RESEARCHARTICLE IdentificationofFungusResistantWild ...ainfo.cnptia.embrapa.br/digital/bitstream/item/... · Table 1. (Continued) Accessions Code Species Brazilian Accessions Code

RESEARCH ARTICLE

Identification of Fungus Resistant WildAccessions and Interspecific Hybrids of theGenus ArachisMarcos Doniseti Michelotto1, Waldomiro Barioni, Jr.2, Marcos Deon Vilela de Resende3,Ignácio José de Godoy4, Eduardo Leonardecz5¤, Alessandra Pereira Fávero2*

1 Pólo Centro Norte, Agência Paulista de Tecnologia dos Agronegócios, Pindorama, São Paulo, Brazil,2 Embrapa Southeast Livestock, São Carlos, São Paulo, Brazil, 3 Embrapa Forestry,Colombo, Paraná,Brazil, 4 Agronomic Institute of Campinas, Campinas, São Paulo, Brazil, 5 Laboratory of ScientificComputing, Campus Planaltina, University of Brasília, Planaltina, Federal District, Brasília

¤ Current address: Laboratory of Computational Biology, Department of Genetics and Evolution, FederalUniversity of São Carlos,–São Carlos, São Paulo, Brazil* [email protected]

AbstractPeanut, Arachis hypogaea L., is a protein-rich species consumed worldwide. A key improve-

ment to peanut culture involves the development of cultivars that resist fungal diseases

such as rust, leaf spot and scab. Over three years, we evaluated fungal resistance under

field conditions of 43 wild accessions and three interspecific hybrids of the genus Arachis,as well as six A. hypogaea genotypes. In the first year, we evaluated resistance to early and

late leaf spot, rust and scab. In the second and third years, we evaluated the 18 wild species

with the best resistance scores and control cultivar IAC Caiapó for resistance to leaf spot

and rust. All wild accessions displayed greater resistance than A. hypogaea but differed in

their degree of resistance, even within the same species. We found accessions with as

good as or better resistance than A. cardenasii, including: A. stenosperma (V15076 and Sv

3712), A. kuhlmannii (V 6413), A. kempff-mercadoi (V 13250), A. hoehnei (KG 30006), and

A. helodes (V 6325). Amphidiploids and hybrids of A. hypogaea behaved similarly to wild

species. An additional four accessions deserve further evaluation: A.magna (V 13751 and

KG 30097) and A. gregoryi (V 14767 and V 14957). Although they did not display as strong

resistance as the accessions cited above, they belong to the B genome type that is crucial

to resistance gene introgression and pyramidization in A. hypogaea.

IntroductionThe oil and protein reach peanut (Arachis hypogaea L.) is consumed both in natura and pro-cessed as oil, constituting the fifth largest oleaginous crop worldwide [1]. his plant, which is na-tive from South America, belongs to a genus with 81 described species distributed in ninetaxonomic sections [2,3]. The Arachis section includes 31 species including the commercialpeanut.

PLOSONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 1 / 17

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Citation: Michelotto MD, Barioni W, Jr., de ResendeMDV, de Godoy IJ, Leonardecz E, Fávero AP (2015)Identification of Fungus Resistant Wild Accessionsand Interspecific Hybrids of the Genus Arachis. PLoSONE 10(6): e0128811. doi:10.1371/journal.pone.0128811

Academic Editor: Randall P. Niedz, United StatesDepartment of Agriculture, UNITED STATES

Received: January 5, 2015

Accepted: April 30, 2015

Published: June 19, 2015

Copyright: © 2015 Michelotto et al. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in anymedium, provided the original author and source arecredited.

Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.

Funding: APF received funds of Embrapa for thiswork, and IJG received funds of CNPq for this work.The funders provided support in the form of salariesfor authors [APF, WBJ], but did not have anyadditional role in the study design, data collection andanalysis, decision to publish, or preparation of themanuscript. The specific roles of these authors arearticulated in the ´author contributions´ section.

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The development of fungus resistance represents one of the main challenges for the im-provement of cultivated peanuts. Some of the most severe fungal foliar diseases include leafspot (Cercosporidium personatum Berk & Curtis Deighton and Cercospora arachidicolaHorii),rust (Puccinia arachidis Speg.), web blotch (Phoma arachidicolaMarasas, Pauer & Boerema),and scab (Sphaceloma arachidis Bit & Jenk). The genus Arachis has long been studied with re-gards to the introgression potential of resistance genes in peanut cultivars [4–9]. Extensivestudies have shown that the A. cardenasii accession GKP 10017 is resistant to diseases [10,8].However, these studies were conducted in greenhouses or laboratories, with detached leaves.Obstacles associated with field research such as the low availability of seeds of wild species, ana-lytical difficulties, and inoculum natural pressure are common in wild Arachis bioassays. Fieldstudies of ancient and recently-collected accessions are necessary, especially in areas close toproduction centers.

The state of São Paulo accounts for 80% of peanut production in Brazil. Leading phytosani-tary threats in the state include late leaf spot (Cercosporidium personatum), early leaf spot(Cercospora arachidicola), rust (Puccinia arachidis), and scab (Sphaceloma arachidis). Further-more, inoculum pressure in São Paulo is consistently high [9], making this a good site for theassessment of genotype resistance to prevailing pathogens.

We evaluated 43 accessions and three interspecific Arachis hybrids with regards to resis-tance to foliar diseases under field conditions in the state of São Paulo. Accessions might belater crossed generating amphidiploids (artificially doubled interspecific hybrids with distinctgenomic backgrounds that might be AABB or might have other genomic combinations) to befurther crossed with cultivars or elite lines of A. hypogaea, generating segregated populationsthat can be selected and backcrossed in a breeding program.

Materials and Methods

Plant cultureBioassays were conducted at the Pólo Apta Centro Norte experimental area in Pindorama, SãoPaulo, Brazil. Seeds were originally provided by the Arachis Germplasm Bank, Embrapa Genet-ic Resources and Biotechnology. Seeds of different genotypes (Table 1) were treated with thefungicide Plantacol

1

(10g/100kg of seeds) and germinated in paper towels in a room with ade-quate temperature, air humidity and light. Seedlings were transplanted to 200-ml plastic cupsfilled with soil and sand (3:1) and placed in a greenhouse. When plants reached a height of 10to 15 cm they were transplanted to the field in soil previously prepared with 250 kg/ha of 8-28-16 NPK.

During the first year, we evaluated 43 accessions belonging to 10 wild species, six A. hypo-gaea genotypes and three interspecific hybrids, including amphidiploids and segregating popu-lations (Table 1). Twenty-five F2 individuals of the progenie by the cross between IAC Caiapóand the amphidiploid An 2 were evaluated. The average of the experimental unit were used foranalyses of variance. In the second and third years, we selected the 18 most resistant accessionsand the IAC Caiapó cultivar as control.

The experiment design was random uncompleted delineated block with four replications.Each block was initially composed of four meters with five plants spaced one meter apart andwith a separation of 1.5 meters between lines. This spacing was needed because of the amplegrowth of these plant species. Just three plants in the middle of the experimental unit were eval-uated. Every block was sprayed twice-monthly with insecticides to avoid infestation. Weedcontrol was performed with the pre-transplantation application of commercially available Tri-fluralin (2.5 l/ha). During plant growth, weed control was performed manually. The

Fungi Resistance of Wild Arachis Species and Hybrids

PLOS ONE | DOI:10.1371/journal.pone.0128811 June 19, 2015 2 / 17

Competing Interests: The authors have declaredthat no competing interests exist.

