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Identification of a soybean rust resistance gene in PI 567104B
†Min Liu, †Shuxian Li, Sivakumar Swaminathan, Binod B. Sahu, Leonor F. Leandro,
Andrea J. Cardinal, Madan K. Bhattacharyya, Qijian Song, *David R. Walker, and
*Silvia R. Cianzio
M. Liu, visiting student, Dep. of Agronomy, Iowa State Univ., Ames, IA, 50011-1010, (current
address: Dep. of Agronomy, Shenyang Agricultural Univ., 120 Dongling Ave., Shenyang,
Liaoning 110866, China); S. Li, USDA-ARS, Crop Genetics Research Unit, Stoneville, MS,
38776; S. Swaminathan, B. B. Sahu, M. K. Bhattacharyya, and S. R. Cianzio, Dep. of
Agronomy, Iowa State Univ., Ames, IA, 50011-1010; L. Leandro, Plant Pathology Dep., Iowa
State Univ., Ames, IA. 50011-1010; A. J. Cardinal, Dep. of Agronomy, North Carolina State
Univ., Raleigh, N.C. 27695, (current address: Genetic Projects Lead Vegetables Seeds R&D,
Syngenta Biotechnology, Inc. 3054 Cornwallis Rd. Research Triangle Park, NC 27709); Q.
Song, USDA-ARS, Soybean Genomics and Improvement Laboratory, Beltsville Agricultural
Research Center, Beltsville, MD 20705; D. R. Walker, USDA-ARS, Soybean/Maize
Germplasm, Pathology and Genetics Research Unit, and Dep. of Crop Sciences, University of
Illinois at Urbana-Champaign, Urbana, IL 61801.
†The first two authors contributed equally and should be considered co-first authors.
*Corresponding authors (scianzio@iastate.edu and walkerdr@illinois.edu)
Ph : 1-515-294-1625 (Cianzio)/ 1-217-244-1274 (Walker) ; Fax : 515-294-3163 (Cianzio)
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Abstract
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Asian soybean rust (SBR), caused by the fungus Phakopsora pachyrhizi Syd. & P. Syd., is one
4
of the most economically important diseases that affect soybean production worldwide. A long-
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term strategy for minimizing the effects of SBR is the development of genetically resistant
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cultivars. The objectives of the study were to identify the location of a rust-resistance (Rpp)
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gene(s) in plant introduction (PI) 567104B, and to determine if the gene(s) in PI 567104B was
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different from previously mapped Rpp loci. The progeny of the cross of ‘IAR 2001 BSR’ x PI
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567104B was phenotyped from field assays of the F2:3 and F4:5 generations and from a growth
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chamber assay of 253 F5:6 recombinant inbred lines (RILs). For the growth chamber, the
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phenotyping was conducted by inoculation with a purified 2006 fungal isolate from Mississippi.
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A resistance gene locus on PI 567104B was mapped to a region containing the Rpp6 locus on
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chromosome 18. The high level of resistance of F1 seeds from two other crosses with PI
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567104B as one of the parents indicated that the gene from PI 567104B was dominant. The
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interval containing the gene is flanked by the simple sequence repeat (SSR) markers Satt131
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and Satt394, and includes the SSR markers BARCSOYSSR_18_0331 and
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BARCSOYSSR_18_0380. The results also indicated that the resistance gene from PI 567104B
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is different from the Rpp1 to the Rpp4 genes previously identified. To determine if the gene
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from PI 567104B is different from the Rpp6 gene from PI 567102B, additional research will be
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required.
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Keywords: Asian soybean rust, Phakopsora pachyrhizi, disease resistance, SSR markers, gene
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mapping.
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Author contribution statement: M. L. and S. S. conducted molecular, statistical and linkage
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analyses of phenotypic data. S. L. conducted phenotypic screenings (field and Conviron
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growth chamber), provided guidance to molecular analysis, wrote the sections on growth
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chamber screening, preparation of inoculum, inoculation description, designed the severity
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rating scale and contributed to data summary. B. B. S. provided initial guidance to map of
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resistance in PI 567104B. Q. S. analyzed the data to produce the final linkage and LOD score
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maps. D. R. W., L. F. L. and A. C. planted and conducted field screenings. D. R. W. supplied F1
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seeds. M. K. B. provided guidance on molecular analyses and marker used. S. R. C. planned
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the research, developed the populations, and supervised the project. M. L., D. R. W. and S. R.
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C. prepared the manuscript.
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Key message: Using a combination of phenotypic screening and molecular, statistical, and
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linkage analyses, we have mapped a dominant resistance gene to Phakopsora pachyrhizi in
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soybean PI 567104B.
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Abbreviations: DAI, days after inoculation; LG, linkage group; MG, maturity group; PI, Plant
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Introduction; RB, reddish brown lesions; RCBD, randomized complete block design; RILs,
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recombinant inbred lines; SBR, Asian Soybean Rust; TAN, tan lesions; IM, immune.
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Introduction
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Asian soybean rust (SBR), caused by Phakopsora pachyrhizi Syd. & P. Syd., is one of the most
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economically important soybean [Glycine max (L.) Merr.] disease worldwide, capable of
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causing yield losses ranging from 10% to 80% (Kawuki et al. 2003; Kumudini et al. 2008;
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Sinclair and Hartman 1999). When environmental conditions favor pathogen development on
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susceptible genotypes, rust lesions can cover most of the leaf surface, at times occurring on the
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stem and pods as well (Sinclair and Hartman, 1999). Premature defoliation reduces
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photosynthesis and the number of days to maturity (Kumudini et al. 2008). SBR-infected plants
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may have few pods and light seeds which are often of poor quality.
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P. pachyrhizi was first identified in Japan in 1902 (Hennings 1903). It is endemic to
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eastern Asia and possibly northern Australia, but over time it has gradually spread to soybean-
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growing countries around the world (Bromfield 1984; Pretorius et al. 2001; Rossi 2003; Wang
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and Hartman 1992; Yorinori et al. 2005). It was first reported in the U.S.A. in Hawaii in 1994
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(Killgore and Heu 1994), and in the continental U.S.A. in Louisiana in 2004 (Schneider et al.
