**3. Participation of biotechnology to support and improve the breeding process in the INIA potato program**

**Figure 1** shows an organization chart about the role of biotechnology in the potato breeding program in Chile. In first place, the germplasm bank is the source of genetic diversity for controlled crosses. This in vitro gene banks hold the varieties developed, advanced breeding lines, and imported breeding material that can be used of donor of some characters and native landraces. With this system, the material is preserved free from pathogens and suitable to be transferred to foreign countries in case of any need of varieties and breeding lines.

Techniques to introduce the material to in vitro conditions and thermotherapy to obtain pathogen-free in vitro plants are applied. Thermotherapy in combination with previous chemotherapy can be employed successfully, but efficiency is variable depending on virus types to remove. Results of DAS ELISA test before and after thermotherapy of a group of potato accessions from the field strongly infected by different viruses are shown in **Table 1**, indicating that in the case of PVY, 51% of the materials could be cleaned after two rounds of thermotherapy.

including native potatoes, commercial varieties, and valuable breeding lines. It is important to notice that SSR markers must be polymorphic enough to distinguish between the different varieties that it is necessary to discriminate. In the case of using molecular markers in the process of plant propagation of potato, as a tool to assure the identity of the commercial varieties that are being propagated in vitro, it is convenient to determine the set of markers that allow to produce different allele phenotypes (band patterns) for all the commercial varieties that are multiplied by the seed program, in order to differentiate them. In the case of wild material collected and kept in germplasm bank, or breeding lines with unknown pedigree, the SSR markers could discriminate different allele phenotypes, but it depends on the numbers of markers and polymorphism detected. Always it is possible that different genotypes could not be differentiated because no polymorphism in the regions of the genomes are being analyzed, but once a different band pattern is found between some plant materials, it is proven that they

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

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7

**Figure 1.** Organization chart about the role of biotechnology in the Chilean potato breeding program.

The materials stored in germplasm bank are analyzed by SSR markers to characterize them by molecular fingerprint. Currently, eight SSR markers are used. These markers are employed as a routine test for varietal identification since all the varieties are released with a described molecular profile that allows tracking of the varieties in the markets after available to farmers and to solve problems as mixture of varieties. These markers have been used in an overview of the genetic diversity and genotype numbers in germplasm bank of Chilean collection, The Use and Impact of Biotechnology in Potato Breeding: Experience of the Potato Breeding… http://dx.doi.org/10.5772/intechopen.72961 7

The main objectives of the program are:

• Conformation and appearance of tuber

international demand

6 Potato - From Incas to All Over the World

• Industrial uses

• Specific objectives:

○ PVY resistance

field conditions.

thermotherapy.

○ Late blight resistance

○ Golden nematode resistance

• Good performance for different end uses (fresh market and processing) for national or

• Good agronomic characteristics: high yield and wide adaptation to agro-climatic zones

To achieve these objectives, the activities of the program involve controlled crosses every year (around 100), with 30,000 novel genotypes that are evaluated in multiyear and locations in

**Figure 1** shows an organization chart about the role of biotechnology in the potato breeding program in Chile. In first place, the germplasm bank is the source of genetic diversity for controlled crosses. This in vitro gene banks hold the varieties developed, advanced breeding lines, and imported breeding material that can be used of donor of some characters and native landraces. With this system, the material is preserved free from pathogens and suitable to be

Techniques to introduce the material to in vitro conditions and thermotherapy to obtain pathogen-free in vitro plants are applied. Thermotherapy in combination with previous chemotherapy can be employed successfully, but efficiency is variable depending on virus types to remove. Results of DAS ELISA test before and after thermotherapy of a group of potato accessions from the field strongly infected by different viruses are shown in **Table 1**, indicating that in the case of PVY, 51% of the materials could be cleaned after two rounds of

The materials stored in germplasm bank are analyzed by SSR markers to characterize them by molecular fingerprint. Currently, eight SSR markers are used. These markers are employed as a routine test for varietal identification since all the varieties are released with a described molecular profile that allows tracking of the varieties in the markets after available to farmers and to solve problems as mixture of varieties. These markers have been used in an overview of the genetic diversity and genotype numbers in germplasm bank of Chilean collection,

transferred to foreign countries in case of any need of varieties and breeding lines.

