**5. Biotechnology techniques to support clone selection procedures to pyramiding resistance genes to late blight**

Biotechnology can be easily combined with classical breeding methods with the objective to pyramiding resistance genes (R genes) to avoid the breakdown of resistance in the case of fast evolving pathogens. We will describe below our experience in developing a strategy to pyramiding R genes for late blight resistance in breeding lines.

For this purpose, we are using the Ma*R8* and Ma*R9* genotypes as sources of resistance to late blight in the Chilean potato breeding program.

#### **5.1. Late blight as a major threat for potato production and food security**

The potato late blight caused by *Phytophthora infestans* (Mont.) de Bary is a major challenge to potato production worldwide [20]. Reliance on susceptible potato cultivars in commercial agriculture has meant that fungicides are widely used to control late blight. However, such materials have significant monetary and environmental costs to society [21–23]. To control this disease, up to 14 applications of fungicides may be needed for a crop season.

*P. infestans* can infect the entire plant, including the stems, leaves, and tubers. When left unchecked, it can quickly destroy a potato crop within a few days. The success of this pathogen is not only due to its elevated virulence but also to its remarkable capacity of rapidly adapt to resistant plants. Therefore, new resistant potato varieties with multiple resistance genes must be produced, as the use of varieties with genetic resistance to late blight is essential for growing low-cost, healthy, and environmentally sustainable potatoes.

However, only three of the 25 potato varieties used in Chile have some intermediate level of resistance to late blight. Notably, while *S. tuberosum* lacks significant resistance, wild potato species are rich sources of late blight resistance genes. *Solanum demissum*, a hexaploid Mexican wild *Solanum* species, is an important source of resistance to late blight. The major resistance (R) genes from *S. demissum* have late blight race specificity.

Eleven R gene differentials containing R genes introgressed into *S. tuberosum* from *S. demissum* were collected by Mastenbroek [24] and are referred to as the Mastenbroek differential set: Ma*R1* to Ma*R11*. In Ma*R8* and Ma*R9*, at least four (*R3a*, *R3b*, *R4*, and *R8*) and seven (*R1*, *Rpi-abpt1*, *R3a*, *R3b*, *R4*, *R8*, and *R9*) R genes were present, respectively [15, 25]. This set can be used to simultaneously introduce multiple R genes. However, since this set has a low agronomic value, crosses must be made with elite breeding material to obtain breeding lines suitable for propagation.

Significantly, the resistance provided by the R genes is background dependent as genes that suppress R genes can also be segregated into F1 offspring plants [26]. Thus, more knowledge is needed about how R genes perform in different genetic backgrounds. Research has also focused on creating GM organisms carrying constructions with the described R genes [27]. However, this class of plant material is not allowed in many parts of the world for human or animal consumption including Europe and Chile. Furthermore, society is suspicious about its sustainable utilization because of the health, environmental, and social implications of GMOs. For this reason, it is necessary to investigate the applications of the natural stacking of several R genes to overcome *P. infestans.*

The objectives of our work are:

• Screening methods based on next-generation sequencing technologies promise to revolutionize screening for desired genotypes, but it is necessary to solve the problem of distinguish between three different heterozygous genotypes (AAAB, AABB, and ABBB) in traits

• In order to make more precise the addressing of breeding procedures to improve specific traits (i.e., compounds with nutritional value or to eliminate undesired characters), methods as the new biotechnological techniques (NBTs) could be promising in countries where GMOs are not allowed. These new technologies as CRIPSR/Cas can be used to develop a

**5. Biotechnology techniques to support clone selection procedures to** 

Biotechnology can be easily combined with classical breeding methods with the objective to pyramiding resistance genes (R genes) to avoid the breakdown of resistance in the case of fast evolving pathogens. We will describe below our experience in developing a strategy to

For this purpose, we are using the Ma*R8* and Ma*R9* genotypes as sources of resistance to late

The potato late blight caused by *Phytophthora infestans* (Mont.) de Bary is a major challenge to potato production worldwide [20]. Reliance on susceptible potato cultivars in commercial agriculture has meant that fungicides are widely used to control late blight. However, such materials have significant monetary and environmental costs to society [21–23]. To control

*P. infestans* can infect the entire plant, including the stems, leaves, and tubers. When left unchecked, it can quickly destroy a potato crop within a few days. The success of this pathogen is not only due to its elevated virulence but also to its remarkable capacity of rapidly adapt to resistant plants. Therefore, new resistant potato varieties with multiple resistance genes must be produced, as the use of varieties with genetic resistance to late blight is essen-

However, only three of the 25 potato varieties used in Chile have some intermediate level of resistance to late blight. Notably, while *S. tuberosum* lacks significant resistance, wild potato species are rich sources of late blight resistance genes. *Solanum demissum*, a hexaploid Mexican wild *Solanum* species, is an important source of resistance to late blight. The major resistance

Eleven R gene differentials containing R genes introgressed into *S. tuberosum* from *S. demissum* were collected by Mastenbroek [24] and are referred to as the Mastenbroek differential set: Ma*R1*

where plex number affects the character under selection.

12 Potato - From Incas to All Over the World

**pyramiding resistance genes to late blight**

blight in the Chilean potato breeding program.

pyramiding R genes for late blight resistance in breeding lines.

**5.1. Late blight as a major threat for potato production and food security**

this disease, up to 14 applications of fungicides may be needed for a crop season.

tial for growing low-cost, healthy, and environmentally sustainable potatoes.

(R) genes from *S. demissum* have late blight race specificity.

genetic engineering with no transgenic status of the final product.


#### **5.2. Materials and methods**

Controlled crosses were performed between five commercial varieties and the genotypes Ma*R8* and Ma*R9* that hold four and seven genes of resistance, respectively.