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2005). SBR has been reported in up to 20 other states in the U.S.A. in some growing seasons,
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and it is also established in the country of Mexico and all of the soybean producing countries in
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South America (Hershman et al. 2011; Isard et al. 2005). P. pachyrhizi possesses a diverse and
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complex pathogenicity, infecting a wide range of legume genera (Ono et al. 1992; Slaminko et
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al. 2008a, b).
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Although sources of SBR resistance have been known since the 1970s, no U.S. cultivars
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are known to be resistant (Li et al. 2012). Management of SBR in regions and countries where
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P. pachyrhizi is present relies on the application of fungicides, and sometimes on the use of
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early-maturing soybean cultivars to limit economic losses (Raetano et al. 2011). Although
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timely fungicide applications can be effective for managing SBR, they add to production costs
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and suppress beneficial fungi (e.g., those that parasitize insect pests) as well as pathogenic ones.
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In tropical production areas, multiple fungicide applications may be required during a growing
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season (Godoy 2012). In addition, some fungicides cause soybean stems to remain green after
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the pods have matured, which can cause delays in harvest (Bob Kemerait, Univ. of Georgia,
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personal communication, 2013). Sustainable management of SBR epidemics is most likely to
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succeed if genetic resistance to the pathogen can be combined with judicious use of fungicides.
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Six loci with dominant resistant SBR alleles from different G. max plant introductions
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(PIs) have been reported, and multiple resistance alleles have been found at some of the loci
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(Chakraborty et al. 2009; Garcia et al. 2008, 2011). The Rpp1 gene was identified in PI 200492
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(McLean and Byth 1980), and was mapped on chromosome 18 (Linkage Group G; LG G)
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between SSR markers Sat_187 and Sat_064 (Hyten et al. 2007). The Rpp2 gene was identified
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in PI 230970 (Bromfield and Hartwig 1980), and was later mapped to chromosome 16 (LG J)
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(Silva et al. 2008). The Rpp3 gene was identified in PI 462312 (Bromfield and Melching 1982;
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Hartwig and Bromfield 1983), and was subsequently mapped to chromosome 6 (LG C2) (Hyten
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et al. 2009), while the Rpp4 gene was identified in PI 459025B (Hartwig 1986) and mapped to a
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different location on chromosome 18, approximately 26 cM from Rpp1 (Silva et al. 2008),
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based on the marker locations in the Song et al. (2004) integrated linkage map. An Rpp gene in
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PI 200526 and different resistance alleles from other germplasm accessions were mapped to the
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Rpp5 locus on chromosome 3 (LG N) (Garcia et al. 2008). In 2012, the Rpp6 gene was
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identified in PI 567102B and mapped to a third Rpp locus on chromosome 18 approximately 40
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cM from the Rpp4 locus and about 66 cM from the Rpp1 locus (Li et al. 2012). The Japanese
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cultivar Hyuuga was found to have an Rpp gene, Rpp?(Hyuuga), at or near the Rpp3 locus on
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chromosome 6 (Monteros et al. 2007; Monteros et al. 2010). A second resistance gene from
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Hyuuga was later mapped to the Rpp5 locus on chromosome 3 (Kendrick et al. 2011). Garcia et
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al. (2008) discovered recessive resistance genes at different loci in PI 224270 (rpp2[?]) and in
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PI 200456 (rpp5). In addition to the six loci thus far reported in the literature, patents have been
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claimed on at least 10 other loci associated with SBR resistance (Bailey et al. 2014), though the
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use of proprietary markers to map these genes makes it difficult to determine where the loci are
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located relative to markers frequently used in the public sector.
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Resistance reactions are typically characterized by the development of reddish-brown
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(RB) lesions with two or fewer uredinia on infected leaf tissue (Bromfield et al. 1980).
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Immune, or Type 0 reactions are less common, but occur in certain pathotype-Rpp gene
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interactions, typically involving Rpp1. In contrast, susceptible soybean hosts typically develop
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tan (TAN) reactions with three or more uredinia per lesion and a tan color resulting from the
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lesions and/or clumps of urediniospores. The resistance of soybean genotypes to P. pachyrhizi
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can vary temporally and geographically (Akamatsu et al. 2013; Paul et al. 2013; Twizeyimana
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and Hartman 2012; Walker et al. 2011, 2014). Durable resistance would provide an economic
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and environmentally friendly way to protect soybean crops from SBR, but it might likely
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require pyramiding two or more Rpp genes each effective against a majority of the P. pachyrhizi
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pathotypes in the geographical target region for cultivar development and production.
The search for new P. pachyrhizi resistance genes would increase the chances of finding
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novel genes that would condition broader or more durable resistance in pyramids with other
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Rpp genes (Khanh et al. 2013). Field evaluations of germplasm in several states in the southern
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U.S.A. (Miles et al. 2006; Walker et al. 2011, 2014), and in countries in South America (Miles
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et al. 2008) showed that PI 567104B has high levels of resistance to many populations of P.
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pachyrhizi. The resistance of accession PI 567104B to P. pachyrhizi populations from North
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and South America is somewhat unusual, since to date many of the accessions found to be
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resistant in South America (Miles et al. 2008) were susceptible to rust populations in the
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southern United States (Walker et al. 2011, 2014). The SBR resistance of PI 567104B could
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therefore be of value for the development of SBR-resistant soybean cultivars. The objectives of
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this study were to identify the location of a rust-resistance (Rpp) gene or genes in plant
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introduction (PI) 567104B, and to determine if the gene(s) in PI 567104B was different from
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previously mapped Rpp loci.
