Keywords:

Oomycete

Phytophthora infestans

PLRV

PYV

Solanum tuberosum

Identication of regions associated to late blight resistance and viruses in potato germplasm

using molecular markers

Identicación de regiones asociadas a la resistencia del tizón tardío y virus en germoplasma de papa

mediante marcadores moleculares

Identicação de regiões associadas à resistência à plaga tardia e vírus em germoplasma de batata

usando marcadores moleculares

Ariadne Vegas García

1,2*

Yanet Sandrea Rincón

2†

Asia Zambrano

2†

Lourdes González

Martha Osorio

Guillermo Trujillo

Jorge Peralta Calle

Rev. Fac. Agron. (LUZ). 2022, 39(2): e223935

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v39.n3.01

Crop Production

Associate editor: Dr. Francisco Osorio-Acosta

Colegio de Postgraduados Campus

Veracruz, Mexico Tepetates, Veracruz,

MX.

Abstract

The combination of traits of economic interest in new potato cultivars,

such as resistance to late blight, viral diseases, and culinary quality are

important to achieve their adoption by farmers. In the present work,

molecular markers were used to identify regions associated to late blight,

the viruses PVY and PLRV resistance, in 50 materials belonging to the

National Institute of Agricultural Research (INIA-Venezuela): commercial

cultivars, differentials of blight, advanced clones from CIP and hybrids from

the Fundación PROINPA of Bolivia. DNA extraction was carried out from

vitroplants and known microsatellite, SCAR and CAPS molecular markers

were used. Among 96 to 26% of the accessions amplied regions of the

QTL tbr of chromosome XII, associated with resistance to blight. Only the

differential R9 and crc2/P8 from PROINPA amplied the R1 gene region.

Between 18 and 68% of the genotypes presented the regions associated with

the PVY and PLRV resistance genes (Ry

adg

and N genes), respectively; only

10% amplied both regions; while in 24% these genes were not detected,

among them are the commercial varieties Granola, Andinita and Cartayita.

This study generated valuable information to support genebank curators and

breeders in potato genetic improvement programs of this country.

Instituto Nacional de Investigaciones Agropecuarias

(INIAP). Estación Experimental Santo Domingo. Km 38 vía

Santo Domingo – Quinindé. Cantón La Concordia. Santo

Domingo de los Tsáchilas. Ecuador.

Instituto Nacional de Investigaciones Agrícolas (INIA),

Centro Nacional de Investigaciones Agropecuarias

(CENIAP). Unidad de Biotecnología Vegetal. Apartado

Postal 5653, Maracay 2101, Venezuela.

Instituto Nacional de Investigaciones Agrícolas (INIA),

Centro de Investigaciones Agropecuarias del Estado Mérida,

Venezuela.

Applied Biotecnology Laboratory. International Potato

Center (CIP). P.O. Box 1558, Lima 12, Perú.

Universidad Agraria del Ecuador, sede Guayaquil. Av. 25 de

julio. Guayaquil 090104. Ecuador.

Received: 08-08-2021

Accepted: 27-05-2022

Published: 27-06-2022

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Rev. Fac. Agron. (LUZ). 2022, 39(3): e223935. July - September. ISSN 2477-9407.2-6 |

Resumen

La combinación de caracteres de interés económico en nuevos

cultivares de papa, tales como la resistencia al tizón tardío, las

enfermedades virales, y la calidad culinaria son importantes para

lograr su adopción por los agricultores. En el presente trabajo se

usaron marcadores moleculares para identicar regiones asociadas

a la resistencia del tizón tardío y a los virus PVY y PLRV, en 50

materiales pertenecientes al Instituto Nacional de Investigaciones

Agrícolas (INIA-Venezuela): cultivares comerciales, diferenciales del

tizón, clones avanzados del CIP e híbridos de la Fundación PROINPA

de Bolivia. La extracción del ADN se realizó a partir de vitroplantas

y se utilizaron marcadores moleculares tipo microsatélites, SCAR y

CAPS conocidos. Entre el 96 al 26 % de las accesiones amplicaron

regiones del QTL tbr del cromosoma XII, asociado a la resistencia al

tizón. Solo el diferencial R9 y crc2/P8 de PROINPA amplicaron la

región del gen R1. Entre el 18 y 68 % de los genotipos presentaron

las regiones asociadas con los genes de resistencia al PVY y PLRV

(genes Ry

adg

y N), respectivamente; solo el 10 % amplicaron ambas

regiones; mientras que en 24 % no se detectaron estos genes, entre

ellos se encuentran las variedades comerciales Granola, Andinita y

Cartayita. Este estudio generó información valiosa de soporte a los

curadores de los bancos de germoplasma y a los tomejoradores en

los programas de mejoramiento genético de papa del país.

