Characterization of Resistance of Peanut to …...Characterization of Resistance of Peanut to...

© 2013 Plant Management Network.Accepted for publication 23 September 2013. Published 25 November 2013.

Characterization of Resistance of Peanut to Puccinia arachidis

Imana L. Power and Albert K. Culbreath, Department of Plant Pathology, The University of Georgia, Coastal Plain Experiment Station, 2360 Rainwater Road, Tifton, GA 31793; and Barry L. Tillman, North Florida Research and Extension Center, University of Florida, 3925 Highway 71, Marianna, FL 32446

Corresponding author: A. K. Culbreath. [email protected]

Power, I. L., Culbreath, A. K., and Tillman, B. L. 2013. Characterization of resistance of peanut to Puccinia arachidis. Online. Plant Health Progress doi:10.1094/PHP-2013-1125-02-RS.

AbstractPeanut rust, caused by Puccinia arachidis Speg, is an important foliar disease of peanut (Arachis hypogaea L.) in tropical countries. The best option for disease management is host resistance. The objectives of this project included characterizing peanut genotypes for resistance to P. arachidis, assessing the genetic variation of newly developed Collaborative Research and Support Program (CRSP) peanut breeding lines, and assessing genetic variability among P. arachidis populations. In field studies conducted over 2010-2011, several CRSP breeding lines demonstrated varying levels of rust resistance. Detached leaf assays were used to examine the components of resistance to P. arachidis. Few significant differences were observed in these studies. We used SSR markers to characterize newly developed CRSP breeding lines, plant introductions, and commonly grown cultivars. The SSR markers used detected polymorphisms but were not able to distinguish resistant from susceptible peanut genotypes. Sequences of the 5.8S-ITS2-28S region of P. arachidis isolates collected from different regions in the United States and other countries do not indicate high genetic variability among the populations.

IntroductionPeanut rust, caused by Puccinia arachidis Speg, is an important foliar

disease of peanut (Arachis hypogaea L.) in mainly low-input peanut-producing countries with warm, tropical climates; it typically does not cause extensive losses in the southeastern United States. Under normal cultivation conditions, yield losses due to infection by the peanut rust fungus can be considerable (2,24,25,27,29). Subrahmanyam et al. (25,26,27) found yield losses as high as 50% in India. In areas where rust causes frequent problems, management methods include cultural practices to reduce the inoculum source, such as eradicating volunteer plants and allowing fallow periods of at least one month between crops, and multiple fungicidal sprays throughout the season (2,17,23,24). However, chemical applications increase the production costs and moreover, the fungus may develop resistance with frequent fungicide applications (23). In addition, rust is problematic in numerous production areas where fungicidal control is not an option due to cost or availability of fungicides. The use of resistant peanut cultivars is a promising alternative management approach, and can be beneficial to growers across a range of production levels.

Many germplasm accessions have been screened, and several peanut genotypes with resistance to peanut rust have been identified (10,11,12), with sources for resistance mainly originating from Peru, Bolivia, and India (2,24,29,34). However, very little new information on rust resistance has become available in the last two decades. More recently, several breeding lines were developed with a Bolivian landrace as parent, in the UF150 project of the Peanut Collaborative Research and Support Program (Peanut CRSP) as part of

25 November 2013Plant Health Progress

the United States Agency for International Development (USAID). These breeding lines are currently being screened for multiple disease resistance in the United States and several low input peanut producing countries in the Western Hemisphere.

There is no complete resistance to P. arachidis reported in cultivated peanut. Peanut rust resistance is partial and rate reducing, where several polygenic minor genes, the components of resistance, provide varying levels of partial resistance, leading to a reduced rate of the disease epidemic. The components of peanut rust resistance described are incubation period, latent period, infection frequency, pustule size, percent diseased area, spore production, and spore germination (2,3,27). In the 1980s and early 1990s Cook (3) and Subramanyam (27) characterized the components of peanut rust resistance for several genotypes, but little work has been reported on the more recently developed breeding lines.