Table 1. Arachis spp. accessions included in the present study.

Accessions Code Species BrazilianAccessionsCode

CollectionsitesCity

State in Brazilor Country

Lat(W)

Long(S)

Alt(m)

Genome

K 9484 A. batizocoi Krapov. & W. C. Gregory 013315 Parapeti BOL 20°05’

63°14’

700 K

KG 35005 A. benensis Krapov. & W.C. Gregory 037206 Trinidad BOL F

GKP 10017 A. cardenasii Krapov. & W. C.Gregory

013404 Roboré BOL 18°20’

59°46’

200 A

K 7988 A. duranensis Krapov. & W. C.Gregory

013307 Campo Duran ARG 22°19’

63°13’

500 A

VSGr 6389 A. gregoryi C. E. Simpson, Krapov.&Valls

012696 Vila Bela da Ssa.Trindade

MT 15°19’

60°06’

210 B

VOfSv 14760 A. gregoryi C. E. Simpson, Krapov.&Valls


MT 16°08’

59°47’

B

VOfSv 14767 A. gregoryi C. E. Simpson, Krapov.&Valls


MT 16°05’

59°58’

290 B

VS 14957 A. gregoryi C. E. Simpson, Krapov.&Valls


MT 15°22’

60°14’

B

CoSzSv 6862 A. helodes Martius ex Krapov &Rigoni

018619 MT 15°22’

56°13’

175 A

VSGr 6325 A. helodes Martius ex Krapov &Rigoni

012505 S. Antonio doLeverger

MT 15°52’

56°04’

150 A

KG 30006 A. hoehnei Krapov. & W. C. Gregory 036226 Corumbá MS 18°15’

57°28’

A

VRcMmSv 14546 A. hoehnei Krapov. & W. C. Gregory 022641 Corumbá MS 19 °15’

57 °22’

100 A

cv. BR1 A. hypogaea subsp. fastigiata var.fastigiata

033383 AB

cv. IAC Caiapó A. hypogaea 037371 AB

2562 A. hypogaea 037354 AB

IAC Runner 886 A hypogaea subsp. hypogaea var.hypogaea

037389 AB

cv. IAC Tatu-ST A. hypogaea subsp. fastigiata var.fastigiata

011606 Campinas SP AB

V 12549 A. hypogaea subsp. hypopaea var.hypogaea

030716 AB

KGPScS 30076 A. ipaënsis Krapov. & W. C. Gregory 036234 Ipa BOL 21°00’

63°25’

650 B

V 13250 A. kempff-mercadoi Krapov., W. C.Gregory & C. E. Simpson

030643 Sta. Cruz de laSierra

BOL 17°45’

63°10’

280 A

VKSSv 8979 A. kuhlmannii Krapov. & W. C.Gregory

020354 Cáceres MT 15°35’

57°13’

210 A

VPoBi 9243 A. kuhlmannii Krapov. & W. C.Gregory

022560 Corumbá MS 18°52’

56°16’

100 A

VPoJSv 10506 A. kuhlmannii Krapov. & W. C.Gregory

024953 N. Sra. doLivramento

MT 15°48’

56°21’

A

VRGeSv 7639 A. kuhlmannii Krapov. & W. C.Gregory

017515 Miranda MS 20°15’

56°23’

125 A

VSGr 6351 A. kuhlmannii Krapov. & W. C.Gregory

012602 Cáceres MT 15°56’

57°48’

130 A

VSGr 6413 A. kuhlmannii Krapov. & W. C.Gregory

012688 Cáceres MT 15°47’

57°25’

200 A

VSW 9912 A. kuhlmannii Krapov. & W. C.Gregory

022900 Aquidauana MS 20°26’

55°54’

210 A

(Continued)



Table 1. (Continued)

Accessions Code Species BrazilianAccessionsCode

CollectionsitesCity

State in Brazilor Country

Lat(W)

Long(S)

Alt(m)

Genome

KGSSc 30097 A. magna Krapov., W. C. Gregory &C. E. Simpson

036871 San Ignacio deVelasco

BOL 16°22’

60°58’

370 B

VPzSgRcSv 13761 A. magna Krapov., W. C. Gregory &C. E. Simpson


MT 15°21’

60°04’

380 B

VSPmSv 13751 A. magna Krapov., W. C. Gregory &C. E. Simpson

033812 Vila Bela da SsaTrindade

MT 16°16’

59°27’

530 B

VOa 14165 A. monticola Krapov. & Rigoni 036188 Yala, Jujuy ARG 24°07’

65°23’

AB

VSPmSv 13710 A. simpsonii Krapov. & W. C. Gregory 033685 Porto Esperidião MT 15°58’

58°31’

270 A

HLK 408 A. stenosperma Krapov. & W. C.Gregory

013366 Antonina PR 25°24’

48°44’

3 A

Lm 5 A. stenosperma Krapov. & W. C.Gregory

036013 Antonina PR A

SvW 3712 A. stenosperma Krapov. & W. C.Gregory

035254 Cocalinho MT 14°22’

51°00’

220 A

VSStGdW7805-AR

A. stenosperma Krapov. & W. C.Gregory

032476 São Felix doAraguaia

MT 11°38’

50°48’

240 A

VKSSv 9010 A. stenosperma Krapov. & W. C.Gregory

020176 Santo Antonio doLeverger

MT 15°52’

56°04’

150 A

VMiSv 10229 A. stenosperma Krapov. & W. C.Gregory

023001 Cananéia SP 25°01’

47°55’

10 A

VS 13670 A. stenosperma Krapov. & W. C.Gregory

018104 Araguaiana MT 15°33’

52°12’

350 A

VSMGeSv 7379 A. stenosperma Krapov. & W. C.Gregory

016063 Antonina PR 25°26’

48°42’

3 A

VSPmSv 13832 A. stenosperma Krapov. & W. C.Gregory

033961 S. M. doAraguaia/LuizAlves

MT 13°13’

50°34’

280 A

VSPmW 13824 A. stenosperma Krapov. & W. C.Gregory

033936 S. M. doAraguaia/LuizAlves

MT 13°13’

50°34’

280 A

VSSv 13258 A. stenosperma Krapov. & W. C.Gregory

016128 São Sebastião SP 23°45’

45°24’

5 A

VSv 10309 A. stenosperma Krapov. & W. C.Gregory

024830 Rondonópolis MT 16°28’

54°39’

215 A

VArLf 15076 A. stenosperma Krapov. & W. C.Gregory

040266 Matinhos PR A

WPz 421 A. stenosperma Krapov. & W. C.Gregory

033511 Alvorada TO 12°36’

49°20’

310 A

WiDc 1118 A. williamsii Krapov. & W. C. Gregory 036897 Trinidad BOL B

An 2 = (V 6389 x V9401)4x

(A. gregoryi x A. linearifolia)4x AB

An 4 = (KG 30076x V 14167)4x

(A.ipaënsis x A. duranensis)4x AB

IAC Caiapó x An2 A. hypogaea x (A. gregoryi x A.linearifolia)4x

AB

* Collectors: = Ar = A.R. Custodio, Bi = L. B. Bianchetti, Co = L. Coradin, Dc = D. Claure, G = W. C. Gregory, Gd = I. J. Godoy, Ge = M. A. N. Gerin,

Gr = A. Gripp, H = R. Hammons, J = L. Jank, K = A. Krapovickas, L = W.R. Langford, Lf = L. G. Faria,Lm = L. Monçato, M = J. P. Moss, Mi = S.T.S.Miotto,

Mm = M. Moraes, Oa = O.Ahumada, Of = F. O. Freitas, P = J. R. Pietralli, Pm = R. N. Pittmann, Po = A. Pott, Pz = E. Pizarro, R = V. R. Rao, Rc = R.C.