Palabras clave: Oomicete, Phytophthora infestans, PLRV, PYV,

Solanum tuberosum.

Resumo

A combinação de características de interesse econômico em

novas cultivares de batata, como resistência à requeima, doenças

virais e qualidade culinária, são importantes para sua adoção pelos

produtores. No presente trabalho, marcadores moleculares foram

utilizados para identicar regiões associadas à resistência à requeima

e aos vírus PVY e PLRV, em 50 materiais pertencentes ao Instituto

Nacional de Pesquisa Agropecuária (INIA-Venezuela): cultivares

comerciais, diferenciais da requeima, clones avançados do CIP e

híbridos da Fundación PROINPA da Bolívia. A extração de DNA foi

realizada a partir de vitroplantes e foram utilizados microssatélites,

marcadores moleculares SCAR e CAPS conhecidos. Entre 96 a 26

% dos acessos amplicaram regiões do QTL tbr do cromossomo

XII, associadas à resistência à ferrugem. Apenas o diferencial R9 e

crc2/P8 de PROINPA amplicou a região do gene R1. Entre 18 e

68 % dos genótipos apresentaram as regiões associadas aos genes de

resistência PVY e PLRV (genes N e Ry

adg

), respectivamente; apenas

10 % amplicou ambas as regiões; enquanto em 24 % esses genes não

foram detectados, entre eles estão as variedades comerciais Granola,

Andinita e Cartayita. Este estudo gerou informação valiosa para

apoiar curadores de bancos de germoplasma e melhoristas de plantas

nos programas de melhoramento genético da batata do país.

Palavras chave: Oomiceto, Phytophthora infestans, PLRV, PYV,

Solanum tuberosum.

Introduction

In Venezuela, the potato (Solanum tuberosum L.) is considered

a fundamental part of the diet, especially for inhabitants located in

the Andean areas, states of Mérida, Táchira and Trujillo, due to its

high content of carbohydrates, vitamins and minerals; constituting

one of the main sources of economic income for small and medium

farmers in these regions, where more than 80 % of the cultivated area

is planted (González et al., 2017).

Globally, pests and diseases are the biggest problem facing potato

growers, particularly small-scale growers in less developed countries,

where certied seed and agrochemicals are not affordable. Among

the most important diseases are late blight, bacterial wilt, and PVY

(Potato Virus Y), PVX (Potato Virus X) and potato leafroll virus

(PLRV) (Haverkort et al., 2016).

Late blight, commonly referred to as late candelilla or blight, is

caused by the oomycete Phytophthora infestans (Mont.) de Bary.

The disease can destroy foliage and stems, and attack tubers, when

conditions are favorable, at moderate temperatures, 16 to 22 °C,

and high humidity (100 %). In prolonged humid conditions, all the

tender and aerial organs of the plants wither and rot very quickly.

It can be satisfactorily controlled using resistant cultivars, spraying

with chemical compounds applied systematically, and cultural

practices (Agrios, 2005). The oomycete comprises several pathotypes

or physiological races, identied according to their virulence in

differential potato genotypes. Pathotypes are the product of genetic

variation due to mutations, sexual recombination; and possibly

to changes in the ploidy of the pathogen or to hybridization with

other Phytophthora species. New races with greater resistance to

systemic fungicides, greater virulence and parasitic aptitude have

been identied, as well as the existence of oospores resulting from

the sexual reproduction of the pathogen in new agricultural regions

(Alvarez-Morezuelas et al., 2021).

In Venezuela, the disease is present in almost all areas of the

country where potatoes are grown, causing losses up to 100 %, when

the infection occurs at critical times of crop development and before

tuberization. Because the Granola cultivar, widely used in the country

in the areas most affected by blight, has low resistance to candelilla,

producers apply systemic fungicides without restriction (García and

García, 2004). Isolates of P. infestans collected in various regions of

the country have been characterized and are of the A1 compatibility

type, with different susceptibilities to the metalaxyl fungicide

(Rodríguez et al., 2008).