Genetic variability in cultivated peanut is low. This is believed to be the result of the recent hybridization of the two diploid Arachis species A. duranensis and A. ipaensis, followed by chromosome doubling (15,16,18,31). In the last few years, hundreds of simple sequence repeat (SSR) markers have been developed by research groups including the University of Georgia and The International Crop Research Institute for the Semi-Arid Tropics (ICRISAT). Khedikhar et al. (15), Mace et al. (16), Mondal et al. (18), and Varshney et al. (31) identified SSR markers that were able to detect high levels of polymorphism in peanut recombinant inbred lines RILs and peanut genotypes from different geographical regions, of which several were able to distinguish rust resistant from susceptible genotypes. Information on whether these markers can identify peanut rust in the CRSP breeding lines would be beneficial.

The lifecycle of the peanut rust pathogen is incomplete; it is not known whether alternate hosts exist. Instead, the pathogen is highly host specific as there are no reports on hosts outside of the Arachis genus. There are no reports on the presence of basidiospores, pycniospores or aeciospores, and teliospores have been rarely observed (2,24). The asexually produced dikaryotic urediniospores are predominant. Little is known about the diversity of the P. arachidis fungus, and to our knowledge, little research is being conducted on this subject. Therefore, knowledge on the molecular variability of the pathogen will lay the groundwork in the population structure and evolution of the pathogen. Greater knowledge on the variability of the P. arachidis populations and the genetics of resistance to peanut rust will moreover enable us to effectively breed for resistance and thus effectively manage the peanut rust disease on the long run.

Although peanut rust is primarily a disease of the tropics and subtropics and has been sporadic in occurrence in the southeastern United States, global climate change may result in greater problems with this disease in the United States either through greater frequency of tropical storms that move inoculum from sources in the Caribbean to peanut production areas in the United States or by extending the range over which the pathogen can overwinter. Increased potential for rust epidemics should be addressed proactively, because most peanut cultivars in the United States currently grown have low disease resistance (2,9,24,25,26,27,29) or the level of resistance to the rust pathogen is not known.

With this paper we report preliminary results of: (i) rust resistance in newly developed peanut breeding lines; (ii) use of previously identified SSR markers for rust resistance genes to distinguish rust resistant genotypes from susceptible ones using genetic markers; and (iii) the genetic variation among P. arachidis populations.

Rust Resistance in CRSP Breeding Lines: Field Resistance Field studies were conducted at the University of Florida, Plant Science and

Education Unit, Citra, FL, in 2010 and 2011, and at the University of Georgia, Coastal Plain Experiment Station, Tifton, GA, in 2011, to evaluate the field resistance of the CRSP breeding lines. The experiments were organized in a randomized complete block with three replications. Twenty five and 19 genotypes were planted at a seeding rate of 20 seed/m of row, in two row plots


(4.5 m × 6 m) at Citra and Tifton, respectively (Table 1). Disease severity was assessed weekly, using a modified nine-point ICRISAT scale based on lesion density and leaf necrosis (29): 1 = no disease and 9 = more than 50% of foliage damaged by the disease.

Table 1. Characteristics of the peanut genotypes studied in field, growth chamber, and molecular experiments.

GenotypeOrigin/background

Status/generation

Field (F)/components (C) study/Molecular (M)

Resistant/susceptible

97x36-HO2-1-B2G-3-1-2-2 (Entry1)

((89xOL28-)xBayoGrande) CRSP UF150 F (Citra,

Tifton) C M Resistant (this study)

99x33-1-B2G-12-2-1 (Entry2)

BayoGrandexHull CRSP UF150 F (Citra,

Tifton) M Resistant (this study)

99x33-1-B2G-13-1-1 (Entry3)



99x33-1-B2G-2-2-2 (Entry4)



99x8-1-B2G-3-1-1(Entry5)



Altika Cultivar F (Citra) C M Susceptible (this study)

BOL11-b7 Overo Chiquitano/(F84x23) CRSP UF150 F (Citra) Susceptible

(this study)

BOL19-b5 FLMDR98/BG CRSP UF150 F (Citra) C M Susceptible

(this study)

BOL20-b5 FLMDR98/BG CRSP UF150 F (Citra) Susceptible

(this study)


(this study)


(this study)

BOL3-7 (F79x4)/PI656458 CRSP UF150 F (Citra,


BayoGrande Bolivia Landrace F (Citra) C M Susceptible (this study)

DP-1 (80202x81206] CRSP UF150 F (Citra) Resistant (this study)