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Materials and methods
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Plant material
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A population was developed from the cross of ‘IAR 2001 BSR’ × PI 567104B. IAR 2001 BSR
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is a high-yielding SBR-susceptible cultivar released by Iowa State University-ISURF, Docket #
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03542 (Cianzio et al. 2008). PI 567104B is a MG IX accession from Indonesia (Germplasm
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Resources Information Network, http://www.ars-grin.gov/cgi-bin/npgs/acc/). The accession
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was identified by Miles et al. (2006) to be resistant to a mixture of four P. pachyrhizi isolates
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from Brazil, Paraguay, Thailand and Zimbabwe. It was also resistant to a local field SBR
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population at Capitán Miranda, Paraguay (Miles et al. 2008). In the U.S.A., PI 567104B was
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highly resistant to field populations in the states of Alabama, Florida, Georgia, Louisiana, and
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South Carolina in most of the years that it had been evaluated (Walker et al. 2011, 2014).
The cross, designated AX20871, was made in Puerto Rico in March 2006 at the Iowa
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State University research site located at the University of Puerto Rico’s Isabela Substation
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(ISU-PR). It was part of a group of crosses made to transfer P. pachyrhizi resistance to high-
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yielding elite soybean lines of early maturity groups. For AX20871, six F1 seeds were obtained
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and planted in June 2006 in Puerto Rico. Each F1 plant was identified and harvested
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individually in September 2006. The identity of the individual F1 plants was maintained
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throughout the study even though the hybrid nature of the F1 plants was confirmed using flower
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and pubescence colors as morphological markers. After additional results confirming PI
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567104B field resistance to SBR (Miles et al. 2006; Miles et al. 2008), the F2 seeds of the
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population which had been maintained in cold storage, were planted in Puerto Rico in October
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2007, and each individual F2 plant was identified prior to harvest in January 2008. A total of
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253 F2:3 lines were obtained for the population and were maintained in cold storage until the
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next planting. One hundred and thirty F2:3 and F4:5 lines were used in the 2008 and 2009 field
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assays described in the next section.
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F5:6 recombinant inbred lines (RILs) were developed at the ISU-PR research site by
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advancing the 253 original F2:3 lines through single-plant selections of each line from 2008 to
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2011, when each RIL had reached the F6 generation. The RILs were screened for their reactions
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to a 2006 P. pachyrhizi isolate from Mississippi (MS06-1B) in the growth chamber study
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described below. Genotypic and phenotypic data from 114 of the 130 F4:5 lines phenotyped in
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the field in 2008 and 2009, and of the 253 F5:6 RILs phenotyped in the growth chamber were
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used to map the resistance gene in PI 567104B.
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To assess the degree of dominance of the Rpp gene(s) in PI 567104B, putative F1 plants
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from two other crosses with the PI as the SBR-resistant parent were tested for their reactions to
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the MS06-1B isolate. Three putative F1s from a cross of ‘Osage’(Chen et al. 2007) × PI
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567104B and 12 putative F1s from a cross between the University of Georgia breeding line
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G00-3880 and PI 567104B were evaluated in 2014 at Stoneville, MS with the same methods
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used for the growth chamber assay of the RILs.
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P. pachyrhizi resistance evaluations
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Field evaluations. The 130 F2-derived lines mentioned in the previous section were planted at
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the University of Florida’s North Florida Research and Education Center (NFREC) in Quincy,
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FL, during August of 2008 (F2:3 generation) and August of 2009 (F4:5 generation). The
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remaining 123 lines were not included because the number of seeds available was insufficient
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to plant replicated experiments. The purpose of the late field planting was to delay flowering
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until the late summer and early fall, when temperatures and rainfall in north-central Florida
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typically become more favorable for pathogen population development. The parents of the
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cross, as well as PI 200492 (Rpp1), PI 230970 (Rpp2), PI 462312 (Rpp3), and PI 459025B
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(Rpp4) were also included in the tests.
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A randomized complete block design (RCBD) with three replications was used, with
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single-row plots 1.2 m in length and 90 cm apart. Plots were scored for SBR disease severity
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and infection type in November 2008 and in November 2009, when plants in the row were at
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reproductive growth stages R5-R6 (Fehr et al. 1971). A six-point SBR severity scoring scale
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described by Walker et al. (2011) was modified to a 1 to 6 scale in which 1 = no visible lesions;
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2 = light severity in the bottom of the canopy; 3 = light to moderate lesions in the middle
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portion of the canopy; 4 = moderate lesions in the middle portion of the canopy; 5 = abundant
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lesions in the middle of and top of the canopy; and 6 = very high disease severity, equivalent to
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susceptible checks.
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Infection types were classified in the field tests using the criteria described by Bonde et
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al. (2006) and Bromfield et al. (1980). Resistant genotypes had either an immune reaction
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characterized by a lack of obvious symptoms, or reddish brown (RB) lesions, which are
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considered indicative of incomplete resistance. The susceptible genotypes had a tan (TAN)
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infection type, characterized by the presence of abundant urediniospores. Lines with a mixture
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of TAN and RB plants within the same row were considered to be segregating for the resistance
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gene. On the basis of the SBR severity scores and infection types observed, plants were rated as
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resistant or susceptible.
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Growth chamber assay. A growth chamber assay using a purified P. pachyrhizi isolate was
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also conducted to phenotype the 253 F5:6 RIL lines. This assay was conducted in a Conviron
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Model PGR 15 growth chamber (Conviron Inc., Pembina, ND) at the USDA-ARS facility at
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Stoneville, MS in January 2013. The parental lines and the same PIs used as controls in the
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field tests were also included in the growth chamber assay, as well as PI 567102B (Rpp6) (Li et
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al. 2012), which originated from Indonesia, like PI 567104B.
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To prepare the population for screening in the growth chamber, two seeds of each F5:6
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RIL were planted in individual Jiffy Poly-Pak TM pots (Hummert, St. Louis, MO), which were
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placed in a 27 × 52 cm flat that contained 5 × 10 pots. Autoclaved Metro Mix TM 360 medium
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(Sun Grow Horticulture Products, Belleview, WA) was used for filling the pots. The seed were
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planted in the Agronomy Greenhouse at Iowa State University in Ames, IA, on 2 Jan. 2013.