Oliveira, S = C. E. Simpson, Sc = A. Schinini, Sg = A. K. Singh, St = H. T. Stalker, Sv = G. P. Silva, Sz = R. Schultze-Kraft, V = J. F. M. Valls, W = W. L.

Werneck, Wi = D. E. Williams.

doi:10.1371/journal.pone.0128811.t001



experimental design was the same for all three years of evaluations apart from the number ofaccessions analyzed.

Resistance testingIn the first year, fungal diseases evaluated included early leaf spot (Cercospora arachidicola),late leaf spot (Cercosporidium personatum), rust (Puccinia arachidis.) and scab (Sphacelomaarachidis). All diseases except for scab were also evaluated in the second and third years to con-firm resistance of wild species accessions (18 genotypes and IAC Caiapó control). Scab was notevaluated in the second and third years due to its low incidence. A 1–9 visual grade scale fordamage caused at the end of the plant cycle was used in all evaluations.

Resistance data for early leaf spot, late leaf spot and rust were analyzed following the SASGLM procedure [11] taking into account the model cultivar effect (1 to 19) and time (years 1, 2and 3). Data from early and late leaf spot were transformed 1/x and log10(x), respectively, assuggested for the normalization of residues and cultivar variance homogeneity. In the compari-son of averages from cultivars, we adopted Duncan’s test at a significance of 5%. Software Sele-gen-Reml/Blup [12] were used for Restricted Maximum Likelihood/ Best, Linear, UnbiasedPrediction (REML/BLUP) analysis (Model 20 for first year data and Model 29 for three-yeardata).

Data were also subjected to grouping analysis (GA) complemented with principal compo-nent analysis (PCA) to group genotypes according to the variables: late leaf spot, early leafspot, scab, and rust. Genotype GA was performed according to Ward’s method [13], and Eu-clidian distance was considered a measure of dissimilarity. Dendrogram and connection graphswere used to interpret GA results. In PCA the two first principal components (PC1 and PC2)were considered the most important in their respective contributions to total variability. PC1and PC2 allowed for simultaneous visualization of variable and genotype projections as well asdeduction of the linear correlation among the variables: late leaf spot, early leaf spot, scab andrust. The software used for PCA and GA was STATISTICA [14], other analyses were con-ducted with SAS [11] and MS Office Excel.

Analysis of Variance conducted on data for the three different years showed significant dif-ferences between years for all three diseases and the interaction accessions x years for disease(late leaf spot and rust). Therefore, average measurements were used for GA.

Results and DiscussionIn the first year of study, we evaluated resistance to late leaf spot, early leaf spot, rust and scab.Within 50 accessions evaluated at the first year (Table 2) there was a large difference in resis-tance to late leaf spot, with averages ranging from 1.75 to 9. On the other side, for early leafspot, scab and rust, the variation among wild accessions was less significant. It is possible toverify either the difficulty to select accessions based on ANOVA and Duncan test. These resultsjustify the utilization of PCA and grouping analysis.

REML/BLUP analysis were shown in Table 3 for 50 genotypes in first year field assay. Theselection accuracy of genotypes had a high value, as well as PEV value was low for all variables.All CVgi% were higher than CVe% values except for scab variable indicating that the environ-ment had a important effect in the phenotypic pattern of this disease.

Resistance rank of each accession was obtained in individual BLUP analysis, as well as ageneral rank was observed by the sum of all ranks of the three diseases. The highest values arethose with best resistance to the three diseases. Ranks of genotypes in BLUP analysis were verysimilar to Duncan test results (Table 2).



Table 2. Duncan test results for Arachis spp. accessions for resistance to late leaf spot (LLS), early leaf spot (ELS), scab (S) and rust (R) in fieldassay (first year).

Accessions Code Species LLS ELS S R

2562 A. hypogaea 9.00 a1 - - 5.50 b

IAC Tatu-ST A. hypogaea 8.75 a 2.00 efg 3.00 a 5.00 bc

IAC Runner 886 A hypogaea 8.69 a 1.67 efgh 2.33 abc 8.00 a

BR1 A. hypogaea 8.00 ab 5.00 a 2.50 ab 4.67 cd

IAC Caiapó A. hypogaea 7.50 bc 5.19 a 2.44 ab 4.17 d

K 35005 A. benensis 6.75 c 1.00 h 1.00 e 1.00 f

K 9484 A. batizocoi 5.33 d 1.67 efgh 1.67 bcde 1.00 f

K 7988 A. duranensis 5.33 d 3.67 b 1.00 e 1.00 f

V 14165 A. monticola 5.33 d 1.33 fgh 2.33 abc 2.00 e

V 12549 A. hypogaea 5.00 de 3.25 bc 2.50 ab 5.00 bc

V 13761 A. magna 5.00 de 1.00 h 1.00 e 1.33 f

K 30097 A. magna 4.67 def 1.00 h 1.33 de 1.33 f

V 7805-AR A. stenosperma 4.67 def 1.33 fgh 1.00 e 1.00 f

An 4 (A.ipaënsis x A. duranensis)4x 4.50 defg 1.75 efgh 1.50 cde 1.00 f

V 10506 A. kuhlmannii 4.00 efgh 2.25 def 1.00 e 1.33 f

V 8979 A. kuhlmannii 4.00 efgh 3.00 bcd 1.00 e 1.50 ef

V 14767 A. gregoryi 4.00 efgh 1.50 fgh 1.25 de 1.00 f

V 7639 A. kuhlmannii 4.00 efgh 1.33 fgh 1.00 e 1.33 f

IAC Caiapó x An2 A. hypogaea x (A. gregoryi x A. linearifolia)4x 3.78 efghi 1.63 efgh 1.48 de 1.55 ef

K 30076 A. ipaënsis 3.75 efghi 1.25 gh 2.50 ab 2.00 e

V 9243 A. kuhlmannii 3.75 efghi 1.25 gh 1.00 e 1.00 f

V 6351 A. kuhlmannii 3.75 efghi 2.25 def 1.25 de 1.00 f

An 2 (A. gregoryi x A. linearifolia)4x 3.75 efghi 1.00 h 2.00 bcd 1.00 f

W 421 A. stenosperma 3.50 fghij 2.00 efg 1.00 e 1.00 f

Wi 1118 A. williamsii 3.50 fghij 1.50 fgh 1.00 e 2.00 e

V 14546 A. hoehnei 3.50 fghij 2.50 cde 1.25 ed 1.00 f

V 13832 A. stenosperma 3.33 ghijk 1.33 fgh 1.33 de 1.00 f

Co 6862 A. helodes 3.25 ghijk 1.75 efgh 1.00 e 1.00 f

V 9912 A. kuhlmannii 3.25 ghijk 1.00 h 1.00 e 1.00 f

V 13751 A. magna 3.25 ghijk 1.25 gh 1.00 e 1.00 f

V 14957 A. gregoryi 3.00 hijkl 1.00 h 1.25 de 1.00 f

V 6389 A. gregoryi 3.00 hijkl 1.00 h 1.75 bcde 1.00 f

H 408 A. stenosperma 3.00 hijkl 1.67 efgh 1.33 de 1.00 f

V 13824 A. stenosperma 3.00 hijkl 1.67 efgh 1.33 de 1.00 f

V 10309 A. stenosperma 3.00 hijkl 1.67 efgh 1.67 bcde 1.50 ef

V 14760 A. gregoryi 2.75 hijkl 1.00 h 1.50 cde 1.00 f

G 10017 A. cardenasii 2.67 hijkl 1.67 efgh 1.00 e 1.33 f

V 13710 A. simpsonii 2.67 hijkl 2.00 efg 1.00 e 1.00 f

V 7379 A. stenosperma 2.67 hijkl 1.67 efgh 1.67 bcde 1.33 f

V 13670 A. stenosperma 2.50 ijkl 2.00 efg 1.50 cde 1.00 f

V 15076 A. stenosperma 2.33 jkl 1.67 efgh 1.00 e 1.00 f

V 6325 A. helodes 2.33 ijkl 1.33 fgh 1.00 e 1.33 f

Lm 5 A. stenosperma 2.33 jkl 1.67 efgh 1.33 de 1.00 f

V 9010 A. stenosperma 2.33 jkl 1.67 efgh 1.33 de 1.00 f

V 10229 A. stenosperma 2.25 jkl 1.75 efgh 1.50 cde 1.00 f

V 13258 A. stenosperma 2.25 jkl 1.50 fgh 1.50 cde 1.00 f

(Continued)