96x72-HO1-9-1-1-1-1-2-1 (Entry15) ((89xOL2-)x(84x28-)) CRSP UF150 F (Citra,


98x116-5-1-1-1-2-1 (Entry16) ((DP-1)x(89xOL28-)) CRSP UF150 F (Citra,


97x34-HO3-1-B2G-3-1-1-1 (Entry17) [(89xOL28-)x(87x8-)] CRSP UF150 F (Citra,


PI 561685 (Tifrust-10) Tifton ARS F (Citra,

Tifton) M Resistant (12)

PI 561688 (Tifrust-13) Tifton ARS F (Citra,

Tifton) C M Resistant (10)

PT910-2-8-11 VA98R/BayoGrande CRSP UF150 F (Citra) Resistant

(this study)

PTBOL3-3 (F79x4)/PI656458 CRSP UF150 F (Citra,


PTBOL3-4 (F79x4)/PI656458 CRSP UF150 F (Citra,


(continued).


Table 1. (continued).

GenotypeOrigin/background

Status/generation

Field (F)/components (C) study/Molecular (M)

Resistant/susceptible

RP-97F2-B-9-2-2-1-b3-B

[MDR98xBGrande] CRSP UF150 F (Citra) Resistant

(this study)

Southern Runner USA Released cultivar F (Citra, Tifton) C M Resistant

(this study)

York USA Released cultivar F (Citra, Tifton) C M Resistant

(this study)

PI 568164 India Plant introduction C M Resistant (30)

PI 259747 (Tarapoto) Peru Plant introduction C M Resistant (21, 28)

PI 298115 Israel Plant introduction C M Resistant (28)

PI 562530 India Plant introduction C M Resistant (30)PI 540472 China Plant introduction C M Resistant (30)

PI 314817 Plant introduction M Resistant (28)

PI 478856 Plant introduction M Resistant (30)

NC 3033 USAReleased germplasm/RIL parent

C M Unknown

NC 94022 USA Breeding line/RIL parent C M Unknown

Bailey High OL USA Released cultivar/RIL parent C M Unknown

SunOleic 97R USA Released cultivar RIL parent C M Unknown

SPT-06-06 USA RIL parent C M Unknown

GT-C20 China C M Unknown

Georgia Green USA Released cultivar C M Unknown

Florida-07 USA Released cultivar/RIL parent F (Tifton) C M Unknown

Tifguard USA Released cultivar F (Tifton) C M Unknown

Tifrunner USA Released cultivar/RIL parent C M Unknown

Georgia-07W USA Released cultivar F (Tifton) C M Unknown

C-99R USA Released cultivar C M Unknown

Georgia-03L USA Released cultivar F (Tifton) C M UnknownGuyana Jumbo Guyana Cultivar C M Susceptible

TAG24 Plant introduction M Susceptible (15)

TMV2 Plant introduction M Susceptible (18)

Disease severity data were used to calculate the area under disease progress curve (AUDPC) (22) for each plot. The effects of genotype on AUDPC were analyzed using the Proc GLM procedure (SAS v 9.2, SAS Institute Inc., Cary, NC). Replications were considered random effects, and genotype was considered a fixed effect. Differences among genotypes were determined using the "lsd" option included in each main effect. Fisher’s LSD (P ≤ 0.05) was used to determine significant differences between AUDPC. Genotypes with a disease severity score of three or lower were considered resistant (AUDPC < 5.8 in 2010 or AUDPC < 6.7 in 2011).

In 2010, 18 of the 25 genotypes demonstrated resistance to rust (Table 2). Of these genotypes, several could potentially be developed into cultivars, whereas


others would be more suitable in rust resistance breeding programs. Disease severity was low in Citra in 2011, and few differences in rust severity were observed among genotypes. In Tifton, the disease epidemic started too late in the season be high enough to distinguish between genotypes by harvest.

Table 2. Rust resistance in CRSP breeding lines planted at Citra, FL (2010 and 2011) and Tifton, GA (2011).