**3. Participation of biotechnology to support and improve** 

**the breeding process in the INIA potato program**

**Figure 1.** Organization chart about the role of biotechnology in the Chilean potato breeding program.

including native potatoes, commercial varieties, and valuable breeding lines. It is important to notice that SSR markers must be polymorphic enough to distinguish between the different varieties that it is necessary to discriminate. In the case of using molecular markers in the process of plant propagation of potato, as a tool to assure the identity of the commercial varieties that are being propagated in vitro, it is convenient to determine the set of markers that allow to produce different allele phenotypes (band patterns) for all the commercial varieties that are multiplied by the seed program, in order to differentiate them. In the case of wild material collected and kept in germplasm bank, or breeding lines with unknown pedigree, the SSR markers could discriminate different allele phenotypes, but it depends on the numbers of markers and polymorphism detected. Always it is possible that different genotypes could not be differentiated because no polymorphism in the regions of the genomes are being analyzed, but once a different band pattern is found between some plant materials, it is proven that they


**Table 1.** Efficiency of thermotherapy treatments for virus cleaning in a group of potato accessions from the field strongly infected by different viruses.

are different genotypes. This is very useful when studying collections of material with similar morphological features or when not all the descriptors are available to be examined (collections of tubers, in vitro plants or others where no flowers or leaves are available, or material affected by virus that affects the phenotype). In the case of Chilean collection of native potatoes, we have found 320 different allelic phenotypes using four SSR markers, indicating that there are at least 320 different genotypes in the collections. Of these, 158 belonging to the INIA collection were not found in another collection belonging to other Chilean institutions. As expected, different genotypes were known under the same popular name by the farmers. The molecular information is useful to know the genetic structure of the material preserved or used for breeding. For more details of our results, please see [6].

parents by means of study segregation of the marker in an F1 population in order to know the frequency of progeny that can hold the desired character and recognize most efficient parents

**Table 2.** Molecular markers used for routine assays to select breeding lines with resistance genes to potato diseases.

**Gene Marker Resistance to Reference**

*Gro1–4* Gro1–4 [9] Paal et al. *GroV1* U14 [10] Jacobs et al. *Ryadg* Ry3.3.3S/RyADG23R PVY [11] Kasai et al. *Rx2* AC15 PVX [12] Bendahmane et al.

*R1* R1 *P. infestans* [14] Ballvora et al. *R2* R2 [15] Kim et al. *R3a* R3a [16] Huang et al. *R3b* R3b [17] Rietman

*H1* 57R *G. rostochiensis* [7] Finkers-Tomczak

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

TG689 De Jong, W. Cornell

Ask [13] Bendahmane et al.

et al.

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

et al.

University,

(unpublished); [8] Galek

9

Once the new variety is ready to enter to the market, it is necessary to produce the stock of

Then, in the early stages of seed production in certification system, all the mother plant materials for the new varieties are checked by molecular fingerprint, PCR, and ELISA test for pathogen diagnosis, to assure the identity and pathogen-free status of the starting seed material and then micropropagated before entering to the certification system in the field. PVY and *Pectobacterium* are tested by PCR (**Table 3**), and PVX, PVM, PVS, PVA, PLRV, and PVY

In this stage, tissue culture for massive micropropagation of new varieties is still a pivotal

• Today, in the official certification system, all the varieties are propagated via in vitro cul-

• Introduction of new varieties to in vitro condition is essential to produce certificated seed

and makes possible that the varieties be distributed in the market.

seed to support the entrance in the seed certification system.

