**6. Marker-assisted gene pyramiding and marker-assisted recurrent selection**

Marker-assisted gene pyramiding (MAGP) is one of the most important applications of DNA markers to plant breeding. Gene pyramiding has been proposed and applied to en‐ hance resistance to disease and insects by selecting for two or more than two genes at a time. For example in rice such pyramids have been developed against bacterial blight and blast (Huang et al., 1997; Singh et al., 2001; Luo et al., 2012). Castro et al. (2003) reported a success in pyramiding qualitative gene and QTLs for resistance to stripe rust in barley. The advant‐ age of using markers in this case allows selecting for QTL-allele-linked markers that have the same phenotypic effect. To enhance or improve a quantitatively inherited trait in plant breeding, pyramiding of multiple genes or QTLs is recommended as a potential strategy (Richardson et al., 2006). The cumulative effects of multiple-QTL pyramiding have been pro‐ ven in crop species like wheat, barley and soybean (Richardson et al., 2006; Jiang et al., 2007a, 2007b; Li et al., 2010; Wang et al., 2012). Pyramiding of multiple genes/QTLs may be achieved through different approaches: multiple-parent crossing or complex crossing, back‐ crossing, and recurrent selection. A suitable breeding scheme for MAGP depends on the number of genes/QTLs required for improvement of traits, the number of parents that con‐ tain the required genes/QTLs, the heritability of traits of interest, and other factors (e.g. marker-gene association, expected duration to complete the plan and relative cost). Assum‐ ing three or four desired genes/QTLs exist separately in three or four lines, pyramiding of them can be realized by three-way, four-way or double crossing. They may also be integrat‐ ed by convergent backcrossing or stepwise backcrossing. However, if there are more than four genes/QTLs to be pyramided, complex or multiple crossing and/or recurrent selection may be often preferred.

For MABC-based gene pyramiding, in general, there may be three strategies or breeding schemes: stepwise, simultaneous/synchronized and convergent backcrossing or transfer. Supposing one cultivar W is superior in comprehensive performance but lack of a trait of interest, and four different genes/QTLs contributing to the trait have been identified in four germplasm lines (e.g. P1, P2, P3 and P4). Three MABC schemes for pyramiding the genes/ QTLs can be described as follow.

**Scheme 1.** Stepwise Backcrossing

For MABC-based gene pyramiding, in general, there may be three strategies or breeding schemes: stepwise, simultaneous/synchronized and convergent backcrossing or transfer. Supposing one cultivar W is superior in comprehensive performance but lack of a trait of interest, and four different genes/QTLs contributing to the trait have been identified in four germplasm lines (e.g. P1, P2, P3 and P4). Three MABC schemes for pyramiding the genes/

QTLs can be described as follow.

68 Plant Breeding from Laboratories to Fields

**Scheme 1.** Stepwise Backcrossing

In the stepwise backcrossing, four target genes/QTLs are transferred into the recurrent pa‐ rent W in order. In one step of backcrossing, one gene/QTL is targeted and selected, fol‐ lowed by next step of backcrossing for another gene/QTL, until all target genes/QTLs have been introgressed into the RP. The advantage is that gene pyramiding is more precise and easier to implement as it involves only one gene/QTL at one time and thus the population size and genotyping amount will be small. The improved recurrent parent may be released before the final step as long as the integrated genes/QTLs (e.g. two or three) meet the re‐ quirement at that time. The disadvantage is that it takes a longer time to complete. In the simultaneous or synchronized backcrossing, the recurrent parent W is first crossed to each of four donor parents to produce four single-cross F1s. Two of the four single-cross F1s are crossed with each other to produce two double-cross F1s, and these two double-cross F1s are crossed again to produce a hybrid integrating all four target genes/QTLs in heterozygous state. The hybrid and/or progeny with heterozygous markers for all four target genes/QTLs is subsequently crossed back to the RP W until a satisfactory recovery of the RP genome, and finalized by one generation of selfing. The advantage of this method is that it takes the shortest time to complete. However, in the backcrossing all target genes/QTLs are involved at the same time and thus it requires a large population and more genotyping. Convergent backcrossing is a strategy combining the advantages of stepwise and synchronized back‐ crossing. First the four target gene/QTLs are transferred separately from the donors into the recurrent parent W by single crossing followed by backcrossing based on markers linked to the target genes/QTLs, to produce four improved lines (WAA, WBB, WCC, and WDD). Two of the improved lines are crossed with each other and the two hybrids are then intercrossed to integrate all four genes/QTLs together and develop the final improved line with all four genes/QTLs pyramided (i.g. WAABBCCDD). Relatively speaking, convergent backcrossing is more acceptable because in this scheme not only is time reduced (compared to stepwise transfer) but gene fixation and/or pyramiding is also more easily assured (compared to si‐ multaneous transfer).