Genotype

AUDPCx

CITRA 2010LSD=2.33

CITRA 2011LSD=1.82

Tifton 2011LSD=0.94

97x36-HO2-1-B2G-3-1-2-2 2.4 hi 2.7 fg 3.4 cd

99x33-1-B2G-12-2-1 5.3 d-g 4.5 b-f 3.7 b-d

99x33-1-B2G-13-1-1 3.9 f-i 3.7 c-f 3.2 cde

99x33-1-B2G-2-2-2 4.5 e-i 4.7 b-e 3.3 cde

99x8-1-B2G-3-1-1 4.4 e-i 5.3 abc 4.5 ab

Altika 6.6 b-e 3.1 efg -

BOL11-b7 9.0 a 3.5 c-g -

BOL19-b5 9.2 a 4.3 b-f -

BOL20-b5 8.9 ab 3.3 d-g -

BOL21-b5 7.6 abc 4.1 b-g -

BOL22-b5 8.3 ab 3.9 c-g -

BOL3-7 3.0 ghi 2.7 fg 2.8 de

BayoGrande 7.3 a-d 6.8 a -

DP-1 5.7 c-f 5.8 ab -

96x72-HO1-9-1-1-1-1-2-1 4.7 e-h 3.7 c-g 3.7 a-d

98x116-5-1-1-1-2-1 4.6 e-I 5.0 a-d 4.6 a

97x34-HO3-1-B2G-3-1-1-1 3.8 f-I 4.3 b-f 4.4 ab

PI 561685 (Tifrust-10) 3.1ghi 3.9 c-g 2.9 de

PI 561688 (Tifrust-13) 2.4 i 2.3 g 2.4 e

PT910-2-8-11 5.5 c-f 3.9 c-g -

PTBOL3-3 3.5 f-I 2.4 g 3.3 cde

PTBOL3-4 5.3 d-g 3.8 c-g 2.8 de

RP-97F2-B-9-2-2-1-b3-B 5.0 c-f 3.7 c-g -

Southern Runner 4.9 efg 4.3 b-f 3.9 abc

York 5.7 c-f 3.6 c-g 3.0 de

Florida-07 - - 3.4 cd

Tifguard - - 3.5 cd

Georgia -07W - - 3.2 cde

Georgia -03L - - 3.2 cdeWithin columns, means followed by the same letter are not significantly different at P ≤ 0.05x Data are means from three replicates. Means from Proc GLM of area under the disease progress curve

(AUDPC), assessed at weekly intervals


Rust Resistance in CRSP Breeding Lines: Components of Resistance

To assess the components of resistance, a detached leaf experiment was carried out as described by Cook (3). Peanut plants were grown at 25°C from seed in the greenhouse in 15 cm pots filled with potting soil. The youngest fully expanded leaves of six- to eight-week-old plants were collected, the leaflets detached, and placed on sterile damp filters in a Petri dish (15 cm diameter) with the abaxial side up. The leaflets were then inoculated by spraying them for one second using an aerosol sprayer containing an uredinial spore suspension (40,000 spores per ml). Spore suspensions were made with a peanut rust isolate collected from fields in Georgia, and increased in the greenhouse on cultivars Florida-07 and Georgia Green. There were three replicates per genotype. The Petri dishes containing inoculated leaflets were arranged in a randomized complete block, and incubated in darkness for 16 h at 25°C. After inoculation, the closed Petri dishes were incubated at 25°C, 12h photoperiod for 31 days. Leaflets were examined for the numbers of pustules at 20 and 31 days after inoculation (DAI), and pustule size at 31 DAI. Pustule size was determined by measuring the largest diameter of each pustule, using a dissecting microscope at 5× magnification.

The effects of genotype on numbers of pustules and pustule size were analyzed using the Proc GLM procedure (SAS v 9.2, SAS Institute Inc., Cary, NC). Replications were considered random effects, and number of pustules, pustule size, and genotype were considered fixed effects. Differences among genotypes were determined using the "lsd" option included in each main effect. Fisher’s LSD (P ≤ 0.05) was used to determine significant differences among genotypes for numbers of pustules, and pustule size.

There were few significant differences between genotypes for numbers of pustules developed and pustule size in both Spring and Fall (Table 3). In the Spring, genotypes NC3033 and SunOleic 97R had the highest pustule numbers and SunOleic 97R had the largest pustule size. In the Fall genotype PI568164 had both the highest pustule number and largest lesion diameters.


Table 3. Components of peanut rust resistance from detached leaf studies.