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Each line was planted in a randomized complete block design (RCBD) with three replications,
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and plants were watered daily. One week after planting, when the seedlings were at the VE stage
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(Fehr et al. 1971), each line was thinned to one plant per pot. On 10 Jan. the plants were
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transported to the USDA laboratory in Stoneville, MS, where they were maintained in a
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Conviron Model PGR 15 growth chamber (Conviron Inc., Pembina, ND) under a 16-h
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photoperiod, with a light intensity of 433 µE m-2 s-1 at 25°C for continuous growth.
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Inoculum was prepared using a P. pachyrhizi single-uredinium-derived isolate from
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Mississippi (MS06-1B) that was originally isolated from field-collected kudzu [Pueraria lobata
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(Wild.) Ohwi] leaves in 2006 (Li 2009; Li et al. 2007, 2012). The identity of MS06-1B had
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previously been confirmed by microscopy, enzyme-linked immunosorbent assay and
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polymerase chain reactions as previously described (Li et al. 2007). Urediniospores were
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increased on the susceptible soybean cultivar Williams 82 (Bernard and Cremeens 1988) at the
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Stoneville Research Quarantine Facility in Mississippi (Li 2009). Urediniospores of MS06-1B
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were harvested using a Cyclone Surface SamplerTM (Burkard Manufacturing Co. Ltd. UK).
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Inoculation was performed on 21-day old seedlings using freshly collected urediniospores from
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Williams 82 plants. Spore suspensions were made using sterile distilled water containing 0.01%
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Tween TM 20 (Sigma-Aldrich, St. Louis, MO). The suspensions were mixed and then filtered
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through a 100-µm cell strainer (BD Biosciences, Bedford, MA) to remove any debris, and
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clumps of urediniospores. Urediniospore concentrations were quantified using a
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hemocytometer and were diluted to a final concentration of 40,000 urediniospores ml-1.
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Inoculation was at the rate of 1 ml of spore suspension per plant, and the plants were inoculated
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using a Preval sprayer (Yonkers, NY). After inoculation, plants were placed in a dew chamber
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in the dark at 22°C overnight (during 16 h), and then moved to a Conviron Model PGR 15
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growth chamber (Conviron Inc., Pembina, ND) in which temperatures were maintained at 23°C
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during the day and 20°C at night. The plants were grown under a 16-h photoperiod and a light
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intensity of 280 µE m-2 s-1.
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Assessment of infection types was made 14 days after inoculation (DAI). Disease
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severity was evaluated on the basis of the reaction of the first trifoliate leaf on every plant, as
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described by Li (2009). A modified version of the five-point lesion density scale described by
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Miles et al. (2006) was used. The modification involved the inclusion of percentage of infected
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area as part of the scale, such that 1 = no visible lesions; 2 = few lesions (1–20% infected area);
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3 = moderate lesion density (21–50% infected area); 4 = heavy lesion density (51–80% infected
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area); and 5 = very heavy lesion density over most of the leaf (81–100% infected area). This
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rating scale was functionally similar to the six-point severity scale used for the field assays, but
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ratings in this case were made on a few leaves that had been manually inoculated, whereas the
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field ratings took into consideration lesion distribution in the plant canopy as well as lesion
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density. The infection types on each soybean line were also recorded and classified as
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previously described by Bonde et al. (2006) and Bromfield (1984). When a mixture of TAN and
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RB infection types were observed on the same leaf, the reaction was classified as mixed (MIX).
6
7
Assay to determine the degree of dominance of the resistance gene in PI 567104B. Putative
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F1 hybrids from two different crosses were assayed to determine the degree of dominance of the
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Rpp gene present in PI 567104B. Three putative F1 plants from the cross Osage × PI 567104B
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and 12 putative F1 plants from a cross between the University of Georgia line G00-3880 and PI
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567104B (provided by Z. Li, University of Georgia, Athens, GA) were inoculated with the
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MS06-1B isolate using the same methods that had been used to inoculate the RIL population.
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Osage and G00-3880 were included in the assays to confirm that the isolate induced a
14
susceptible reaction on those susceptible parents. The Osage × PI 567104B plants were
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genotyped with the polymorphic SSR marker Satt460 (linked to the Rpp3 locus on Chr 6) to
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confirm that they were hybrids. The plants were rated for rust severity and sporulation using
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scales of 1 to 5, lesion type (TAN, RB or IM) was also recorded.
18
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DNA isolation and genotyping
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At Iowa State University, three seeds of each of the 253 F5:6 RILs, in addition to seeds from the
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two parents and from PIs carrying previously reported genes (Rpp1-Rpp4 and Rpp6), were
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planted in individual Styrofoam cups (240 ml in size) containing 180 ml of Metro Mix TM 360
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medium (Sun Grow Horticulture Products, Belleview, WA). The seeds were placed on the
24
surface of the potting mix and covered with an additional 60 ml of the same medium. Each line
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was planted once, and the planting order of the lines followed the randomization order of a
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randomized complete block design (RCBD). At plant stage V3 (Fehr et al. 1971), a single
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trifoliate leaflet from each of the three plants of each line was collected, frozen in liquid
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nitrogen, and stored at -80°C until they were lyophilized. The sample leaflets from each line
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were ground by hand into a fine powder using a mortar and pestle after pouring liquid nitrogen
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on lyophilized leaf tissues. Pulverized tissue (2-3 ml) from each line was placed into a 15 ml
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FalconTM conical centrifuge tube (BD Biosciences, Bedford, MA), and DNA was extracted
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using the CTAB (hexadecyltrimethy ammonium bromide) protocol of Keim et al. (1988). A
7
similar method was used to extract DNA from the putative F1 plants to confirm the hybrid
8
nature of the plants. Relative concentrations of extracted DNA samples were determined by
9
band intensity following electrophoresis and were adjusted through dilution prior to PCR
10
11
amplification.