*Rx1 y Rx2*

for controlling crosses.

N° 10 11

**Routine markers for resistance genes implemented in PMGP-INIA**

are diagnosed by DAS ELISA test.

ture before being multiplied in the field.

biotechnological technique:

There is a flow of material from the gene bank to the annual scheme of controlled crosses and selection in the plant breeding program. Some genotypes are selected in order to combine characteristics in the progeny through controlled crosses and grown to obtain flowers and used as donor of valuable traits. On the other hand, promissory breeding lines from the field are introduced to in vitro culture and kept in the bank.

During the phases of selecting/discarding clones in field plots, molecular markers are implemented. Molecular markers associated with one or few genes that have a large and heritable effect in important traits are used (e.g., disease resistance in gene per gene model). Molecular markers for golden nematode resistance, virus resistance, late blight resistance, and some markers for flesh color are involved in the battery of markers to assist the selection and verify the combination of several resistance genes (**Table 2**). We investigate the allele dosage in some The Use and Impact of Biotechnology in Potato Breeding: Experience of the Potato Breeding… http://dx.doi.org/10.5772/intechopen.72961 9


**Table 2.** Molecular markers used for routine assays to select breeding lines with resistance genes to potato diseases.

are different genotypes. This is very useful when studying collections of material with similar morphological features or when not all the descriptors are available to be examined (collections of tubers, in vitro plants or others where no flowers or leaves are available, or material affected by virus that affects the phenotype). In the case of Chilean collection of native potatoes, we have found 320 different allelic phenotypes using four SSR markers, indicating that there are at least 320 different genotypes in the collections. Of these, 158 belonging to the INIA collection were not found in another collection belonging to other Chilean institutions. As expected, different genotypes were known under the same popular name by the farmers. The molecular information is useful to know the genetic structure of the material preserved

present per infected accession

**Table 1.** Efficiency of thermotherapy treatments for virus cleaning in a group of potato accessions from the field strongly

There is a flow of material from the gene bank to the annual scheme of controlled crosses and selection in the plant breeding program. Some genotypes are selected in order to combine characteristics in the progeny through controlled crosses and grown to obtain flowers and used as donor of valuable traits. On the other hand, promissory breeding lines from the field

During the phases of selecting/discarding clones in field plots, molecular markers are implemented. Molecular markers associated with one or few genes that have a large and heritable effect in important traits are used (e.g., disease resistance in gene per gene model). Molecular markers for golden nematode resistance, virus resistance, late blight resistance, and some markers for flesh color are involved in the battery of markers to assist the selection and verify the combination of several resistance genes (**Table 2**). We investigate the allele dosage in some

or used for breeding. For more details of our results, please see [6].

are introduced to in vitro culture and kept in the bank.

**Situation prior to thermotherapy treatment**

8 Potato - From Incas to All Over the World

Number of accessions subjected to in vitro thermotherapy

Number of accessions infected with at least one

Average number of viruses present per infected accession

infected by different viruses.

virus

**Number or %**

**Situation after in vitro thermotherapy treatments**

cleaned after two rounds of

157 Number of accessions

% incidence of PVX 26 % incidence of PVX 3.2 88% % incidence of PVY 69 % incidence of PVY 33 51% % incidence of PVS 78 % incidence of PVS 7 91% % incidence of PLRV 86 % incidence of PLRV 21.7 75% % incidence of PVA 8.9 % incidence of PVA 0.6 93% % incidence of PVM 1.2 % incidence of PVM 0 100%

thermotherapy

2.6 Average number of viruses

157 Number of accessions infected with at least one virus

**Number or %**

91

0.63

66 42%

**Efficiency of** 

**FIN × 100**

**thermotherapy (INI-FIN)/**

parents by means of study segregation of the marker in an F1 population in order to know the frequency of progeny that can hold the desired character and recognize most efficient parents for controlling crosses.