Genotype

Spring 2011 Fall 2011

No. of pustules31 DAIx

Pustule size31 DAIby

No. of pustules31 DAIay

Pustule size31 DAIby

99x33-1-B2G-13-1-1 1.3 cd 4.9 cd 0.33 ed 1.2 def

97x36-HO2-1-B2G-3-1-2-2 1.0 cd 5.6 bcd 1.0 cde 1.8 b-f

Altika 2.7 cd 9.9 a-d 3.7 b-e 3.6 a-d

Bailey High OL 2.0 cd 8.1 a-d 3.3 b-e 4.3 abc

BayoGrande 4.7 bcd 13.8 abc 7.0 b 3.6 a-d

BOL 3-7 2.3 cd 10.2 a-d 3.0 cde 2.0 b-f

BOL 19-b5 1.7 cd 9.5 a-d 0.3 de 0.5 ef

C-99R 1.3 cd 3.6 cd 0.0 e 0.0 f

Florida-07 1.3 cd 3.7 cd 0.7 de 1.0 def

Georgia Green 2.0 cd 4.6 cd 1.3 cde 4.4 ab

Georgia-03L 1.0 cd 4.1 cd 1.7 cde 2.7 b-f

Georgia-07W 5.3 bc 11.6 a-d 3.7 b-e 2.6 b-f

GT-C20 0.7 d 2.9 d 2.3 cde 1.6 c-f

Guyana Jumbo - - 2.7 cde 1.7 c-f

PI 259747 (Tarapoto) 1.3 cd 3.0 d 4.0 bcd 2.9 b-e

PI 298115 0.3 d 2.3 d 2.3 cde 2.8 b-e

PI 540472 5.3 bc 11.1 a-d 4.7 bc 4.3 abc

PI 561688 (Tifrust-13) 2.7 cd 5.1 cd 0.3 de 0.9 ef

PI 562530 2.0 cd 3.9 cd 0.0 e 0.0 f

PI 568164 4.0 bcd 17.3 a 11.3 a 5.9 a

NC 3033 12.0 a 12.1 a-d 0.0 e 0.0 f

NC 94022 3.0 cd 10.4 a-d 0.3 de 0.6 ef

SPT-06-06 3.0 cd 15.5 ab 3.0 cde 1.7 c-f

Southern Runner 2.0 cd 5.4 bcd 2.0 cde 3.9 abc

SunOleic 97R 7.7 ab 17.3 a 0.3de 1.1 def

Tifguard 2.0 cd 8.0 a-d 2.7 cde 1.0 def

Tifrunner 3.0 cd 5.5 bcd 1.7 cde 3.0 b-e

York 1.7 cd 8.7 a-d 0.0 e 0.0 f

Within columns, means followed by the same letter are not significantly different at P ≤ 0.05.x Means from Proc GLM of number of pustules assessed 31 days after inoculation (DAI).y Means from Proc GLM of pustule size assessed 31 days after inoculation (DAI).

SSR Markers to Identify Rust Resistance Genes in Peanut Genotypes

Total genomic DNA of 41 genotypes (Table 1) was extracted from fresh unfolded leaves of eight- week-old greenhouse-grown plants, following a CTAB protocol (19). A set of seven SSR markers − GM431, GM457, GM496, GM518, GM553, GM567, and GM591 (5) − previously identified as being able to detect high levels of polymorphism in peanut RILs and peanut genotypes from different geographical regions (15,16,18,31), were used for preliminary characterization of the 41 genotypes. The SSR markers were selected based on


their ability to distinguish between rust resistant and susceptible genotypes in previous reports (16). PCR amplifications were performed in 10 µl total volumes (8) containing 2.5-ng DNA template, using a 64°C-58°C touchdown PCR amplification program (4). PCR products were sent to the Georgia Genomics Facility (Athens, GA) for fingerprinting.

Peak analysis was conducted using GeneMapper software v4.0 (Applied Biosystems, Forest City, CA). The alleles of each SSR locus from the 41 peanut genotypes were scored as presence or absence of the allele. Analysis of molecular variance (AMOVA), principal component analysis and estimates of genetic distances between the genotypes were calculated with GenAlEx6.4 (20) to analyze differences between the resistant and susceptible peanut genotypes. The Jaccard coefficient was used to compute the genetic similarity matrix of the genotypes based on the SSR data using DendroUPGMA (genomes.urv.es/UPGMA) (6). An unweighted pair group method of arithmetic means (UPGMA) dendrogram was constructed using the PhyloWidget program (www.phylowidget.org) (14).