Genetic correlation between the variables LLS, ELS, S and R for the first year assay and forthree years data, based on the REML/BLUP analysis, are shown in Table 4. In the first year,LLS and R was genetically correlated. In the analysis of three years for the 18 wild accessions se-lected as resistant and the control, all variables were correlated.

A first GA was conducted with the 50 genotypes (Fig 1). At cut-off point 10 of this dendro-gram, genotypes are divided into two groups: Group 1 encompasses all six accessions of A.hypogaea, A.monticola (V 14165) and A. ipaënsis (KG 30076). Interestingly, A.monticola is atetraploid species closely related to, and most likely a direct ancestor of, A. hypogaea [15]. Evi-dence also suggests that A. ipaënsis was the B genome species that originated A. hypogaea [16–18]. Group 2 encompasses all other wild genotypes included in the study, indicating that themajority of wild species are very distinct from cultivated peanut with regards to resistance toevaluated fungal diseases. This finding also suggests that many unexplored genes may be pres-ent in these pools that could be introduced into the genome of A. hypogaea. Another importantGA outcome is the grouping of three hybrids (two amphidiploids—An 2 and An 4—and the F2progeny individuals of Caiapó x An 4) in Group 2, as they all kept resistance patterns similar tothose of wild species. This finding shows that resistance is maintained after interspecificcrossings.

If the more susceptible accessions are removed from the analysis, a more detailed picture ofwild accession differentiation emerges (Fig 2): a cut-off point of 11 separated accessions ac-cording to their resistance to scab, whereas a cut-off point of 7 then discriminated between fivegenotype groups. The genotypes with least resistance to scab were subdivided as to their resis-tance to rust. The ones more resistant to scab were further subdivided as to their resistance toearly leaf spot. Among the early leaf spot resistant genotypes, another division was possiblewith regard to resistance to late leaf spot. Therefore, the joint evaluation of four diseases indi-cates that Group 2 of Fig 2 provides the most resistant accessions and might be the best one formultiple selections. The seven accessions that comprise this group are V 15076 (A. stenos-perma), V 6413 (A. kuhlmannii), V 13250 (A. kempff-mercadoi), Sv 3712 (A. stenosperma), KG30006 (A. hoehnei), V 6325 (A. helodes), and GKP 10017 (A. cardenasii). All of these accessionsare of A genome type, and apparently A genome species are more resistant to fungal diseasesthan species with other genomes in the Arachis section. Validating this observation is difficultdue to the smaller number of B genome sensu lato accessions evaluated in this report, whichmakes it difficult to investigate true variability when compared to the number of A genomeaccessions.

Another important aspect of resistance is the variability observed among accessions of a sin-gle species. Pande and Rao [8] have previously emphasized the importance of evaluating reac-tions at the individual level. We show that A. stenosperma accessions are present in everygroup, whereas A. kuhlmannii are present in three, A. hoehnei in two, and A. gregoryi in twogroups. A wider distribution of A. stenospermamay be a result from a larger number of

Table 2. (Continued)

Accessions Code Species LLS ELS S R

V 13250 A. kempff-mercadoi 2.00 kl 1.50 fgh 1.00 e 1.00 f

V 6413 A. kuhlmannii 2.00 kl 1.67 efgh 1.00 e 1.00 f

Sv 3712 A. stenosperma 2.00 kl 1.00 h 1.00 e 1.00 f

K 30006 A. hoehnei 1.75 l 1.25 gh 1.25 ed 1.00 f

1.Distinct letters indicate significant differences among accessions according to Duncan’s test (p<0,05)




Tab

le3.

Estim

atives

ofc

omponen

tsofv

ariance

(Individual

REML)a

ndtheco

mponen

tsofa

verage(Individual

BLUP)fortheva

riab

lesresistan

ceto

late

leaf

spot(LLS),ea

rlyleaf

spot(ELS),sc

ab(S)a

ndrust

(R)a

nd50

gen

otypes

,infirsty

earoffield

assa

y.

Componen

tsofav

erag

e(Individual

BLUP)