Two types of resistance to late blight have been identied in potato

and related wild species, one based on major dominant genes (R

genes), and the other polygenic, based on a Quantitative Trait Locus

(QTL). The rst type of resistance was introduced into cultivated

potatoes through the introgression of R genes from the wild species

S. demissum, of which 11 (R1-R11) have been identied. However,

these genes were overtaken by newer races of the oomycete, and

they were found not to provide durable resistance, either alone or in

combination. For this reason, R genes present in other wild species,

S. berthaultii, S. bulbocastanum, S. guerreroense, S. neoantipoviczi

and S. pinnatisectum, among others, which have been incorporated

in a pyramidal combination to S. tuberosum, were searched for

(Ballesteros et al., 2010; Fadina et al., 2017; Zoteyeva et al., 2014).

It has been argued that the best strategy to achieve longer-lasting

resistance, possibly exerting less selection pressure on the pathogen,

is the introgression of R genes that confer quantitative eld resistance,

as in the case of genes R8, R9a, R10 and Rpi-blb1 (Fadina et al., 2017;

Jiang et al., 2018). Other breeders have chosen to select genotypes

with quantitative eld resistance, controlled by various non-race-

specic genes, which has been described as horizontal, incomplete,

and broad-spectrum (Fadina et al., 2017; Jiang et al., 2018). However,

this type of resistance depends on external factors and has shown

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Vegas et al. Rev. Fac. Agron. (LUZ). 2022, 39(3): e2239353-6 |

a strong correlation with late maturity. Today it is known that, in

general, QTLs correspond to groups of R genes (Jiang et al., 2018;

Rubio et al., 2016).

Ballvora et al. (2002) cloned the rst late blight resistance gene,

called R1, located on chromosome V, in a dense region, where other

resistance genes have also been found. To date, genes R1, R2, R3, and

R8 have been cloned (Jiang et al., 2018).

On the other hand, QTL or regions of the genome whose

phenotypic effect is measurable on a continuous scale have been

identied to identify potato genotypes resistant to late blight (Jiang

et al., 2018; Trujillo, 2004). In this sense, Trujillo (2004) developed

CAPS (Cleaved Amplied Polymorphic Sequences) and SCAR

(Sequence Characterized Amplied Region) markers, which amplied

regions of the QTL tbr of chromosome XII, from a segregating

population of S.

phureja (diploid, with resistance to late blight) x S.

tuberosum (dihaploid, susceptible to the disease). This QTL of the

parent S. tuberosum had a high contribution to eld resistance to late

blight, which also provided phenotypic variation of up to 43 % and

was associated with markers linked to candidate genes involved in

biochemical defense pathways, such as phenylalanine ammonia lyase

(PAL) and chalcone isomerase (CHI) enzymes, WRKY transcription

factors, osmotin, and cytochrome P450 induced by P. infestans.

Regarding viruses, in the Andean region of Venezuela, the potato crop is

affected by a series of viruses, PLRV, PVY, PVX, Potato Virus S, Andean

Mottled Virus (Potato Virus M, PVM) and Potato Virus A (PVA), indicated

by García et al. (2005) and Pichardo et al. (2013).

Potato virus Y (PVY), from the Potivirus group, is one of the

most important viruses in several Solanaceae species, including

potato, tomato, tobacco and paprika (Scholthof et al., 2011; Thomas-

Sharma et al., 2016). It is transmitted from infected seed potato

tuber and by at least 20 species of aphids in a non-persistent

manner and causes high yield reduction in most potato areas.

Symptoms range from moderate to severe mottling to streaking or

coalescing streaking of the leaves (Agrios, 2005). It has been

reported that new strains of the virus have increased in incidence,

which severely damage tubers (Kamangar et al., 2014). The most

efficient protection to control potato viral diseases is achieved

through the production of resistant cultivars. Several PVY

resistance genes have been found in potato and its wild relatives.

The Ry genes in Solanum tuberosum group Andigena (Ry

adg

), S.

stoloniferum (Ry

sto

), S. chacoense (Ry

chc

), and S. tuberosum group

Phureja (Ry (o)

phu

) are known to provide high levels of resistance to

PVY, the first two located on chromosomes XI and XII, and the

latter on chromosome IX (Herrera et al., 2018, Torrance et al.,

2020).Kasai et al. (2000) developed the SCAR marker RYSC3 from a

potato plant containing the Ry

adg

gene, and a 320 bp fragment was

amplied only in genotypes with this gene, becoming a powerful tool

in marker-assisted selection in breeding programs.