A total of 15 polymorphic alleles were generated for the seven loci across the peanut genotypes, with an average of 2.1 alleles per locus. Although polymorphisms were detected no distinction between resistant and susceptible peanut genotypes was observed. No distinct resistant or susceptible clades were observed in the neighbor joining analysis (Fig. 1). A similar lack of grouping of resistant or susceptible genotypes was observed in the principal components analysis (Fig 2). These results are in contrast with those reported by Mace et al. (16), who found SSR markers associated with rust resistance genes. In their study, GM431, GM496, GM 553, and GM567 were present only in resistant genotypes and absent in susceptible ones. They also found marker GM518 absent in resistant and present in susceptible genotypes. In our study, no marker alleles were consistently associated with resistant or susceptible genotypes. The AMOVA indicated that a low percentage of the genetic variation was associated with disease resistance and the genotypes. Fifteen% of the observed variation is accounted for by "among resistant and susceptible populations," whereas 85% is accounted for "within resistant and susceptible groups" (Table 4). The observed low polymorphism is consistent with previous reports (15,16,18,31).


Fig. 1. Dendrogram of 41 cultivated peanut genotypes based on genetic differences in resistance to peanut rust, calculated from 7 SSR markers. The Jaccard coefficient was used to compute the genetic similarity matrix of the genotypes. Bootstrap values on tree branches represent the percent appearance of a given branch from 100 replications. Only values higher than 50% are shown.


Fig. 2. Principal Component Analysis plot of 41 cultivated peanut genotypes based on genetic differences in resistance to peanut rust, calculated from 7 SSR markers.

Table 4. Results from the analysis of molecular variance (AMOVA) for rust resistance in 41 peanut genotypes based on genetic differences in resistance to peanut rust, calculated from 7 SSR markers Source df SS MS Est. Var. %

Among Pops 2 14.187 7.093 0.414 15%

Within Pops 38 86.301 2.271 2.271 85%

Total 40 100.488 2.685 100%

Stat Value P(rand >= data)

PhiPT 0.154 0.004Probability, P(rand>=data), for PhiPT is based on permutation across the full data set.PhiPT = AP / (WP + AP) = AP / TOT, where AP = Est. Var. Among Pops, WP = Est. Var. Within Pops

Genetic Variation Among P. arachidis PopulationsWe used P. arachidis field isolates, consisting of a collection of spores,

collected in North America, South America, Central America and Asia (Table 5), in different years. Genomic DNA of isolates collected in GA was extracted from 10-25 mg of urediniospores per field isolate, by grinding the spores in a bead beater for 5 min with glass beads, followed by the Omniprep for fungi extraction kit (G-Biosciences, St. Louis, MO) according to the instructions. DNA of isolates collected outside of the state of GA, was extracted using the Qiagen REPLI-g Ultrafast mini kit (Qiagen, Valencia, CA), as described by Wang et al. (33) with minor revisions: Several spores per isolate were added to a 2.5 µl mixture that contained 1 µl phosphate buffered saline (PBS) and 1.5 µl denaturing buffer (D2). After ice-incubation, the manufacturer’s protocol was followed. DNA quality was examined on a 1% agarose gel, and the quantity was determined with nanodrop.


Table 5. Geographical origin of 33 Puccinia arachidis isolates studied Isolatenr Country