Once the new variety is ready to enter to the market, it is necessary to produce the stock of seed to support the entrance in the seed certification system.

Then, in the early stages of seed production in certification system, all the mother plant materials for the new varieties are checked by molecular fingerprint, PCR, and ELISA test for pathogen diagnosis, to assure the identity and pathogen-free status of the starting seed material and then micropropagated before entering to the certification system in the field. PVY and *Pectobacterium* are tested by PCR (**Table 3**), and PVX, PVM, PVS, PVA, PLRV, and PVY are diagnosed by DAS ELISA test.

In this stage, tissue culture for massive micropropagation of new varieties is still a pivotal biotechnological technique:



areas with potato cultivation in northern part of the country. For this pest, it is not possible to conduct field trials in south of Chile, so molecular marker implementation is crucial to track resistance in the progeny from controlled crosses with appropriate donor parents able to pro-

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

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

11

The program has implemented six biotechnological techniques; these are applied in the stages of characterization of the gene bank, selection of parents, marker-assisted selection, characterization of varieties, and propagation of material for seed production. One hundred percent of the varieties have been released involving biotechnology, especially by the use of in vitro culture techniques to produce pathogen-free material for initial stages of seed production of advanced lines. Biotechnological techniques have participated in the improvement of 2 of the 13 main characteristics associated with the program objectives. Two of the 11 varieties were characterized by molecular fingerprint at the time of their release. Biotechnological techniques such as in vitro culture, molecular fingerprint, and molecular diagnosis of diseases are used to produce primary multiplication of reproductive material for 100% of the varieties

Some important facts about the use of biotechnology in breeding and development of variet-

• Tissue culture is essential in the maintenance of varieties with the same genotype and ini-

• Molecular fingerprint is important for varietal identification: vital in traceability of stock plants during micropropagation, and it has possibilities to be used to track the presence of

• Molecular markers associated with one or few genes that have a large and heritable effect

• Molecular markers allow to have more precision to choose parents for crossing in order to

• Markers for multigenic traits such as stress tolerance or cold tolerance have not been developed yet and remain as a big challenge to develop molecular genetic tools for multigenic traits.

In potato breeding, the selection of desirable phenotypes from a large breeding population

• Automatic, low-cost, and high-throughput phenomic technologies would be a valuable

in important traits are used (e.g., disease resistance in gene per gene model).

**4. Conclusions about the role of biotechnology in Chilean potato** 

duce offspring with different resistance genes.

released by INIA currently present on the market.

tial steps of seed production system.

varieties in the market.

will remain essential.

combine or pyramiding genes.

tool for massive screening of phenotypes.

**breeding program**

ies are:

**Table 3.** Procedures used for molecular diagnosis for *Pectobacterium* spp. and PVY in potato plants.

Currently our program keeps in vitro the 11 INIA varieties, 134 advanced breeding lines from INIA program, and 32 foreign varieties and breeding lines with research purposes.

Molecular fingerprints have been done to characterize 61 varieties, 25 advanced breeding lines, and 823 native landraces. We use the CIP identity kit for molecular profiling of the most valuable material for reliable identification and traceability during breeding process and seed production and in the future to track the presence in the market.

We use 11 molecular markers for marker-assisted selection, and at the date, we have analyzed 461 breeding lines and 33 varieties. The most important advantage of applying these markers is to allow more precision to choose parents for crossing in order to combine or pyramiding genes.

In **Table 4**, we can see the markers associated with resistance genes and light yellow flesh color present in the released varieties. It is possible to see that many varieties hold markers associated to golden nematode resistance, a quarantine pest in Chile but present in some


**Table 4.** Molecular markers associated with resistance genes and light yellow flesh color present in the released varieties.

areas with potato cultivation in northern part of the country. For this pest, it is not possible to conduct field trials in south of Chile, so molecular marker implementation is crucial to track resistance in the progeny from controlled crosses with appropriate donor parents able to produce offspring with different resistance genes.