Potato leaf curl, caused by PLRV, is one of the most important

viral diseases and is distributed throughout the world, causing large

production losses. It only affects this crop, causing prominent leaf

curling and stunting of plant growth. In some varieties, the phloem

necroses in the leaves and tubers. It is transmitted through infected

seed tubers and in the eld by 10 aphid species in a persistent manner,

but is not mechanically transmitted (Agrios, 2005; Thomas-Sharma

et al., 2016). It can cause between 20 and 60% yield reduction, and

when it occurs in co-infection with PVY or PVX, its effects can

be greater (Mesa et al., 2016, Pichardo et al., 2013). The spread

of PLRV can be controlled with insecticide applications, however,

the most economical and ecologically acceptable way is the use of

resistant cultivars. There are two types of resistance to PLRV, one

that works against infection by the viruliferous aphid and another that

limits the multiplication and accumulation of the virus. Resistance

to PLRV has been described as polygenically controlled, whereas

resistance to PLRV accumulation appears to be under the control

of a dominant gene (Barker and Solomon, 1990). The major QTL,

PLRV.1, located on chromosome XI in the dense resistance region,

contains several genes for qualitative and quantitative resistance to

viruses and other potato pathogens. This QTL explains between 50

and 60 % of the phenotypic variance. The SCAR NI27

1164

marker was

developed, which is closely linked to this region and allows to assist

in the selection of cultivars with resistance to PLRV (Marczewski et

al., 2001).

Molecular markers developed in potato, from known regions

(microsatellites, genes, QTL) associated with resistance to pathogens

are useful tools for the genetic study of germplasm collections, even

when functional active genes cannot be distinguished from structurally

homologous inactive ones, and allow the identication of promising

genetic materials for genetic improvement, when they present the

combination of various resistances, in addition to complementing

eld evaluations (Fadina et al., 2017; López, 2013).

The objective of this work was to identify regions associated with

resistance to late blight (Phytophthora infestans) and to the PVY and

PLRV viruses, in 50 promising commercial potato genetic materials

for local use, belonging to the INIA – Mérida in vitro germplasm

bank, using developed molecular markers (microsatellites, SCAR and

CAPS).

Materials and methods

Plant Material, DNA Extraction and Amplication

The research was carried out in the Unidad de Biotecnología

Vegetal, INIA-CENIAP. In the extraction of total genomic DNA,

the methodology described by Zambrano et al. (2002) was used

on in vitro plant samples of 50 potato genetic materials, including:

12 commercial cultivars (Andinita, Capiro, Caribay, Cartayita,

Costanera, Fripapa INIA, Granola, Guadalupe, INIAFRIT, María

Bonita, Monserrate, Única); 15 late blight differentials (R1, R2, R4,

R5, R6, R7, R8, R9, R10, R11, R1R4, R2R3, R2R4, R3R4, R2R3R4),

a population of 11 advanced CIP clones with high levels of horizontal

resistance to late candelilla, high tuber yields and adaptation

to different environments (391002.6, 391011.17, 391580.30,

392633.54, 393085.5, 393280.57, 393280.64, 393280.82, 393349.68,

393371.58) and I-1062; and 12 hybrids of Fundación PROINPA,

Bolivia (Crc3/80, Crc3/84, Crc2/P8, Crc2/P9, UB6/69, 14okagran6,

14okagran7, 14okagran8, 14okagran11, 14okagran12, 14okagran17

y 14okagran20; which are part of the in vitro bank of germplasm

from INIA-Mérida, Venezuela. The DNAs were resuspended in TE

(pH 8). The integrity was determined by visualization in agarose gels

Promega

at 0.8 %, and the concentration and purity, with the use of

a nanoDrop One®. Amplication was performed in a MJResearch-

PC200 thermocycler, in a total volume of 25 μl containing 2.5

mM MgCl

, Buffer B 1X, 200 μM DNTPs, 1 μM primers, 1U Taq

Polymerase Promega

, 25 ng genomic DNA. The PCR conditions

were in accordance with the protocols developed for microsatellite

markers, SCAR and CAPS, according to Ballvora et al (2002), Kasai

et al (2000), Marczewski et al. (2001), Milbourne et al (1998) and

Trujillo (2004). To amplify the QTL tbr region of chromosome XII,

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Rev. Fac. Agron. (LUZ). 2022, 39(3): e223935. July - September. ISSN 2477-9407.4-6 |

associated with resistance to late blight, six SCAR and CAPS type

markers were used: e35m48.m, e32m61.w, e46m42.g, e44m42.j,

e45m59.o and D15.16rr with specic sequences (Trujillo, 2004).