State/Place Location

Collectiondate

1 USA Gainesville, FL CITRA research station Aug 2010



4 USA Attapulgus, GA UGA Tifton research station Sep 2011

5 USA Attapulgus, GA UGA Tifton research station Sep 2012

6 USA Tifton, GA UGA Tifton research station, Lang farm Sep 2010

7 USA Tifton, GA UGA Tifton research station, Gibbs farm Sep 2010

8 USA Tifton, GA UGA Tifton research station, Lang farm Sep 2012

9 USA Tifton, GA UGA Tifton research station, RDC farm Sep 2012

10 USA Tifton, GA UGA Tifton research station, Gibbs farm #916 Sep 2012



13 USA Tifton, GA UGA Tifton research station, Gibbs farm#1059 Sep 2012

14 Guyana Annai CRSP demoplot Sep 2011

15 Guyana Annai Farmer’s field, Mr. Hamilton Sep 2010

16 Guyana Annai CRSP demoplot Sep 2011

17 Bolivia Saveedro Research field Mar 2011

18 Bolivia Puerto Fernandez ANAPO Research field Mar 2011

19 Bolivia Santa Cruz ANAPO Research station Dec 2011

20 Bolivia Santa Cruz ANAPO Research station Apr 2012

21 Bolivia Puerto Fernandez ANAPO Research field Apr 2012

22 Bolivia 26 de Augusto ANAPO Research field Apr 2012

23 Haiti Tovar Farmer’s field Sep 2011

24 Haiti Limbe Ag School Sep 2011

25 Haiti Bas Limbe Farmer’s field Jun 2012

26 Nicaragua La Libertad Farmer’s field Sep 2011

27 Nicaragua Malpaislo Farmer’s field Sep 2011

28 Nicaragua San Antonio Farmer’s field Sep 2011

29 Nicaragua San Jose Farmer’s field Sep 2011

30 Nicaragua El Ojachal Farmer’s field Sep 2011

31 Nicaragua Ceiba Mocha Farmer’s field Sep 2011

32 Nicaragua Lourdes Farmer’s field May 2012

33 The Philippines Bukidnon Farmer’s field May 2012

Duplo Nicaragua La Libertad Farmer’s field Sep 2011

The primers Rust2inv (1) and LR6 (32) were used to amplify the complete ribosomal 5.8S subunit, the internal transcribed spacer region 2 (ITS 2) and the 28S subunit, in 25 µl total volumes (1). Cleaned PCR products were sequenced with Rust2inv by Eurofins MWG Operon (Albany, GA). To confirm rust


specificity, all DNA sequences were subjected to BLAST search. Nucleotide sequences generated were aligned and analyzed using the software Geneious v6.05 using default settings.

We have collected 33 isolates from the US, Bolivia, Guyana, Haiti, Nicaragua and the Philippines (Table 5), and extracted genomic DNA, PCR-amplified and sequenced the ribosomal 5.8S-ITS2-28S region of 33 isolates. Based on the preliminary results from the sequenced region, no distinct, well-supported groups could be identified, as there was no distinction among geographic regions or collection dates (Fig. 3). This high degree of genetic similarity in the ITS region of the isolates studied, indicates low molecular variability within the populations, which may indicate that the isolates shared a common origin. The homogeneity within these populations may furthermore indicate a lack of sexual recombination, as is suspected due to the absence of the sexual teliospores.

Fig. 3. Phylogenetic relation of 33 Puccinia arachidis isolates collected from cultivated peanut, in the US, Bolivia, Guyana, Haiti, Nicaragua, and the Philippines, as derived from neighbor joining analysis of the ribosomal 5.8S-ITS2-28S region, after multiple alignment with Geneious. The confidence level of the nodes were tested by bootstrapping 1000 replications. Scale bar indicates a distance of 0.06 (6 base pair changes per 100 nucleotide positions).


Conclusion We conducted field experiments, growth chamber experiments, and genetics

of P. arachidis and its host to characterize peanut rust resistance. Though preliminary, this research indicates the existence of field resistance to the peanut rust disease in the newly developed CRPS breeding lines. Including more components will most likely enable us to better explain the mechanism behind the peanut rust resistance.

All SSR markers used were polymorphic, however, very few alleles were present per locus, which is consistent with low polymorphism reported in peanut (15,16,18,31). No distinction between resistance and susceptibility was observed in the studied genotypes. We will include more polymorphic SSR markers to allow us to better distinguish peanut rust resistant genotypes from susceptible ones, on the genetic level.

ITS regions are useful for identifying molecular variability within populations of the same species (7,13). Our data indicate that P. arachidispopulations are highly homogeneous for those regions. Other loci will be examined to determine levels of variability among isolates within P. arachidis. To our knowledge, no information on the population structure of P. arachidishas ever been published.

AcknowledgmentsThe authors thank USAID, Peanut CRSP, the National Peanut Board and the

APS Foundation for funding the research; Dr. Timothy Brenneman, Dr. Robert Kemerait Jr., Dr. Katherine Stevenson, Pablo Navia, Marian Luis, and Abraham Fulmer for collection of P. arachidis isolates; Michael Heath, Ronald Hooks, Matthew Wiggins, Samuel Holbrook, Patricia Hilton, Miranda Goodman, Stephen Mullis, Justin McKinney, Dr. Sergio Morichetti, Mike Giomillion, Dr. Graeme Wright, Jeff Tatnell, and Alyssa Cho for assistance in field and growth chamber studies; Kippy Lewis, Dr. Venkatsan Parkunan, Dr. Bhabesh Dutta, Dr. Peggy Ozias-Akins, Dr. Ye Chu, and Rattandeep Gill for molecular studies and molecular data analysis.

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