Theoretical issues and efficiency of MABC for gene pyramiding have been investigated through computer simulations (Ribaut et al., 2002; Servin et al., 2004; Ye and Smith, 2008). Practical application of MABC to gene pyramiding has been reported in many crops, includ‐ ing rice, wheat, barley, cotton, soybean, common bean and pea, especially for developing durable resistance to stresses in crops. However, there is very limited information available about the release of commercial cultivars resulted from this strategy. Somers et al. (2005) implemented a molecular breeding strategy to introduce multiple pest resistance genes into Canadian wheat. They used high throughput SSR genotyping and half-seed analysis to process backcrossing and selection for six FHB resistance QTLs, plus orange blossom wheat midge resistance gene *Sm1* and leaf rust resistance gene *Lr21*. They also used 45-76 SSR markers to perform background selection in backcrossing populations to accelerate the re‐ storation of the RP genetic background. This strategy resulted in 87% fixation of the elite ge‐ netic background at the BC2F1 on average and successfully introduced all (up to 4) of the chromosome segments containing FHB, *Sm1* and *Lr21* resistance genes in four separate crosses(Somers et al., 2005). Joshi and Nayak (2010) and Xu (2010) recently reviewed the techniques and practical cases in marker-based gene pyramiding.

Similar to the simultaneous/synchronized backcrossing scheme, marker-assisted complex or convergent crossing (MACC) can be undertaken to pyramid multiple genes/QTLs. In partic‐ ular, MACC is a proper option of breeding schemes for gene pyramiding if all the parents are improved cultivars or lines with good comprehensive performance and have different or complementary genes or favorable alleles for the traits of interest. The difference from si‐ multaneous backcrossing is that selfing hybrid and progenies replaces backcrossing hybrid to the recurrent parent. In MACC, the hybrid of convergent crossing is subsequently selfpollinated and marker-based selection for target traits is performed for several consecutive generations until genetically stable lines with desired marker alleles and traits have been de‐ veloped. In order to reduce population size and to avoid loss of most important genes/QTLs, different markers may be used and selected in different generations, depending on their rel‐ ative importance. The markers for the most important genes/QTLs can be detected and se‐ lected first in early generations and less important markers later. Once homozygous alleles of the markers for a gene/locus are detected, they may not be necessarily detected again in the subsequent generations. Instead, phenotypic evaluation should be conducted if condi‐ tions permit.

In the stepwise backcrossing, four target genes/QTLs are transferred into the recurrent pa‐ rent W in order. In one step of backcrossing, one gene/QTL is targeted and selected, fol‐ lowed by next step of backcrossing for another gene/QTL, until all target genes/QTLs have been introgressed into the RP. The advantage is that gene pyramiding is more precise and easier to implement as it involves only one gene/QTL at one time and thus the population size and genotyping amount will be small. The improved recurrent parent may be released before the final step as long as the integrated genes/QTLs (e.g. two or three) meet the re‐ quirement at that time. The disadvantage is that it takes a longer time to complete. In the simultaneous or synchronized backcrossing, the recurrent parent W is first crossed to each of four donor parents to produce four single-cross F1s. Two of the four single-cross F1s are crossed with each other to produce two double-cross F1s, and these two double-cross F1s are crossed again to produce a hybrid integrating all four target genes/QTLs in heterozygous state. The hybrid and/or progeny with heterozygous markers for all four target genes/QTLs is subsequently crossed back to the RP W until a satisfactory recovery of the RP genome, and finalized by one generation of selfing. The advantage of this method is that it takes the shortest time to complete. However, in the backcrossing all target genes/QTLs are involved at the same time and thus it requires a large population and more genotyping. Convergent backcrossing is a strategy combining the advantages of stepwise and synchronized back‐ crossing. First the four target gene/QTLs are transferred separately from the donors into the recurrent parent W by single crossing followed by backcrossing based on markers linked to the target genes/QTLs, to produce four improved lines (WAA, WBB, WCC, and WDD). Two of the improved lines are crossed with each other and the two hybrids are then intercrossed to integrate all four genes/QTLs together and develop the final improved line with all four genes/QTLs pyramided (i.g. WAABBCCDD). Relatively speaking, convergent backcrossing is more acceptable because in this scheme not only is time reduced (compared to stepwise transfer) but gene fixation and/or pyramiding is also more easily assured (compared to si‐