To determine whether the resistance gene in PI 567104B was at any of the previously
12
reported Rpp gene loci (Rpp1-Rpp6), 33 SSR markers linked to these six loci (Supplemental
13
Table 1), were used to genotype the two parental lines and also the same 130 F5:6 RILs that had
14
been descended from the F2-derived lines included in the 2008 and 2009 field tests. Of the 33
15
SSR markers, 12 were polymorphic between the two parents of the population. Williams 82
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was used as positive control for amplification. The 12 polymorphic markers were distributed on
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Chr 3 (LG N), with two markers flanking the Rpp5 locus; on Chr 6 (LG C2), with two markers
18
flanking the Rpp3 locus; on Chr 16 (LG J), with two markers flanking the Rpp2 locus; and on
19
Chr 18 (LG G), with two markers flanking the Rpp1 locus. In addition, two markers linked to
20
the Rpp4 locus and two markers surrounding the Rpp6 locus were also used for a total of 12
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SSR markers (Supplemental Table 1). The PCR primer sequences for the markers were
22
obtained from SoyBase (http://soybase.org/resources/ssr.php).
23
After evidence was found for a statistically significant association between resistance
24
and the genotype at some markers on Chr 18 (LG G), 19 polymorphic SSR markers from the
25
region between 24.96 cM (Satt235) and 104.01 cM (Sat_372) on Chr 18 (SoyBase
1
http://soybase. org/resources/ssr.php), were used to genotype the 130 F5:6 RILs (Supplemental
2
Table 2). Genotypic data from 19 of the markers were then used to construct a linkage map for
3
that region of Chr 18. Later one of the SSR marker Sat_315 was omitted due to distorted
4
segregation in the populations amounting to 18 SSR markers.
5
To further narrow down the possible location of the Rpp gene in PI 567104B, 64 SSR
6
markers between the region Sat_131 (at 31.33 cM in the Song et al. 2004 map) and Satt394 (at
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43.38 cM) were tested (Supplemental Table 3). The 12 polymorphic markers identified within
8
the group of the 64, were then used to genotype the set of 130 F5:6 RILs and the two parents
9
(Supplemental Table 3). The primer sequences and the genomic locations of the SSR markers
10
were obtained from the BARCSOYSSR_1.0 soybean SSR database
11
(http://soybase.org/BARCSOYSSR/index.php) (Song et al. 2010). Among the polymorphic
12
markers were BARCSOYSSR_18_0331 (abbreviated to “BARC331” in tables and figures) and
13
BARCSOYSSR_18_0380 (abbreviated to “BARC380” in tables and figures).
14
BARCSOYSSR_18_0331 (r = 0.905, P<0.001) and BARCSOYSSR_18_0380 (r = 0.892, P <
15
0.001) were identified as having the highest significant Pearson correlation coefficient values
16
with the phenotypic data of the 253 RILs (data not shown).
17
Polymerase chain reaction (PCR) amplifications were performed in 10 µl reaction
18
volumes on a MyCycle™ Cycler (Biorad, Hercules, CA) using the cycling conditions
19
recommended for specific simple sequence repeat (SSR) primer pairs (Cregan et al. 1994;
20
Narina et al. 2011). The PCR program consisted of an initial denaturation step at 94oC for 2
21
minutes, following by 40 cycles of 94oC for 30s, a primer annealing step at temperatures
22
between 47 and 57oC, depending on the optimum annealing temperature for each primer pair,
23
and 30 seconds of extension at 72oC. A touchdown protocol was used for some primer pairs.
24
The PCR programs ended with a final 10-minute extension at 72oC. The PCR products were
25
analyzed on a 4% agarose gel stained with ethidium bromide as indicated by McMullen (2003).
1
Molecular biology grade high resolution agarose (Low EEO; DNase and RNase free; Fisher
2
Scientific, Pittsburgh, PA) was used for preparing the gels.
3
4
5
Genetic mapping and statistical analysis
Since the experimental error in the growth chamber assay was expected to be lower than
6
that of the field assays, the genotypic and phenotypic data for the F5:6 RILs in the growth
7
chamber experiment were analyzed for genotype-phenotype associations using a two-way
8
contingency table (Gilula 1983). The association between molecular marker genotype and
9
disease severity ratings was tested by Fisher’s exact test (Sokal and Rohlf 1995) using PROC
10
FREQ procedure of SAS ver. 9.2 (SAS institute, Cary, NC). After significant associations were
11
found with markers on Chr18, a genetic linkage map was created using JoinMap 4.0 (Van
12
Ooijen 2006). A LOD significance threshold of above 3 was used to cluster the markers into a
13
linkage group following elimination of any marker loci with segregation distortion (P < 0.001).
14
Recombination frequencies were converted to genetic distances using the Kosambi (1944)
15
mapping function.
16
In order to construct this genetic map, in addition to the 18 polymorphic SSR markers
17
described before between the region 24.96 cM (Satt235) and 104.01 cM (Sat_372), two of the
18
highly significant markers, BARCSOYSSR_18_0331 and BARCSOYSSR_18_0380 were also
19
used to genotype the 130 F5:6 RILs. In total 20 SSR markers were used finally for genotyping
20
the lines (Supplemental Fig. 1).
21
Quantitative trait loci (QTL) analysis was conducted to confirm and map the possible
22
location of the Rpp gene on Chr 18 in PI 567104B by using the composite interval mapping
23
(CIM) function in Windows QTL Cartographer V2.5 (Wang et al. 2007), with a step size of 1
24
cM. A LOD significance threshold at the P = 0.01 level was obtained on the basis of 1000
25
random permutations of the phenotypic and genotypic data. QTL analysis was done using
26
growth chamber assay data from 114 F5:6 RILs that descended from the same F2:3 and F4:5 lines
1
that previously were rated in the field experiments of 2008 and 2009, respectively. Data from
2
16 of the 130 lines were not used because they had haplotypes that could only be explained by
3
double crossovers between tightly linked markers, an event that rarely may occurs in plants
4
Kosambi (1944). Since inaccuracies in genotyping were a more plausible explanation for the
5
“mosaic” haplotypes (varying fungus population between 2008 and 2009 natural infection), and
6
the 16 lines were omitted from the mapping analysis. The total number of lines included in the
7
CIM analysis was therefore 114.