For the CAPS marker (e46m42.g), the PCR products were digested

with the enzyme EcoRI and incubated at 37 ºC overnight before

electrophoresis. In all cases, 1 % agarose gels (Promega

) were

used. Two other microsatellite markers STM0003 and STM0030,

present on chromosome XII, associated with resistance to late

blight were included (Milbourne et al., 1998). For the amplication

of the R1 gene, primers 76-2sf2 and 76-2SR, developed by Ballvora

et al. (2002) were used.

The molecular markers associated with blight resistance used

are known to be codominant, except for the markers e44m42.j,

e45m59.o and R1 (Ballvora et al., 2002; Kasai et al., 2000;

Marczewski et al., 2001, Milbourne et al., 1998; Trujillo, 2004).

Two positive controls, donated by CIP, were used: DNA from

two individuals of the diploid population (PD), from the cross

between the female diploid parent (2n=2X=24) Solanum tuberosum

Phureja (P) clone CHS-625 (2n) with resistance to late blight

and the dihaploid male parent (n=2X=24) Solanum tuberosum

Andigena (D) clone PS-3 susceptible to the disease; and DNA from

two individuals of the tetraploid population B1B3, product of the

cross between a genotype of the population B1 Solanum tuberosum

Andigena, female, and a genotype of the population B3, male,

which in turn comes from population A. The latter is characterized

because it contains major genes introduced from S. demisum, and

on the other hand, population B presents horizontal resistance but

with the absence of R genes (Rodríguez et al., 2008).

To identify the Ry

adg

gene, which confers resistance to PVY,

DNAs were amplied with the primers SCAR 3.3.3s and ADG23R,

associated with this resistance (Kasai et al., 2000). Potato cultivars

Bintje, DTO-33 and Perrichioli, susceptible to PVY, were used as

negative controls; and the cultivars TXY.11, DXY.7, and TXY.2,

as positive controls, because they are resistant to the disease. The

controls were donated by CIP. The PCR was programmed according

to Kasai et al. (2000). To identify resistance to PLRV, a region of the

N gene was amplied using primers NL27f and NL27r. The PCR

was programmed according to Marczewski et al. (2001).

The amplication products were separated by horizontal

electrophoresis in 1 % agarose gels (Promega

) and two repetitions

of the amplications were performed to corroborate the results. The

100 bp DNA ladder (Promega

) was used to estimate the size of the

amplication products in the agarose gels. The presence or absence

of amplication products associated with resistance to late blight

and the PVY and PLRV viruses, in the 50 INIA potato genotypes,

plus the positive and negative controls mentioned, were recorded,

and analyzed using a Chemidoc® and the Quantity One® program.

Results and discussion

Molecular markers for resistance to Phytophthora infestans

The markers e35r48m, e32m61w, e45m59o and D1516rr were

monomorphic in the agarose gels for the 50 genetic materials studied

and the expected fragments of 339, 201, 123, 188 bp, respectively,

were amplied (table 1). With the e44m42j marker, polymorphism

was observed in agarose and the 155 bp fragment was taken as

positive. In the results with the CAPS e46m42g marker, a single

fragment of 172 bp product of the PCR could be observed, and

two fragments after digestion with the restriction enzyme EcoRI.

Positive controls PD (diploid population) and B1B3 (tetraploid

population) gave the respective fragments mentioned (Data not

shown). The markers e46m42g, e35m48.m and e45m59.o amplied

in 96 to 92 % of the accessions, while the rest were present between

84 to 26 %, being the marker D15.16rr the least frequent (table

1). These results conrmed those obtained by Trujillo (2004) with

respect to these markers associated with the QTL tbr.

On the other hand, the microsatellite markers STM0003 and

STM0030 were highly polymorphic for the genetic materials

studied, and the amplications of the 141 and 147 bp fragments,

respectively, were taken as positive, which were present in most of

the materials, standing out STM0030 with 90 % (table 1).