Theoretical issues and efficiency of MABC for gene pyramiding have been investigated through computer simulations (Ribaut et al., 2002; Servin et al., 2004; Ye and Smith, 2008). Practical application of MABC to gene pyramiding has been reported in many crops, includ‐ ing rice, wheat, barley, cotton, soybean, common bean and pea, especially for developing durable resistance to stresses in crops. However, there is very limited information available about the release of commercial cultivars resulted from this strategy. Somers et al. (2005) implemented a molecular breeding strategy to introduce multiple pest resistance genes into Canadian wheat. They used high throughput SSR genotyping and half-seed analysis to process backcrossing and selection for six FHB resistance QTLs, plus orange blossom wheat midge resistance gene *Sm1* and leaf rust resistance gene *Lr21*. They also used 45-76 SSR markers to perform background selection in backcrossing populations to accelerate the re‐ storation of the RP genetic background. This strategy resulted in 87% fixation of the elite ge‐ netic background at the BC2F1 on average and successfully introduced all (up to 4) of the chromosome segments containing FHB, *Sm1* and *Lr21* resistance genes in four separate crosses(Somers et al., 2005). Joshi and Nayak (2010) and Xu (2010) recently reviewed the

techniques and practical cases in marker-based gene pyramiding.

multaneous transfer).

70 Plant Breeding from Laboratories to Fields

Using markers to select or pyramid for multiple genes/QTLs is more complex and less proven. Recurrent selection is widely regarded as an effective strategy for the improvement of poly‐ genic traits. However, the effectiveness and efficiency of selection are not so satisfactory in some cases because phenotypic selection is highly dependent upon environments and geno‐ typic selection takes a longer time (2-3 crop seasons at least for one cycle of selection). Markerassisted recurrent selection (MARS) is a scheme which allows performing genotypic selection and intercrossing in the same crop season for one cycle of selection (Fig. 1). Therefore, MARS could enhance the efficiency of recurrent selection and accelerate the progress of the proce‐ dure (Jiang et al., 2007a), particularly helps in integrating multiple favorable genes/QTLs from different sources through recurrent selection based on a multiple-parental population.

**Figure 1.** General procedure of marker-assisted recurrent selection (MARS)

For complex traits such as grain yield, biotic and abiotic resistance, MARS has been pro‐ posed for "forward breeding" of native genes and pyramiding multiple QTLs (Ragot et al., 2000; Ribaut et al., 2000, 2010; Eathington, 2005; Crosbie et al., 2006). As defined by Ribaut et al. (2010), MARS is a recurrent selection scheme using molecular markers for the identification and selection of multiple genomic regions involved in the expression of complex traits to assemble the best-performing genotype within a single or across related populations. Johnson (2004) presented an example to demonstrate the efficiency of MARS for quantitative traits. In their maize MARS programs, a large-scale use of markers in biparental populations, first for QTL detection and then for MARS on yield (i.e. rapid cy‐ cles of recombination and selection based on associated markers for yield), could allow increased efficiency of long-term selection by increasing the frequency of favorable alleles (Johnson, 2004). Eathington (2005) and Crosbie et al. (2006) also indicated that the genetic gain achieved through MARS in maize was about twice that of phenotypic selection (PS) in some reference populations. In upland cotton, Yi et al. (2004) reported significant effec‐ tiveness of MARS for resistance to *Helicoverpa armigera*. The mean levels of resistance in improved populations after recurrent selection were significantly higher than those of pre‐ ceding populations.