8
Statistical analyses were conducted to calculate heritability estimates in the broad sense
9
(Chapman et al. 2003; Lewers et al. 1999; SAS 2009, NC). In addition, Skewness and Kurtosis
10
values were obtained from the Excel calculations (Microsoft 2003). The heritability estimates
11
were calculated for each field experiment conducted at Quincy, Florida, which had been planted
12
as indicated in a randomized complete block design with three replications. Replications and
13
lines were considered random effects. For the heritability estimates in the growth chamber
14
experiment conducted at Stoneville, Mississippi, the experimental arrangement was that of a
15
completely randomized design, with three replications. In the growth chamber environment,
16
lines were also considered random effect.
17
18
19
Results
20
PI 567104B was highly resistant to the P. pachyrhizi field populations in north-central Florida,
21
U.S.A., in 2008 and 2009, while IAR 2001 BSR was highly susceptible (Table 1). Accessions
22
with different Rpp genes that were included in the test had variable reactions, depending on the
23
gene and the year of evaluation. All accessions with known genes had higher SBR severity in
24
2008, indicating that there was more disease pressure in 2008 or that the fungal population
25
included pathotypes that were more virulent or aggressive on plants with resistance genes. PI
26
200492 (Rpp1) was highly resistant both years, while SBR severity on PI 459025B (Rpp4) was
Phenotypic evaluation of resistance to SBR
1
similar to the susceptible parent IAR 2001 BSR in 2008, and more resistant to it in 2009. The
2
Rpp2 and Rpp3 accessions appeared to have been more susceptible to the pathotypes present or
3
most abundant in 2008. The higher disease severity on IAR 2001 BSR in 2008 also suggests
4
that environmental conditions were more favorable for the pathogen in 2008 or that the strains
5
of the pathogen were more aggressive that year. The disease severity ratings of the progeny
6
lines showed a similar pattern to that of the parents and accessions with known genes, having
7
differential interactions between the resistance gene(s) and the P. pachyrhizi field populations in
8
each of the two years (Table 1, Fig. 1).
9
The broad heritability values were similar in both years, although the highest value was
10
observed in 2008. The observations agree with the information previously mentioned, in that
11
variations in disease pressure and on the pathotypes in the fungal populations might be the
12
cause of the variability in disease reactions. The Skewness values which characterize the
13
degree of asymmetry of the frequency distribution of the segregating lines around the mean,
14
indicated that in each of the years the distributions tended to be bimodal (Fig. 1). In 2009 the
15
frequency of resistant lines appeared higher than in 2008. Again, an indication of the variability
16
and or aggressiveness of the Asian soybean rust fungal populations in each of the years. The
17
Kurtosis values also indicated departure of the observed frequency distributions from normality.
18
In the year 2009 the departure from normality was more pronounced than in 2008.
19
Disease severity on most of the F2:3 and F4:5 lines in the field assays was intermediate
20
between rust severity of the two parents (Fig. 1). The progeny lines did not fall into discrete
21
phenotypic classes. The distribution of the quasi-quantitative rating data impeded to carry out a
22
Chi-square test to determine the likely number of segregating Rpp genes. The F4:5 field data
23
from 2009 formed two relatively distinctive peaks close to the mean severities of each the
24
parents of the cross.
25
The initial concerns about experimental error in the field data prompted the decision to
1
conduct the growth chamber assay with highly inbred soybean lines, that will have greater
2
environmental and experimental control, and was done using a single purified (i.e.,
3
pathogenically homogeneous) SBR isolate (Table 2; Fig. 2). In the growth chamber assay PI
4
567104B as well as PIs containing the Rpp1 through Rpp4 and Rpp6 genes, were classified as
5
resistant to the MS06-1B isolate, while IAR 2001 BSR was classified as susceptible (Table 2).
6
Of the 253 F5:6 RILs in the assay, the three individual plants of each of the 116 RILs were
7
resistant, the individual plants from 71 of the lines were susceptible, and 66 lines had a mixture
8
of resistant and susceptible plants (Fig. 2). The results indicate that PI 567104B requires
9
further characterization to understand the nature and durability of the resistance trait in the
10
11
accession.
The heritability value calculated under the growth chamber experiment was the highest of
12
the three environments (Table 2). Skewness had a positive value compared to field tests (Table
13
1) indicating that the asymmetry was towards the resistant side of the curve. The Kurtosis was
14
negative, and higher in absolute value than for the field experiments. This indicated that the
15
observations under the growth chamber environment had a more pronounced departure from
16
normality than in the field.
17
The reactions of the confirmed F1 plants challenged in the study showed that the Rpp
18
gene from PI 567104B is dominant. Two of the three putative F1 plants from the cross between
19
PI 567104B and Osage developed a RB-type resistant reaction to the isolate MS06-1B, while
20
the third plant developed a TAN (susceptible) reaction. The SSR marker data confirmed that the
21
susceptible plant was a self, whereas the two resistant plants were confirmed as true F1 hybrids.
22
Of the 12 putative F1 plants from the G00-3880 × PI 567104B cross, five developed RB lesions,
23
four had a TAN (susceptible) reaction, and three developed tan-colored lesions which had no
24
visible sporulation. The plants with the TAN reactions presumably resulted from self-
25
pollination of G00-3880, rather than from true F1 hybrids. The observations that the two
1
confirmed F1s from one cross developed RB reactions and that eight of the putative F1’s from
2
an independent cross also developed either RB reactions or non-sporulating tan-colored lesions
3
demonstrates the dominance of the Rpp gene from PI 567104B in its interaction with the MS06-
4
1B isolate used in the growth chamber assays.