Gen

otype

LLS

ELS

SR

Ran

kg

u+g

GG

Na

Ran

kg

u+g

GG

Na

Ran

kg

u+g

GG

Na

Ran

kg

u+g

GG

Na

GR

2562

14.89

168.80

544.89

168.80

54-

--

--

--

--

-2

3.76

215.41

594.97

416.62

793

IAC

Tatu-ST

24.65

128.56

54.77

148.68

526

1.00

182.78

281.72

153.50

241

1.03

192.47

081.03

192.47

084

3.14

404.79

794.09

675.75

0513

IAC

Run

ner86

63

4.59

168.50

544.71

158.62

535

1.00

182.78

281.86

543.64

647

0.64

52.08

390.78

812.22

71

6.18

617.84

006.18

617.84

0016

BR

14

3.86

187.77

564.49

918.41

292

2.54

424.32

522.75

514.53

605

0.72

472.16

360.83

612.27

498

0.31

371.96

762.81

044.46

4319

IAC

Caiap

00F3

53.44

907.36

284.28

908.20

291

2.96

594.74

692.96

594.74

694

0.77

582.21

470.86

392.30

286

2.47

494.12

873.63

815.29

2016

V12

549

101.04

464.95

852.91

796.83

184

1.27

883.05

982.08

133.86

232

0.82

392.26

280.92

792.36

683

3.29

454.94

844.41

426.06

8119

K94

847

1.32

065.23

443.64

197.55

5719

-0.150

21.63

080.65

752.43

8410

0.20

261.64

150.63

972.07

8620

-0.643

81.01

010.96

962.62

3456

V14

165

91.32

065.23

443.12

617.03

9935

-0.432

71.34

830.24

282.02

386

0.69

162.13

050.81

202.25

097

0.34

081.99

473.16

714.82

0957

V89

7918

-0.038

23.87

561.79

455.70

837

0.87

472.65

571.60

053.38

1531

-0.242

81.19

610.18

091.61

9811

-0.193

01.46

082.04

033.69

4167

IAC

Caiap

óxAn2

19-0.132

53.78

131.69

35.60

6818

-0.134

91.64

610.70

242.48

3318

0.02

881.46

770.39

391.83

2810

-0.144

11.50

982.26

363.91

7565

K30

076

20-0.157

53.75

631.60

055.51

4338

-0.462

21.31

870.18

801.96

893

0.82

392.26

280.89

332.33

215

2.96

724.62

103.87

085.52

4666

K79

888

1.32

065.23

443.35

187.26

563

1.53

633.31

722.34

884.12

9734

-0.288

31.15

060.13

951.57

8422

-0.643

81.01

010.82

292.47

6867

V10

309

35-0.976

2.93

780.66

284.57

6617

-0.121

81.65

920.75

162.53

2612

0.17

761.61

640.56

472.00

3612

-0.193

01,46

081,85

423,50

876

An4

140.56

384.47

762.29

446.20

8215

-0.026

91.75

400.86

172.64

2715

0.04

751.48

630.46

451.90

3334

-0.643

81.01

010.30

521.95

9178

V14

546

24-0.398

03.51

581.29

755.21

138

0.62

602.40

691.47

873.25

9627

-0.146

71.29

220.23

661.67

5526

-0.643

81.01

010.59

732.25

1185

V10

506

160.08

293.99

672.01

795.93

189

0.40

832.18

931.35

983.14

0747

-0.340

81.09

810.01

451.45

3416

-0.316

41.33

751.31

152.96

5488

Wi1

118

26-0.507

13.40

671.16

295.07

6731

-0.327

31.45

360.33

002.11

0930

-0.242

81.19

610.19

511.63

399

0.29

671.95

062.53

114.18

596

V73

7939

-1.218

22.69

560.47

244.38

6227

-0.150

21.63

080.41

822.19

9111

0.20

261.64

150.59

992.03

8819

-0.316

41,33

751,05

452,70

8396

V76

3917

0.05

123.96

501.90

235.81

6132

-0.432

71.34

830.30

622.08

7133

-0.288

31.15

060.15

251.59

1414

-0.316

41.33

751.54

413.19

7996

V63

5122

-0.157

53.75

631.44

075.35

4510

0.40

832.18

931.26

463.04

5629

-0.146

71.29

220.21

021.64

9036

-0.643

81.01

010.25

251.90

6497

K30

097

130.68

714.60

092.42

756.34

1348

-0.713

1.06

790.01

491.79

5820

-0.043

81.39

510.35

021.78

918

-0.316

41.33

751.13

062.78

4599

V14

767

150.08

293.99

672.14

696.06

0828

-0.244

61.53

640.39

452.17

5528

-0.146

71.29

220.22

291.66

1829

-0.643

81.01

010.46

892.12

2710

0

GKP10

017

37-1.218

22.69

560.56

384.47

7620

-0.150

21.63

080.61

712.39

8032

-0.288

31.15

060.16

631.60

5213

-0.316

41,33

751,68

723,34

1110

2

V78

05-AR

120.68

714.60

092.57

256.48

6333

-0.432

71.34

830.28

382.06

4735

-0.288

31.15

060.12

731.56

6227

-0.643

81.01

010.55

132.20

5110

7

V13

832

27-0.584

73.32

911.09

815.01

1936

-0.432

71.34

830.22

412.00

5024

-0.043

81.39

510.28

451.72

3448

-0.643

81.01

010.02

851.68

2313

5

K35

005

62.72

776.64

154.02

887.94

2641

-0.679

81.10

110.13

511.91

6042

-0.340

81.09

810.05

681.49

5721

-0.643

81.01

010.89

272.54

6611

0

Co68

6228

-0.638

43.27

541.03

614.94

9914

-0.026

91.75

400.92

522.70

6243

-0.340

81.09

810.04

761.48

6428

-0.643

81,01

010,50

862,16

2511

3

V13

761

111.00

394.91

772.74

396.65

7747

-0.713

1.06

790.03

031.81

1339

-0.288

31.15

060.08

471.52

3617

-0.316

41.33

751.21

582.86

9611

4

V13

670

40-1.445

2.46

890.42

454.33

8313

0.07

341.85

430.99

852.77

9413

0.09

511.53

400.52

861.96

7549

-0.682

80,97

100,01

391,66

7811

5

V63

8932

-0.878

93.03

50.81

184.72

5644

-0.679

81.10

110.07

951.86

059

0.24

161.68

050.68

822.12

7130

-0.643

81,01

010,43

182,08

5611

5

V14

760

36-1.119

32.79

450.61

334.52

7143

-0.679

81.10

110.09

721.87

8114

0.04

751.48

630.49

421.93

3124

-0.643

81,01

010,70

072,35

4511

7

HLK

408

33-0.901

43.01

240.75

994.67

3724

-0.150

21.63

080.48

922.27

0221

-0.043

81.39

510.33

141.77

0341

-0.643

81,01

010,14

321,79

7111

9

V10

229

45-1.600

22.31

360.20

514.11

8916

-0.026

91.75

40.80

622.58

7116

0.04

751.48

630.43

841.87

7342

-0.643

81,01

010,12

451,77

8311

9

V14

957

31-0.878

93.03

50.86

634.78

0142

-0.679

81.10

110.11

571.89

6625

-0.146

71.29

220.26

731.70

6123

-0.643

81,01

010,75

912,41

3012

1

Lm5

43-1.537

32.37

650.28

764.20

1423

-0.150

21.63

080.51

702.29

8019

-0.043

81.39

510.37

091.80

9839

-0.643

81,01

010,18

361,83

7412

4

W42

125

-0.398

03.51

581.22

975.14

3511

0.13

021.91

111.16

152.94

2449

-0.340

81.09

810.00

001.43

8940

-0.643

81.01

010.16

291.81

6812

5

V63

2541

-1.537

32.37

650.37

664.29

0434

-0.432

71.34

830.26

272.04

3636

-0.288

31.15

060.11

581.55

4715

-0.316

41,33

751,42

003,07

3912

6

An2

23-0.157

53.75

631.37

125.28

5046

-0.679

81.10

110.04

651.82

748

0.43

571.87

460.74

412.18

3050

-0.682

80.97

100.00

001.65

3912

7

V13

824

34-0.901

43.01

240.71

14.62

4826

-0.150

21.63

080.44

002.22

1023

-0.043

81.39

510.29

881.73

7746

-0.643

81,01

010,05

771,71

1512

9

V90

1044

-1.537

32.37

650.24

614.15

9925

-0.150

21.63

080.46

372.24

4622

-0.043

81.39

510.31

431.75

3243

-0.643

81,01

010,10

661,76

0513

4

V13

710

38-1.218

22.69

560.51

694.43

0712

0.13

021.91

111.07

562.85

6540

-0.288

31.15

060.07

541.51

4244

-0.643

81,01

010,08

961,74

3413

4

V92

4321

-0.157

53.75

631.51

685.43

0639

-0.462

21.31

870.17

131.95

2245

-0.340

81.09

810.03

031.46

9232

-0.643

81.01

010.36

462.01

8413

7

V13

258

46-1.600

22.31

360.16

594.07

9730

-0.244

61.53

640.35

192.13

2817

0.04

751.48

630.41

541.85

4345

-0.643

81,01

010,07

331,72

7113

8

K30

006

50-2.081

1.83

280.00

003.91

3837

-0.462

21.31

870.20

551.98

6526

-0.146

71.29

220.25

131.69

0225

-0.643

81,01

010,64

692,30

0813

8

V15

076

42-1.537

32.37

650.33

114.24

4922

-0.150

21.63

080.54

742.32

8338

-0.288

31.15

060.09

451.53

3438

-0.643

81,01

010,20

541,85

9214

0

V64

1348

-1.854

2.05

980.08

23.99

5821

-0.150

21.63

080.58

062.36

1537

-0.288

31.15

060.10

481.54

3735

-0.643

81,01

010,27

811,93

2014

1

V99

1229

-0.638

43.27

540.97

844.89

2245

-0.679

81.10

110.06

261.84

3646

-0.340

81.09

810.02

221.46

1133

-0.643

81,01

010,33

401,98

7915

3

V13

751

30-0.638

43.27

540.92

454.83

8340

-0.462

21.31

870.15

551.93

6448

-0.340

81.09

810.00

711.44

637

-0.643

81,01

010,22

831,88

2215

5

V13

250

47-1.840

62.07

320.12

324.03

729

-0.244

61.53

640.37

252.15

3444

-0.340

81.09

810.03

871.47

7631

-0.643

81,01

010,39

712,05

0915

1

(Con

tinue

d)



Tab

le3.

