linked to the target genes (Xa4, xa5 and Xa21) were used for MAB and the markers were polymorphic between the donor parent IRBB57 and recurrent parent Mangeumbyeo. The validated markers could thus be used successfully to pyramid and confirm the three resistance genes in advanced backcross lines. Finally, we also analyzed the genetic background of the three selected ABL (BC3 progenies) with high background genome recovery. Conventional backcross breeding has difficulty in confirming the several resistance genes combined in breeding lines using phenotypic selection with Xoo inoculation (Rajpurohit et al.2011; Shanti et al.2010; Sundaram et al.2008). The best strategy to pyramid or introduce multiple genes and recover a maximum recurrent parent background effect in the shortest time will be to take up the transfer of genes simultaneously, generate a large backcross population and select the target genes through foreground selection and flanking marker analysis to reduce the persistent linkage drag (Rajpurohit et al.2011; Ye,2010). However, if we select backcross lines with target genes using molecular markers, linkage drag often occurs in indica/japonica. So, we selected the backcross progenies in each backcross and segregating generation through foreground and phenotypic selection simultaneously to reduce the linkage drag. This expensive, cumbersome and time-consuming background selection can be avoided and substituted by another backcross with the recurrent parent, if necessary. Final backcross progenies could be confirmed with the substituted chromosome segments by background analysis using genome-wide molecular markers. On the basis of comprehensive foreground selection, phenotypic selection for morphological and quality traits, and background genotyping, three BC3F5 gene-pyramid lines with pyramided genes homozygous at all three target loci were derived from the donor parent. The three R-gene-derived ABL exhibited high resistance upon inoculation with Xoo strains and had nearly the average expected 93.75% background genome recovery.
In an earlier study, it was reported that the favorable characteristics of Pusa Basmati 1 with two BB resistance genes could be recovered using MAS just in BC1 because of stringent phenotypic selection without any background selection only in segregating generations (Joseph et al.2004). Similarly, BC4 pyramided lines of Sambha Mahsuri with three BB resistance genes (xa5, xa13 and Xa21) were developed by simultaneous foreground and background selection and the selected lines recovered 97% recurrent parent background, exhibiting a broad-spectrum resistance against multiple Xoo isolates (Sundaram et al.2008). In this study, we selected elite ABL with three BB resistance genes in the BC3 generation because BC1 and BC2 progenies were having some undesirable phenotypic traits such as awns, shattering and spikelet sterility. It is possible to recover the recurrent parent phenotype in one or two backcrosses if we introduce multiple resistance genes from indica to indica cultivars (Joseph et al.2004; Rajpurohit et al.2011; Singh et al.2001) and we may also need at least two backcrosses to introduce one resistance gene from indica to japonica cultivars (Suh et al.2009b; Suh et al.2011). However, our results suggest that at least three backcrosses are essential to recover the phenotype of the recurrent parent if multiple resistance genes such as Xa4, xa5 and Xa21 are transferred from an indica cultivar into a japonica cultivar for broad-spectrum BB resistance.
Three BB resistance-gene-derived ABL were evaluated for their resistance to BB under glasshouse conditions with the 18 isolates of Xoo prevalent in Korea. One of these isolates, called HB01009, belongs to the new race K3a (Noh et al.2003). The Xa21 and xa5 genes and their combinations conferred strong resistance to the K3a isolate (Suh et al.2009a,2009b). Variable reactions of the Xoo isolates to Xa4, xa5 and Xa21 suggest that xa5 and Xa21 are more effective in resistance to 14 isolates than Xa4 because Xa4 showed resistance to 8 isolates only. However, the cumulative effect of the three resistance genes (Xa4 + xa5 + Xa21) in the ABL in the Mangeumbyeo genetic background exhibited very high resistance to all 18 isolates of Xoo, including the most virulent isolate of race K3a. The results indicated that the genes in combinations were more effective against the pathogen strains than a single resistance gene alone. The resistance appears to be more durable if different resistance genes are combined (Jeung et al.2006; Kim et al.2009; Singh et al.2001; Suh et al.2009a). This indicates that there is some kind of quantitative complementation with the presence of multiple resistance genes having an additive effect on the overall level of resistance. Accumulating major genes for resistance in an elite genotype by conventional breeding is laborious, time-consuming and very difficult when two or more of the resistance genes are pyramided into an elite cultivar. However, marker-assisted backcrossing with accurate phenotypic selection is the most effective method for a selective transfer or pyramiding of resistance genes into elite rice cultivars free from linkage drag, eventually restoring the recurrent parent genotype (Joseph et al.2004; Shanti et al.2010; Singh et al.2001; Suh et al.2011). The ABL with the three resistance genes in combination have a practical breeding value by providing a wider spectrum of resistance against most of the existing BB isolates in the region and will have a high impact on the yield stability and sustainability of the rice crop in the region. The grain quality characteristics of the three resistance-gene-derived ABL are not significantly different from those of the parent Mangeumbyeo. This indicates that the BB resistance-gene combinations are not closely linked with any negative allele controlling grain quality. It is also reported that Xa1, Xa2 and Xa3 genes have no negative effect for the traits associated with grain quality and the taste of cooked rice (Shin et al.2006). The recurrent parent greatly influenced the determination of grain quality, milling characteristics and cooking and eating qualities. Therefore, the choice of the recurrent parent plays a critical role in backcross breeding programs (Shin et al.2006; Ye2010). The yield and agronomic traits of the ABL in this study are also similar to those of Mangeumbyeo, indicating that there is no apparent agronomic trait penalty associated with the presence of the resistance genes.
In our study, an additional backcross with the recurrent parent was required to recover the desirable phenotype in the BC3 progenies. Three BC3F5 progenies were mostly homozygous for the target traits based on MAS with agronomic traits similar to those of the recurrent parent, Mangeumbyeo, with high resistance to bacterial blight. The background genotype recovery varied from 92.1 to 94.5%. Even though the three ABL showed highly recovered chromosome segments, they could not exhibit a similar phenotype with the recurrent parent because the insertion of small chromosome segments also affected phenotype. Theoretically, with three backcrosses, the average background genotype recovery should be 93.75%, a background recovery rate similar to that of the selected ABL in this study. On the contrary, the background recovery of the recurrent wheat parent during the introgression of stripe rust resistance without marker-assisted background selection was only 82% in BC4F7 progenies (Randhawa et al.2009). However, 97% of the background genotype was obtained in BC2F2:3 progenies by using foreground selection of the target traits, background selection for flanking markers, non-carrier chromosome markers and whole-marker screens during two successive backcrosses in a large backcross population. A high rate of background genotype recovery of the recurrent parent was 86.72% in the BC1F3 generation using MAS and phenotypic selection during the introgression of two BB resistance genes in indica/indica crosses (Joseph et al.2004). In our study, a similar strategy of simultaneous foreground and phenotypic selection was followed for higher background genotype recovery in the japonica/indica cross in three backcrosses. This approach is very useful to reduce the cost and time required for the recovery of desirable recombinants to a considerable extent with target resistance genes in japonica/indica crosses. Therefore, it can be directly developed in a commercial variety. Introgression of resistance with a penalty in yield and grain quality characters would be a futile exercise, as the developed lines would not be accepted by farmers. The three-gene pyramided ABL developed in our study without a penalty in yield and grain quality would be of great advantage to rice farmers in BB-endemic rice areas.