Ten progenies from each cross were randomly selected for molecular analysis and phenotypic evaluations in order to assay for the presence of R genes. The performance of these genotypes was monitored under pathogenic pressure in field conditions.

During the 2013–2014 season, 90 randomly selected progenies were evaluated for pathogen resistance. We calculated the area under disease progress curve (AUDPC) in individual plants under natural infections in the field. To promote infections, the progeny was watered by a spray system twice a week although the natural inoculum was high in Osorno, Chile.

For the molecular analysis, DNA extractions and PCR amplification were performed using genetic markers as described in **Table 2** for *P. infestans* to track the presence or absence of R genes in the Ma*R8* or Ma*R9* crossed with elite material progeny.

Progeny was also phenotypically evaluated. We determined the percentage of leaves affected by late blight during plant development by visually estimating the green and non-green portions of the leaves. The estimations were integrated into the AUDPC or area under the disease progress curve. AUDPC is obtained by the repeated visual inspections and estimation of the percentage of the leaf affected in a set of plants. The value is calculated by the formula used by Jo et al. [28]. Percentages of damaged foliage are plotted through a period of time. AUDPC was calculated for the 10 randomly selected individuals from each cross in the field.

For the following 2014–2015 season, the tubers of 71 genotypes that were not damaged by late blight were harvested. The experiment was designed with three replicates in randomized blocks. We planted three plants of each genotype that hold R genes in front of three plants of the susceptible Atlantic cultivar (susceptible control) per each replicate.

**5.4. Conclusions about pyramiding R genes for resistance to late blight**

blight damage and for large-scale screening of breeding lines.

Manuel Andrés Muñoz\*, Julio César Kalazich, Carolina Verónica Folch,

Institute of Agricultural Research INIA, Regional Center Remehue, Chile

scheme of the potato breeding program.

Sandra Valeska Orena and Annelore Winkler

\*Address all correspondence to: manuel.munozd@inia.cl

Agricultura del gobierno de Chile; 2017. 17 p

Science Publishers; 2011. pp. 20-40

2016. DOI: 10.1007/s11540-016-9328-6

**Acknowledgements**

**Author details**

**References**

2015;**65**:3-16

The Ma*R8* and Ma*R9* crosses were successful and generated hybrid genotypes harboring at least four different R genes that are now available for breeding. The progeny carrying R genes has been selected as parents for backcrosses or early clonal selection step and entered to the

The Use and Impact of Biotechnology in Potato Breeding: Experience of the Potato Breeding…

http://dx.doi.org/10.5772/intechopen.72961

15

A major challenge remains to develop an efficient and reliable system of phenotyping for late

Financial support: Subsecretary of the Chilean Ministry of Agriculture through Potato Breeding Program project and Conservation of Genetic Resources program (501453-70). CORFO (Corporación de Fomento a la Producción) through INNOVA-Public Goods 14BPC4-28525.

[1] Monneveux P, Ramírez D, Pino MT. Drought tolerance in potato (*S. tuberosum* L) can we learn from drought tolerance research in cereals? Plant Science. 2013;**205-206**:76-86 [2] Oficina de estudios y políticas agrarias ODEPA. Boletin de la papa. Ministerio de

[3] Carputo D, Frusciante L. Classical genetics and traditional breeding. In: Genetics, Genomics and Breeding of Potato. Enfield, New Hampshire: Ed: Braaden and Kole,

[4] Mori K, Asano K, Tamiya S, Nakao T, Mori M. Challenges of breeding potato cultivars to grow in various environments and to meet different demands. Breeding Science.

[5] Eriksson D, Carlson-Nilssen V, Ortiz R, Andreasson E. Overview and breeding strategies of table potato production in Sweeden on the fennoscandian region. Potato Research.

Rows of the susceptible Atlantic cultivar were also planted in the border and interspersed in the complete area of the assay. AUDPC values were calculated for all genotypes and susceptible control plots. A pairwise comparison was performed between each genotype and the respective control plot. We calculated the AUDPC from the visual estimation of the percentage of infected foliage.

#### **5.3. Results**

In the 2013–2014 season, an evaluation of randomly selected individual plants from the progeny of Ma*R8* and Ma*R9* crossed with the commercial varieties indicated that progeny carrying the *R2* gene had less foliage damage represented by lower AUDPC values. Furthermore, higher AUDPC values were found in plants that did not contain R genes. We were not able to perform statistical analysis as there were different numbers of clones holding R genes.

In the second season, we utilized undamaged plants from the first season. Most of the plants carrying R genes again had lower AUDPC values than the control plots. Interestingly, plants carrying the *R3* gene did not have different AUDPC values compared to the susceptible control plots.

In conclusion, we found that plants carrying R genes were only slightly affected by late blight in conditions of high pathogenic pressure, with the exception of the *R3a* and *R3b* genes (**Table 5**). Out of the 11 genotypes that did not show differences compared to the susceptible control, nine were holders of genes *R3a* and *R3b,* suggesting that these R genes are less resistant to late blight and, therefore, should not be used in breeding programs in Chile.


**Table 5.** Genotypes harboring different numbers of R genes and % showing an AUDPC value significantly lower than the susceptible control plots.

#### **5.4. Conclusions about pyramiding R genes for resistance to late blight**

The Ma*R8* and Ma*R9* crosses were successful and generated hybrid genotypes harboring at least four different R genes that are now available for breeding. The progeny carrying R genes has been selected as parents for backcrosses or early clonal selection step and entered to the scheme of the potato breeding program.

A major challenge remains to develop an efficient and reliable system of phenotyping for late blight damage and for large-scale screening of breeding lines.