The amplied fragment of 1.4 Kb, corresponding to the region

of the R1 gene for specic resistance to late blight, was only present

in the differential R9, and in Crc 2/P8 of PROINPA (table 1) and it is

conrmed that in population B from CIP, the R1 gene is absent. This

is explained because the accessions studied originally came from

Population B developed at the CIP, without major genes (González

et al., 2017; Jiang et al., 2018).

Under eld conditions in 11 localities of the Mérida state, under

natural infection of P. infestans, INIA accessions were evaluated

during ve consecutive years (2006-2010). The commercial

varieties Granola, Capiro, Andinita and Montañita, showed the

highest values of area under the progress curve of the candelilla

disease, which indicates that they behaved as materials with low

resistance, presenting the Granola variety, along with Fripapa INIA,

Capiro and Única, the lowest yields, between 11.8 and 4.1 t.ha

-1

. On

the other hand, the advanced clones of CIP, among them, 301002.6,

391580.30, 393280.17, 393280.82, 392633.54, 393349.68,

393085.5, 393371.58 and 393080.57, stood out for their high tuber

yields, between 49.6 and 14.9 t.ha

-1

and resistance to candelilla

(González et al., 2011; González et al., 2019). Commercial cultivars

released by INIA between 1987 and 2010 have been identied as

tolerant to P. infestans, Cartayita, Fripapa INIA, INIAFRIT and

Tibisay; moderately resistant cultivars Andinita and María Bonita;

and Caribay is considered resistant. María Bonita has occupied

an important place in the south of the Mérida State and localities

of the Trujillo state. However, Granola continues to be the main

cultivar in the Andean region, with high acceptance by farmers due

to its precocity and post-harvest handling (González et al., 2017;

González et al., 2019).

Molecular markers for resistance to PVY and PLRV viruses

The 321 bp amplied DNA fragment, which identies the Ry

adg

gene region, for PVY resistance was present in cultivars TXY.11,

DXY.7 and TXY.2 (PVY resistant controls), but not in cultivars

Bintje, DTO-33 and Perrichioli (controls susceptible to PVY) (Data

not shown). This fragment was present in nine of the 50 samples:

INIAFRIT, Capiro, Monserrate, Costanera, Caribay, 393280.57,

R2, R4 and R5. On the other hand, in the Granola cultivar, which

is highly susceptible to PVY, the fragment was absent in all the

tests that were carried out. This cultivar could be used as susceptible

material in the identication of the PVY resistance gene region.

Other susceptible cultivars were FRIPAPA INIA, Guadalupe and

María Bonita, several of the differentials, and PROINPA materials

(table 1). These results are corroborated by Herrera et al. (2018), for

positive and negative controls, and commercial materials, such as

Costanera and Cartayita (identied by CIP as I-1039), which were

positive and negative, respectively. However, there is controversy

for the commercial variety Única, which did not present the

fragment associated with the Ry

adg

gene in our study.

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Vegas et al. Rev. Fac. Agron. (LUZ). 2022, 39(3): e2239355-6 |

The 1.2 Kb fragment corresponding to the region of resistance

to the PLRV virus (N gene) was present in 33 of the 50 materials

evaluated, including: INIAFRIT, Capiro, Fripapa INIA, María

Bonita, Guadalupe, 10 of the differentials of blight and materials

from PROINPA Bolivia (table 1). Only in ve genetic materials

fragments were observed for both regions of resistance to the viruses

evaluated: INIAFRIT, Capiro, 393280.57, R2 and R5. In Granola and

Andinita the studied fragments were absent (table 1). In this research,

amplication of the PLRV resistance region was observed in 68 %

of the genetic materials evaluated, while only 18 % showed the PVY

resistance region and 10 % amplied the two resistance regions for

both viruses (table 1). These results are partially corroborated by

Pichardo et al. (2013) who evaluated the presence of PVY, PLRV,

PVM and PVS viruses in vitro plants and potato tubers, by the DAS-

ELISA technique in Venezuela, nding that the Granola variety was

the most affected material and presented mixed infections of the four

viruses, followed by María Bonita which tested positive for the PVY

and PVM viruses. Among the other varieties that were infected by

only one of the viruses were: Andinita by PVM, INIAFRIT for PLRV,

Capiro and Costanera for PVY.

Conclusions

The results show that the studied germplasm belonging to INIA

Venezuela, composed of 50 potato genetic materials, including

commercial cultivars, blight differentials, advanced CIP clones

and hybrids from the Fundación PROINPA of Bolivia, have alleles

Table 1. Amplied alleles associated with resistance to late blight and the PYV and PLRV viruses in 50 genetic materials from the INIA-

Mérida potato germplasm bank.