(Con

tinue

d)

Componen

tsofav

erag

e(Individual

BLUP)

Gen

otype

LLS

ELS

SR

Ran

kg

u+g

GG

Na

Ran

kg

u+g

GG

Na

Ran

kg

u+g

GG

Na

Ran

kg

u+g

GG

Na

GR

Sv37

1249

-1.854

2.05

980.04

253.95

6349

-0.713

01.06

790.00

001.78

0941

-0.288

31.15

060.06

651.50

5447

-0.643

81,01

010,04

281,69

6618

6

Com

pone

ntsof

varia

nce

(Ind

ividua

lREML)

Vg=3.26

25Vg=0.71

96Vg=0.19

00Vg=2.12

06

Ve=0.64

88Ve=0.53

51Ve=0.27

35Ve=0.13

28

Vf=

3.91

13Vf=

1.25

47Vf=

0.46

35Vf=

2.25

35

h2g=0.83

41+-

0.17

34h2

g=0.57

35+-

0.14

64h2

g=0.41

00+-

0.12

38h2

g=0.94

11+-

0.20

06

h2mc=0.96

18h2

mc=0.87

05h2

mc=0.77

65h2

mc=0.98

46

Acclon=0.98

07Acclon=0.93

30Acclon=0.88

12Acclon=0.99

23

CVgi%

=46

.150

7CVgi%

=47

.630

6CVgi%

=30

.293

9CVgi%

=88

.051

1

CVe%

=20

.579

8CVe%

=41

.074

9CVe%

=36

.343

5CVe%

=22

.037

4

CVr=2.24

25CVr=1.15

96CVr=0.83

355

CVr=3.99

55

PEV=0.12

48PEV=0.09

317

PEV=0.04

247

PEV=0.03

27

SEP=0.35

33SEP=0.30

52SEP=0.20

608

SEP=0.18

08

GA=3.91

38GA=1.78

09GA=1.43

89GA=1.65

39

*g:

geno

typicefec

t,u+g:

geno

typicav

erag

e,GG:g

eneticga

in,N

a=ne

wav

erag

e,GR:g

eneral

rank

,Vg:

geno

typicva

rianc

e,Ve:

residu

alva

rianc

e,Vf:ph

enotyp

icva

rianc

e,h2

g=plot

heritab

ility

inbroa

dse

nse,

h2mc=av

erag

ege

notype

heritab

ility,A

cclon=se

llectionge

notype

accu

racy,C

Vgi%

=individu

alco

efficien

tofa

dditive

varia

nce,

CVe%

=co

efficien

tof

expe

rimen

talvariatio

n,CVr=co

efficien

tofrelativeva

riatio

n,PEV=pred

ictio

nerrorva

rianc

eof

geno

typicva

lues

,SEP=stan

dard

deviationof

geno

typicva

lue,

GA=ge

nerala

verage




accessions in this species. If there were more accessions in the other species, we might have ob-served a similarly ample distribution. As it was observed the wide variability at the specieslevel, research efforts are necessary in the identification of resistances in accessions, notin species.

The two amphidiploids were grouped in Group 5. Amphidiploid An 2 remained very closeto one of its progenitors, V 6389 (A. gregoryi). The other progenitor, V 9401 (A. linearifolia),was not included in the study due to an insufficient number of seeds. Amphidiploid An 4 re-sulted from a cross between A. ipaënsis x A. duranensis V 14167 followed by artificial polyploi-dization. The female progenitor fell into Group 1, along with accessions of A. hypogaea. The A.duranensis accession was not included in the study for lack of seeds. Interestingly, some ramifi-cations within Group 5 included amphidiploids and other B and K genome species, but no Agenome accessions. In another subdivision of Group 5, only A genome accessions were segre-gated. Non-A genomes were also concentrated in Groups 3 and 4. The F2 progeny of IACCaiapó x An 4, such as A. stenosperma V 10309, were situated in Group 4, exhibiting partial re-sistance when compared to wild genotypes.

Four accessions that showed special potential for future studies are the A.magna accessionsV 13751 and KG 30097 and the A. gregoryi accessions V 14767 and V 14957. While they werenot the best in terms of resistance, they belong to the B genome type that is crucial for resis-tance-gene introgression and pyramidization in A. hypogaea.

Table 4. Genetic correlation between variables (resistances to late and early leaf spots–LLS, ELS–scab–S, and rust—R) in first year for 50 accessions and in three years field assay for 18 selected ac-cessions and one control.

Genetic correlation

1st year 3 years

Variable LLS ELS S LLS ELS

LLS

ELS 0.3986 0.9519

S 0.2779 0.5447

R 0.7058 0.3095 0.4062 0.9615 0.9801


Fig 1. Distribution of wild Arachis genotypes and A. hypogaea controls with respect to resistance toearly leaf spot, late leaf spot, rust, and scab in the first year of study.Cut-off point = 10 (arrow) indicatesgenotype segregation into two groups.

doi:10.1371/journal.pone.0128811.g001



Similarly to the GA with a cut-off point of 10, a two-group division was observed throughPCA (Fig 3), where the two first components explained 81.41% of variation. Again, Group 1(red circle) was formed by the same eight genotypes as in Fig 1, whereas the other wild acces-sions were tightly connected in Group 2 (green circle). Arrows point towards accessions, in-cluding those of Group 1, which were more susceptible to the diseases evaluated. Some Group1 genotypes such as IAC-Caiapó and BR-1 were more susceptible to early leaf spot, whereascultivars IAC-Tatu-ST and IAC-Runner 886 were more strongly associated with late leaf spot.V12549 was more susceptible to scab. Finally, accessions of A. hypogaea 2562, A.monticolaV14165 and A. ipaënsis 30076 were more strongly correlated with rust. Accession V14165 wasalmost equidistant from Group 1 and 2 accessions.

Group 2 encompassed accessions and hybrids that were opposite to the arrows, indicating atrend to multiple resistances of wild genotypes. Again, the two amphidiploids (An 2 and An 4)and the F2 progeny individuals of Caiapó x An 4 grouped with wild species. Because Group 2genotypes were very closely associated, a more refined analysis to define which one would bepreferred for genetic improvement required re-running PCA without Group 1 accessions.

Fig 4 shows the PCA re-run without Group 1 accessions. The two main components explain57.17% of variation. When the accessions in Fig 4 were divided into the five GA groups ob-tained from Fig 2, these groups tended to disperse, with few intersections. Group 1 genotypes,in green, showed less resistance to early leaf spot; and Group 4, in yellow, was the least resistantto rust. Group 3, in black, was more closely associated to late leaf spot and rust; and Group 5,in blue, was in the same direction as the scab arrow, but showing a fair amount of internal vari-ation. For example, Group 5 amphidiploid An 2 had lower resistance to scab whereas Lm 5 wason the border with Group 2, distant from each one of the arrows. In fact, amphidiploids An 2and An 4 had lower resistance to scab but were more resistant to early leaf spot, rust and lateleaf spot. On the other hand, the F2 of Caiapó x An 2 showed reduced resistance to rust, lateleaf spot and scab, but greater resistance to early leaf spot. An important obstacle to the selec-tion of progenies from interspecific crossings targeting disease resistance is the risk of losingimportant alleles as a result of backcrossing.