5
6
Mapping of an Rpp gene in PI 567104B to Chr 18
7
8
Results of the Fisher’s exact test (Sokal and Rohlf 1995) using soybean rust severity
9
data from the growth chamber assay, identified the SSR markers Sat_131 (P < 0.001), and
10
Satt394 (P < 0.001), which flank the Rpp6 locus on Chr 18, as having the lowest F values
11
among all of the markers flanking the known Rpp loci (Fig. 3 and Fig. 4). The results thus
12
indicated that PI 567104B has a P. pachyrhizi resistance gene at or near the Rpp6 locus,
13
previously discovered by Li et al. (2012). The region covered by markers on Chr18 was
14
extended and a linkage map was constructed with 20 SSR markers. The polymorphic markers
15
covered the region between 21.9 cM (Satt235) and 107.7 cM (Sat_372) on the Song et al.
16
(2004) map of Chr 18 (Supplemental Fig. 1). This region includes intervals containing the loci
17
for Rpp6 (33.3 to 43.4 cM), Rpp4 (76.8 to 80.4) and Rpp1 (96.6 to 108.7 cM).
18
The maximum LOD score occurred in nearly the identical location on the linkage map
19
of Chr 18 when CIM was conducted using the disease severity ratings from the 2009 field assay
20
(Fig. 3), even though the lines were in an earlier generation (F4:5) than the F5:6 plants from
21
which DNA was extracted. The average percentage of lines expected to have plants
22
heterozygous at a given locus would have been 12.50% in the F4 generation and 6.25% in the
23
F5. This difference did not appear to have much of an effect on shifting the position of the
24
maximum LOD score peak when the 2009 field ratings were used.
25
26
Omission of the 16 lines resulted in linkage map interval distances similar to those
calculated in the consensus map of Chr 18 (Song et al. 2004) (Fig. 4). The results of CIM using
1
the growth chamber disease severity ratings to test for significant genotype-phenotype
2
associations involving SSR markers linked to the Rpp4 and Rpp6 loci (Supplemental Table 3)
3
indicated the presence of a major resistance gene within or adjacent to the interval flanked by
4
markers BARCSOYSSR_18_0331 and BARCSOYSSR_18_0380 (Fig. 3 and Fig. 4). This
5
corresponds to an 879 kb region in the Williams 82 reference genome (Schmutz et al. 2010),
6
and is in the region of Chr. 18 reported to contain the Rpp6 locus (Li et al. 2012).
7
8
9
Discussion
The research was conducted to identify the location of a rust-resistance (Rpp) gene(s) in
10
the accession PI 567104B, and to compare its genomic location with previously mapped Rpp
11
loci. A resistance gene locus was mapped to a region also containing the Rpp6 locus on
12
chromosome 18, the resistance gene in PI 567104B appears to be dominant. The interval
13
containing the gene(s) is flanked by the SSR markers Satt131 and Satt394, and includes the
14
SSR markers BARCSOYSSR_18_0331 and BARCSOYSSR_18_0380. The resistance gene
15
from PI 567104B is different from the known genes Rpp1 - Rpp4 previously identified.
16
Additional research will be required to establish the relation of the resistance gene in PI
17
567104B with that of the resistance gene Rpp6.
18
19
Field assessment of PI 567104B
20
The field assays conducted confirmed that PI 567104B was resistant. These and the
21
previous data collected for PI 567104B (Harris et al. 2015; Miles et al. 2006; Walker et al.
22
2011, 2014) indicate that resistance conditioned by the Rpp gene from PI 567104B may be of
23
value for breeding programs. Some caution, however needs to be exerted in the interpretation of
24
the field data. The natural development, reproduction and spread of P. pachyrhizi in the field
25
are highly influenced by the environmental conditions, among them temperature and the
1
amount and duration of moisture on the surfaces of leaves on the hosts. Additionally, the
2
pathotype composition of the pathogen field populations and the variation in the virulence of P.
3
pachyrhizi populations observed remains unknown. Even though PI 567104B has been resistant
4
to most field populations of P. pachyrhizi in the United States since 2006, low levels of
5
sporulation have sometimes been observed on field infected leaves (Harris et al. 2015; Miles et
6
al. 2006; Walker et al. 2011, 2014). These observations may suggest that resistance to some
7
endemic P. pachyrhizi pathotypes might be incomplete. These factors might have complicated
8
the efforts to classify some reactions into discrete Mendelian segregation ratios.
9
An additional problem with the field early generations tested is that although the gene
10
from PI 567104B was found dominant in the F1 progeny of crosses between PI 567104B and
11
two other susceptible elite lines, the degree of dominance over the allele from IAR 2001 BSR is
12
not known and might not have been complete. It has been observed by Garcia et al. (2011), that
13
the degree of dominance of some Rpp genes can vary considerably depending on the allele or
14
genetic background of the susceptible parent used in the cross. In the assay conducted to
15
determine whether the Rpp gene from PI 567104B was dominant, PI 567104B plants from two
16
different seed sources exhibited both IM and RB reactions. Plants from one source were
17
predominantly immune with a single plant developing RB lesions, while plants from the second
18
source were predominantly RB, with a single plant lacking any visible lesions. The results
19
obtained with a single-spore isolate under the growth chamber conditions demonstrate that even
20
the reactions of homozygous PI 567104B plants to a purified P. pachyrhizi isolate can vary to
21
some degree. Additionally all of the putative F1 plants that had a resistance reaction developed
22
RB lesions, none were immune.
23
Incomplete dominance could in part account for the continuous distribution of the
24
reaction that was observed in the field assays, particularly if PI 567104B was itself
25
incompletely resistant to the field populations of the fungus. A high percentage of the progeny
1
lines with a majority of individuals heterozygous at the resistance gene locus potentially
2
interacting with more than one pathotype in the field populations, might have resulted in the
3
large number of intermediate reactions observed in the progeny lines. Possible explanations to
4
these reactions include heterozygosity of lines or among plants within the line, heterogeneity in
5
the fungal populations combined with differential host reactions, incomplete dominance of the
6
Rpp gene(s) against some pathotypes, and even possible epistatic or genetic background effects.
7
Additionally, the plants in the field would have had lesions of varying ages, whereas the lesions
8
of the plants in the growth chamber assay were evaluated exactly two weeks after inoculation.