Marker Percentage (*) Accessions that amplied with the molecular marker (**)

e35m48.m

XII(***), 339 pb (****)

92 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27.28, 29, 30, 31, 32, 33, 34, 35, 36, 37,

38, 39, 40, 42, 43, 44, 45, 46, 48, 49, 50

e32m61.w

XII; 201 pb

84 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,

38, 39, 40, 44, 48, 49, 50

e46m42.g

XII; 172 pb

96 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,

38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50

e44m42.j

XII, 155 pb

78 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 20, 21, 25, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45,

46, 47, 48, 49

e45m59.o

XII, 123 pb

92 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17,18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,

40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50

D15.16rr

XII, 188 pb

26 1, 5, 6, 9, 10, 11, 28, 29, 32, 34, 39, 40, 43

STM0003

XII, 141 pb

44 7, 14, 16, 18, 19, 20, 21, 22, 24, 25, 26, 27, 34, 37, 39, 40, 41, 42, 45, 47, 48, 49

STM0030

XII, 147 pb

90 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 42, 43, 44, 46, 47, 48, 49

V, 1.4 Kb

4 20, 41

Gen Ryadg

XI, 321 pb

18 2, 3, 5, 9, 11, 14, 15, 16, 33

Gen N

XI, 1.2 Kb

68 2, 6, 8, 9, 10, 12, 13, 14, 16, 17, 18, 20, 22, 24, 25, 26, 27, 28, 29, 31, 32, 33, 35, 36, 37, 38, 40, 41, 42, 43, 45, 46, 49, 50

(*) Percentage of accessions that amplied with the corresponding molecular marker; (**) Number corresponding to the accession: Andinita (1), Capiro (2), Caribay (3),

Cartayita (4), Costanera (5), Fripapa INIA (6), Granola (7), Guadalupe (8 ), INIAFRIT (9), Maria Bonita (10), Monserrate (11), Única (12), R1 (13), R2 (14), R4 (15), R5

(16), R6 (17), R7 ( 18), R8 (19), R9 (20), R10 (21), R11 (22), R1R4 (23), R2R3 (24), R2R4 (25), R3R4 (26), R2R3R4 (27), 391002.6 (28), 391011.17 (29), 391580.30 (30),

392633.54 (31), 393085.5 (32), 393280.57 (33), 393280.64 (34), 393280.82 (35), 393349.68 (36), 393371.58 (37), I- 1062 (38), Crc3/80 (39), Crc3/84 (40), Crc2/P8 (41),

Crc2/P9 (42), UB6/69 (43), 14okagran6 (44), 14okagran7 (45), 14okagran8 (46), 14okagran11 (47), 14okagran12 (37), 14okagran17 (49), and 14okagran20 (50); (***)

Localization of molecular markers on chromosomes; (****) Molecular weights of the amplication products.

associated with the resistance to late blight and to the PVY and PLRV

viruses, located on chromosomes V, XI and XII.

In the case of markers developed for resistance to late blight, the

accessions amplied regions located in the QTL tbr of chromosome

XII, while only two materials did so for the R1 gene. In the case of

resistance to PLRV and PVY viruses, a higher percentage of the genetic

materials amplied the region corresponding to the PLRV resistance

gene, and a lower percentage amplied regions that corresponded

to resistance to PVY or to both viruses. This study generated

valuable information to support the curators of germplasm banks

and plant potato breeders in Venezuela, however, the development

and implementation of markers that manage to locate the greatest

number of genes or QTLs associated with blight and virus resistance

is necessary, with their respective validation through greenhouse and

eld evaluations to associate the specic allelic variants with the

phenotypes.

Acknowledgement

The authors are grateful for the technical-scientic support

of doctors Rosario Herrera, Marc Ghislain and Juan Landeo, from

the International Potato Center, including donation of the control

genotypes provided for this study. The nancing of this work was

through the activity “Use of molecular markers to identify resistance

genes to late blight (Phytophthora infestans) and the potato virus”.

Project “Strengthening potato production in the highlands of

Venezuela, through the use of Biotechnological Tools”. No. 26104,

nanced by BID-FONACIT.

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Vegas et al. Rev. Fac. Agron. (LUZ). 2022, 39(3): e2239356-6 |

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