Accessions in Group 2 of Fig 2, in red, were distant from all arrows, and therefore are morelikely to have multiple resistances. Overall, PCA validated the GA results.

Fig 2. Distribution of wild Arachis genotypes according to resistance to late leaf spot (LLS), early leafspot (ELS), rust and scab, in the first year of study, excluding susceptible groups (accessions of A.hypogaea and two closely related wild species).Cut-off point = 7 (arrow) indicates genotype segregationinto five groups.




Table 5 shows average values from the studies conducted in three consecutive years evaluat-ing resistance to fungal diseases in 18 wild accessions and in the cultivar IAC Caiapó. Therewas a difficulty of selection of the best accessions for the three diseases, justifying again thePCA and grouping analysis utilization. It was also observed that there were differences amongyears. ANOVA results showed the interaction between accessions x years.

Of the seven accessions identified as the most resistant (Group 2 of Fig 2), six are shown inbold in Table 5; only accession KG 30006 was not included, because at the time it was not be-lieved to be an A genome species [2]. Additionally, previous tests had failed in crossing andgenerating fertile amphidiploids from this species. Currently, it is known that this species hasthe A genome [19], and further work is needed to validate its potential as a male progenitor ininterspecific crossings and generation of new amphidiploids.

During GA with all 18 genotypes and the control, only two groups were obtained, becauseIAC Caiapó was considered susceptible when compared to the wild accessions. Therefore, weremoved the control from the analysis to evaluate isolated behavior among the accessions.

Fig 5 shows GA where a cut-off point of 0.7 forms three groups. All species has A genome,except A. gregoryi accession V 14767. Rust resistance was not relevant to discriminate between

Fig 3. Distribution of wild Arachis accessions and A. hypogaea controls according to their resistanceto late leaf spot, early leaf spot, rust and scab in the first year of study. PCA with the two firstcomponents explaining 81.41% of variation. Group 1 (red circle) and Group 2 (green circle) includesusceptible and resistant accessions respectively.




accessions, as they all had low grades, i.e., low infection rates. Comparing data from Table 5with Fig 5, we may conclude that Group 1 accessions had lower resistance to late leaf spot,whereas Groups 2 and 3 showed greater resistance to this disease. The distinguishing featurebetween Groups 2 and 3 was that the former included accessions with lower resistance to earlyleaf spot, compared to the latter.

Therefore, our data suggest that Group 2 accessions (Fig 5) have the greatest potential foruse in genetic improvement programs. However, given that late leaf spot is the most importantdisease in the field, and that the difference in resistance to early leaf spot was small betweenGroups 2 and 3, both of these groups should be considered in improvement programs. Again,only A genome species were selected, except for one B genome accession, A. gregoryi V 14767,which segregated to Group 1. V14767 may not be considered the best resistance genotype, butmight prove to be an excellent allele donor for gene pyramiding. We must again point out thatdifferences observed among data from Table 5, Figs 2 and 5 result from the fact that, in the firstyear, we evaluated scab resistance whereas in later years the disease occurred at a very low rateand could not be quantified. Overall, the data show that the best accessions regarding multipleresistance to diseases in this study conditions are V 15076 (A. stenosperma), V 6413 (A. kuhl-mannii), V 13250 (A. kempff-mercadoi), Sv 3712 (A. stenosperma), V 6325 (A. helodes), GKP10017 (A. cardenasii) (Table 5 - bold).

The individual REML analysis (Table 6) of 19 genotypes used in three years assays detectedthat the environmental variance value was low, allowing the discrimination of genotypes.Based on individual BLUP (Table 6), resistance ranking of each accession was obtained, as wellas a general ranking was observed by the sum of all ranks of the three diseases. The highest val-ues are those with the best resistance to the three diseases. Accessions in bold in Table 5 hadGR values higher than 32 in Table 6, corroborating the results of Duncan Test, PCA and GA.

Variance analysis showed that A. kuhlmannii (V 6413) had the lowest average degrees of ob-servation of late leaf spot, whereas A. stenosperma (Sv 3712) and A. kuhlmannii (V 9912) hadthe lowest incidence (lowest grade) of early leaf spot. All wild genotypes showed resistance torust at the natural inoculum pressure used (Table 5).

Fávero et al. [9] utilized detached leaves to show that Arachis hypogaea and Arachis monti-cola were susceptible to late leaf spot, early leaf spot and rust, as we reproduced here in the

Fig 4. Distribution of wild Arachis accessions according to their resistance to late leaf spot, early leafspot, rust and scab in the first year of study. PCA with the two first components explaining 57.17% ofvariation. Green, red, black, yellow and blue groups means groups 1, 2, 3, 4 and 5 of Fig 2 respectively.




Table 5. Average grades of resistance to late leaf spot, early leaf spot and rust of genotypes evaluatedduring three consecutive years and differences among years averages.

Species/Accessions Late Leaf Spot Early Leaf Spot Rust Fig 2 Group Fig 5 Group

A. simpsonii V 13710 2.22 bcdefg1 1.83 b 1.00 b 1 2

A. helodes Co 6862 2.17 defgh 1.53 bcd 1.03 b 1 2

A. kuhlmannii V 6413 1.70 h 1.64 bcd 1.00 b 2 2

A. stenosperma V 15076 1.91 gh 1.46 bcd 1.12 b 2 2

A. kempff-mercadoi V 13250 1.97 fgh 1.44 bcd 1.00 b 2 2

A. cardenasii GKP 10017 2.27 bcdefg 1.27 cd 1.09 b 2 3

A. helodes V 6325 2.18 bcdefg 1.36 cd 1.09 b 2 3

A. stenosperma Sv 3712 2.00 efgh 1.27 d 1.27 b 2 3

A. gregoryi V 14767 2.83 b 1.58 bcd 1.03 b 3 1

A. kuhlmannii V 9912 2.57 bcd 1.31 d 1.00 b 3 1

A. stenosperma V 13832 2.65 bc 1.51 bcd 1.00 b 3 1

A. stenosperma V 10309 2.17 cdefg 1.79 bc 1.10 b 4 2

A. stenosperma V 7379 2.00 fgh 1.64 bcd 1.09 b 4 2

A. stenosperma V 13670 2.57 bcde 1.43 cd 1.00 b 5 1

A. stenosperma HLK 408 2.27 bcdefg 1.54 bcd 1.03 b 5 2

A. helodes Lm5 2.29 bcdefg 1.39 cd 1.24 b 5 3

A. stenosperma V 9010 2.33 bcdef 1.27 cd 1.00 b 5 3

A. stenosperma V 13258 2.31 bcdefg 1.42 bcd 1.00 b 5 3

A. hypogaea IAC Caiapó 6.82 a 5.63 a 5.92 a * *

Year 1 3.00 a 1.79 b 1.25 b

Year 2 2.31 c 1.32 c 1.33 b

Year 3 2.97 b 2.62 a 2.01 a

Accessions displaying multiple resistance in bold.1.Distinct letters indicate significant differences among accessions according to Duncan’s test (p<0,05)

* Not included in GA


Fig 5. Wild Arachis genotypes segregated according to their resistance to late leaf spot (LLS), earlyleaf spot (ELS), and rust after three years of study, excluding IAC Caiapó control.Cut-off point = 0.7(arrow) indicates genotype distribution into three groups.