9
10
Novel Rpp gene from PI 567104B located in the same locus of Rpp6
11
The mapping of the Rpp gene suggests future research leads. One question is whether
12
PI 567104B has a second rust resistance gene at a different Rpp locus. In the present study, a
13
resistance gene in addition to the Rpp6 gene might be possibly located in the same region. The
14
bulked segregant analysis conducted by Harris et al. (2015), however, implicated the possible
15
presence of a resistance allele at both the Rpp4 and Rpp6 locus for the same PI. The finding
16
from the present study and the implication mentioned by Harris et al. (2015), related to the
17
occurrence of two genes at the same resistance locus had been suggested before. Two Rpp
18
genes were eventually identified in the Japanese cultivar Hyuuga (Kendrick et al. 2011). The
19
Rpp?(Hyuuga) gene was initially mapped to Chr 6, at or near the Rpp3 locus (Monteros et al.
20
2007; Monteros et al. 2010), and it was thought to be a different allele because of differences in
21
the reactions of Hyuuga and PI 462312 to certain SBR populations and isolates. When the
22
mapping population was phenotyped using a different rust isolate, a second gene was later
23
detected that mapped to Chr 3 (LG-N), at or near the Rpp5 locus (Kendrick et al. 2011). In the
24
present study after applying CIM to the field data and to the growth chamber data, there were
25
no indications of a resistance gene at the Rpp4 locus inherited from PI 567104B. Although the
26
lines were challenged with different sources of P. pachyrhizi inoculum in each assay, the actual
1
Rpp4 gene from PI 459025B has not conditioned high levels of resistance to U.S. isolates and
2
field populations. It is conceivable then, that the gene or another resistance allele at the same
3
locus could have gone undetected in assays with P. pachyrhizi isolates or strains that were able
4
to defeat the resistance conditioned by the second gene. Further research might clarify this
5
issue.
6
In the present study, the Chr 18 haplotypes of 10 random resistant and 10 random
7
susceptible F5:6 RILs at 20 SSR loci on Chr 18 showed alleles on the resistant lines of PI
8
567104B at the SSR loci around the Rpp6 locus, e.g., Sat_131, BARCSOYSSR_18_0331 and
9
BARCSOYSSR_18_0380, and Satt394. The susceptible RIL typically had susceptible parent
10
alleles at those loci. In this research, the haplotypes further support the hypothesis that a gene
11
or genes could have been inherited from PI 567104B probably located in the interval containing
12
the Rpp6 locus between 32.88 cM (Sat_131) and 43.27 cM (Satt394) of the Song et al. (2010)
13
consensus linkage map. Significant marker-phenotype associations in the remnant 123 F6 RILs
14
also confirmed the location of the gene(s).
15
The estimation of the degree of similarity between the Rpp gene(s) at or near the Rpp6
16
locus in PI 567104B and the actual Rpp6 gene from PI 567102B was not possible in this study.
17
The Rpp6 gene was identified by Li et al. (2012) after this research had been conducted.
18
Additional research, however, informed that the distance between PI 567104B and PI 567102B
19
based on the allelic difference between 34,142 SNPs genotyped with SoySNP50K Beadchip
20
was 0.23, suggesting that the genetic background of the two accessions was different (Song et
21
al. 2015). The similarity and/or dissimilarities between the two plant introductions, however,
22
will require further research including the development of a mapping population derived from
23
the cross of the two PIs, and the corresponding phenotyping under controlled conditions along
24
with the genomic characterization of the segregating progeny. It is important to note that the
25
two accessions were obtained from the same research station in Indonesia, and they resemble
1
one another in morphological traits. These facts indicate that it is important to elucidate the
2
similarity/dissimilarity nature of the genetic resistance to SBR of each of the two accessions.
3
The research communicated and conducted on PI 567104B, both in field and growth
4
chamber experiments indicate that PI 567104B is an important source of SBR resistance.
5
Already PI 567104B has been crossed to several elite lines adapted to the southern and northern
6
USA, and agronomical promising rust-resistant lines selected from the populations could
7
become useful sources of SBR resistance rather than the use of the original plant introduction.
8
9
Conflict of Interest:
10
The authors declare that they have no conflict of interest.
11
12
Acknowledgments
13
The study was funded by grants from the Iowa Soybean Association (ISA), ISU Project 4403
14
and the United Soybean Board (USB), Grant # 528, ISU Project Number 4403. The study was
15
also partially supported by the USDA-ARS Projects 6402-21220-012-00D, Crop Genetics
16
Research Unit, Stoneville, Mississippi, and by the Home Economic and Agricultural
17
Experiment Station, College of Agriculture, Iowa State University. Appreciation is extended to
18
the following collaborators of Iowa State University: Dr. Michelle Graham (ARS-USDA), and
19
to graduate students Chantal Liepold, Alexander Luckew, Jordan Baumbach and to Assistant
20
Researchers Peter Lundeen for their help, advice and support during the conduct of this work,
21
to Gregory G. Gebhart for driving the plants to the USDA lab in Stoneville, Mississippi for
22
SBR resistance screening, and to Nieves Rivera-Velez, for the scoring of the plants in the field
23
tests conducted at Quincy FL. We also appreciate the help that David Wright and Jim Marois of
24
the University of Florida provided in planting and managing the plots in Quincy, FL., and to Dr.
25
Zenglu Li, Dr. Donna Harris, of the University of Georgia for providing molecular markers, and
26
F1 hybrid seed obtained with PI 567104B. Appreciation is also extended to Drs. James Buck,
27
Dan Phillips, and Roger Boerma of the University of Georgia for providing suggestions and
28
reviews of the manuscript. Our gratitude also goes out to Drs. Perry Cregan and David Hyten,
29
who were both at the USDA-ARS Soybean Genomics and Improvement Laboratory in
30
Beltsville, MD at the time this research was conducted, and provided support in the molecular
1
analysis of the RILs.
2
3
4
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