Tab

le6.

Estim

atives

ofc

omponen

tsofv

ariance

(Individual

REML)a

ndtheco

mponen

tsofa

verage(Individual

BLUP)fortheva

riab

lesresistan

ceto

late

leaf

spot(LLS),

earlyleaf

spot(ELS)a

ndrust

(R)a

nd19

gen

otypes

,duringthreeco

nse

cutive

years.

Componen

tsofav

erag

e(Individual

BLUP)

Gen

otype

MP

MC

F

Ran

kg*

u+g

GG

Na

Ran

kg

u+g

GG

Na

Ran

kg

u+g

GG

Na

GR

IAC

Caiap

ó1

4.37

416.88

284.37

416.88

281

3.59

135.28

93.59

135.28

901

4.30

855.62

534.30

855.62

533

V14

767

20.31

232.82

102.34

324.85

196

-0.106

21.59

160.58

802.28

5710

-0.269

01.04

780.26

571.58

2518

V10

309

13-0.321

82.18

690.24

122.74

983

0.07

871.77

641.25

072.94

845

-0.204

71.11

210.76

502.08

1921

Lm5

8-0.202

12.30

660.55

863.06

7214

-0.284

61.41

310.13

081.82

863

-0.063

31.25

351.40

362.72

0525

V13

832

30.16

742.67

611.61

84.12

667

-0.129

21.56

860.48

552.18

3318

-0.298

11.01

880.01

671.33

3628

V73

7916

-0.479

22.02

940.11

562.62

434

-0.059

01.63

870.92

322.62

108

-0.210

11.10

670.39

931.71

6228

HLK

408

10-0.216

92.29

180.40

362.91

228

-0.143

51.55

420.40

692.10

4611

-0.269

01.04

780.21

701.53

3929

V13

710

11-0.275

32.23

340.34

192.85

052

0.08

211.77

981.83

673.53

4416

-0.298

11.01

880.05

611.37

2929

Co68

6214

-0.329

02.17

970.20

052.70

919

-0.158

41.53

940.34

412.04

189

-0.269

01.04

780.32

511.64

1932

V15

076

18-0.564

61.94

400.04

272.55

1410

-0.228

1.46

980.28

691.98

464

-0.181

11.13

571.00

742.32

4332

V99

124

0.11

192.62

061.24

153.75

0116

-0.327

51.37

030.07

451.77

2313

-0.298

11.01

880.13

781.45

4633

GKP10

017

9-0.216

02.29

260.47

252.98

1219

-0.397

81.29

990.00

01.69

786

-0.210

11.10

670.60

251.91

9334

V63

2512

-0.303

22.20

550.28

812.79

6815

-0.312

51.38

530.10

131.79

917

-0.210

11.10

670.48

641.80

3234

Sv37

1215

-0.477

52.03

120.15

532.66

3918

-0.397

1.30

080.02

211.71

992

-0.034

31.28

262.13

713.45

3935

V13

670

50.06

012.56

881.00

523.51

3812

-0.252

31.44

540.19

831.89

6019

-0.301

11.01

570.00

001.31

6836

V13

258

7-0.195

92.31

270.66

723.17

5913

-0.262

81.43

50.16

281.86

0617

-0.298

11.01

880.03

521.35

2137

V90

106

-0.159

42.34

930.81

113.31

9717

-0.397

01.30

080.04

681.74

4515

-0.298

11.01

880.07

971.39

6538

V64

1319

-0.769

41.73

920.00

002.50

875

-0.059

01.63

870.72

682.42

4614

-0.298

11.01

880.10

671.42

3538

V13

250

17-0.515

81.99

290.07

852.58

7111

-0.237

11.46

070.23

921.93

7012

-0.298

11.01

880.17

411.49

140

Com

pone

ntsOfV

arianc

e(Ind

ividua

lREML)

Vg=1.24

44Vg=0.83

51Vg=1.13

22

Vpe

rm=0.06

27Vperm

=0.07

51Vperm

=0.04

45

Ve=0.52

81Ve=0.62

29Ve=0.37

77

Vf=1.83

52Vf=1.53

31Vf=1.55

45

h2g=0.67

81+-

0.15

96h2

g=0.54

47+-0.14

27h2

g=0.72

84+-0.16

78

r=0.71

22+-

0.16

36r=0.59

37+-

0.14

90r=0.75

70+-

0.17

10

c2perm

=0.03

414

c2perm

=0.04

897

c2perm

=0.02

86

h2mg=0.86

47h2

mg=

0.78

35h2

mg=

0.89

07

GA=2.50

87GA=1.69

78GA=1.31

68

*g:

geno

typicefec

t,u+g:

geno

typicav

erag

e,GG:g

eneticga

in,N

a=ne

wav

erag

e,GR:g

eneral

rank

,Vg:g

enotyp

icva

rianc

e,Vperm:v

arianc

eof

thepe

rman

ente

nviro

men

tal

effects,

Ve:res

idua

lvarianc

e,Vf:ph

enotyp

icva

rianc

e,h2

g=plot

heritab

ility

inbroa

dse

nse,

r:plot

repe

atab

ility,c

2perm

=en

viromen

tdeterminationco

efficien

t,h2

mg:g

enotyp

e

averag

ehe

ritab

ility,G

A-Gen

eral

Ave

rage




field. Similarly, our results agree with those of Fávero et al. [9] with regards to V9243 suscepti-bility to late leaf spot, and Wi 1118 and V13824 susceptibility to rust. However, in contrast tothat previous work, we show that in three years of field evaluation the Sv 3712 accession was re-sistant to rust. Yet another distinct new finding of our study is the susceptibility of A. batizocoito scab; Fávero et al. [9] found this accession to be highly resistant for late and early leaf spotsbut did not test it for scab.

Pande and Rao [8] also identified late leaf spot resistance in an A. hoehnei accession collect-ed at a site near the collection site of the species used in our study, and they reported the sameresult for their KG 30006 accession from the same region. In both studies, A.monticola acces-sions were susceptible to late leaf spot and rust.

ConclusionsWe have found accessions with greater resistance to disease than A. cardenasii. The mostpromising accessions with multiple resistance to late leaf spot, early leaf spot, rust and scab inour study conditions were V 15076 (A. stenosperma), V 6413 (A. kuhlmannii), V 13250 (A.kempff-mercadoi), Sv 3712 (A. stenosperma), KG 30006 (A. hoehnei), V 6325 (A. helodes) andGKP 10017 (A. cardenasii). Amphidiploids and A. hypogaea x amphidiploid hybrids behavedsimilarly to wild species. Four accessions that should be further evaluated are the A.magna ac-cessions V 13751 and KG 30097 and the A. gregoryi accessions V 14767 and V 14957. Althoughthey did not show specifically high resistance, they belong to the B genome type that is crucialto resistance gene introgression and pyramiding in A. hypogaea.

Supporting InformationS1 Table. Raw data of 50 Arachis genotypes evaluated for resistance to late leaf spot, earlyleaf spot, rust and scab in field assays.(DOC)

Author ContributionsConceived and designed the experiments: MDM IJG APF. Performed the experiments: MDMIJG APF. Analyzed the data: WBJ EL MDVR. Contributed reagents/materials/analysis tools:IJG APF. Wrote the paper: APF MDMWBJ.

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RESEARCHARTICLE IdentificationofFungusResistantWild ...ainfo.cnptia.embrapa.br/digital/bitstream/item/... · Table 1. (Continued) Accessions Code Species Brazilian Accessions Code