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Theor Appl Genet (2016) 129:863–877DOI 10.1007/s00122-015-2651-5

ORIGINAL ARTICLE

Identification of a soybean rust resistance gene in PI 567104B

Min Liu1,2 · Shuxian Li3 · Sivakumar Swaminathan1 · Binod B. Sahu1 · Leonor F. Leandro4 · Andrea J. Cardinal5,6 · Madan K. Bhattacharyya1 · Qijian Song7 · David R. Walker8 · Silvia R. Cianzio1

Received: 5 August 2015 / Accepted: 8 December 2015 / Published online: 7 March 2016 © Springer-Verlag Berlin Heidelberg (outside the USA) 2016

Abstract Asian soybean rust (SBR), caused by the fun-gus Phakopsora pachyrhizi Syd. and P. Syd., is one of the most economically important diseases that affect soybean production worldwide. A long-term strategy for minimiz-ing the effects of SBR is the development of genetically resistant cultivars. The objectives of the study were to iden-tify the location of a rust-resistance (Rpp) gene(s) in plant introduction (PI) 567104B, and to determine if the gene(s) in PI 567104B was different from previously mapped Rpp loci. The progeny of the cross of ‘IAR 2001 BSR’ × PI 567104B was phenotyped from field assays of the F2:3 and F4:5 generations and from a growth chamber assay of 253 F5:6 recombinant inbred lines (RILs). For the growth cham-ber, the phenotyping was conducted by inoculation with a purified 2006 fungal isolate from Mississippi. A resistance gene locus on PI 567104B was mapped to a region con-taining the Rpp6 locus on chromosome 18. The high level of resistance of F1 plants from two other crosses with PI 567104B as one of the parents indicated that the gene from PI 567104B was dominant. The interval containing the gene is flanked by the simple sequence repeat (SSR) mark-ers Satt131 and Satt394, and includes the SSR markers BARCSOYSSR_18_0331 and BARCSOYSSR_18_0380. The results also indicated that the resistance gene from PI 567104B is different from the Rpp1 to the Rpp4 genes previously identified. To determine if the gene from PI 567104B is different from the Rpp6 gene from PI 567102B, additional research will be required.

Abstract Key message Using a combination of phenotypic screening and molecular, statistical, and linkage analy-ses, we have mapped a dominant soybean rust resist-ance gene in soybean PI 567104B.

Communicated by V. Hahn.

M. Liu and S. Li contributed equally and should be considered co-first authors.

The use of trade, firm, or corporation names on this page is for the information and convenience of the reader. Such use does not constitute an official endorsement of approval by the USDA Agricultural Research Service, NAL or BIC of any product or service to the exclusion of others that may be suitable.

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Electronic supplementary material The online version of this article (doi:10.1007/s00122-015-2651-5) contains supplementary material, which is available to authorized users.

* David R. Walker david.walker@ars.usda.gov; walkerdr@illinois.edu

* Silvia R. Cianzio scianzio@iastate.edu

1 Department of Agronomy, Iowa State University, Ames, IA 50011-1010, USA

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AbbreviationsDAI Days after inoculationLG Linkage groupMG Maturity groupPI Plant introductionRB Reddish brown lesionsRCBD Randomized complete block designRILs Recombinant inbred linesSBR Asian soybean rustTAN Tan lesionsIM Immune

Introduction

Asian soybean rust (SBR), caused by Phakopsora pach-yrhizi Syd. and P. Syd., is one of the most economi-cally important soybean [Glycine max (L.) Merr.] dis-ease worldwide, capable of causing yield losses ranging from 10 to 80 % (Kawuki et al. 2003; Kumudini et al. 2008; Sinclair and Hartman 1999). When environmental conditions favor pathogen development on susceptible genotypes, rust lesions can cover most of the leaf sur-face, at times occurring on the stem and pods as well (Sinclair and Hartman 1999). Premature defoliation reduces photosynthesis and the number of days to matu-rity (Kumudini et al. 2008). SBR-infected plants may have few pods and light seeds which are often of poor quality.

P. pachyrhizi was first identified in Japan in 1902 (Hen-nings 1903). It is endemic to eastern Asia and possibly northern Australia, but over time it has gradually spread to soybean-growing countries around the world (Bromfield

1984; Pretorius et al. 2001; Rossi 2003; Wang and Hart-man 1992; Yorinori et al. 2005). It was first reported in the USA in Hawaii in 1994 (Killgore and Heu 1994), and in the continental USA in Louisiana in 2004 (Schneider et al. 2005). SBR has been reported in up to 20 other states in the USA in some growing seasons, and it is also established in Mexico and all of the soybean producing countries in South America (Hershman et al. 2011; Isard et al. 2005). P. pachyrhizi possesses a diverse and complex pathogenicity, infecting a wide range of legume genera (Ono et al. 1992; Slaminko et al. 2008a, b).

Although sources of SBR resistance have been known since the 1970s, no USA cultivars are known to be resist-ant (Li et al. 2012). Management of SBR in regions and countries where P. pachyrhizi is present relies on the application of fungicides, and sometimes on the use of early-maturing soybean cultivars to limit economic losses (Raetano et al. 2011). Although timely fungicide applica-tions can be effective for managing SBR, they add to pro-duction costs and suppress beneficial fungi (e.g., those that parasitize insect pests) as well as pathogenic ones. In trop-ical production areas, multiple fungicide applications may be required during a growing season (Godoy 2012). In addition, some fungicides cause soybean stems to remain green after the pods have matured, which can cause delays in harvest (Bob Kemerait, University of Georgia, personal communication, 2013). Sustainable management of SBR epidemics is most likely to succeed if genetic resistance to the pathogen can be combined with judicious use of fungicides.

Six loci with dominant resistant SBR alleles from dif-ferent G. max plant introductions (PIs) have been reported, and multiple resistance alleles have been found at some of the loci (Chakraborty et al. 2009; Garcia et al. 2008, 2011). The Rpp1 gene was identified in PI 200492 (McLean and Byth 1980), and was mapped on chromosome 18 (Link-age Group G; LG G) between SSR markers Sat_187 and Sat_064 (Hyten et al. 2007). The Rpp2 gene was identi-fied in PI 230970 (Bromfield and Hartwig 1980), and was later mapped to chromosome 16 (LG J) (Silva et al. 2008). The Rpp3 gene was identified in PI 462312 (Bro-mfield and Melching 1982; Hartwig and Bromfield 1983), and was subsequently mapped to chromosome 6 (LG C2) (Hyten et al. 2009), while the Rpp4 gene was identified in PI 459025B (Hartwig 1986) and mapped to a different location on chromosome 18, approximately 26 cM from Rpp1 (Silva et al. 2008), based on the marker locations in the Song et al. (2004) integrated linkage map. An Rpp gene in PI 200526 and different resistance alleles from other germplasm accessions were mapped to the Rpp5 locus on chromosome 3 (LG N) (Garcia et al. 2008). In 2012, the Rpp6 gene was identified in PI 567102B and mapped to a third Rpp locus on chromosome 18 approximately 40 cM

2 Present Address: Department of Agronomy, Shenyang Agricultural University, 120 Dongling Ave, Shenyang, Liaoning 110866, China

3 Crop Genetics Research Unit, USDA-ARS, Stoneville, MS 38776, USA

4 Plant Pathology Department, Iowa State University, Ames, IA 50011-1010, USA

5 Department of Agronomy, North Carolina State University, Raleigh, NC 27695, USA

6 Present Address: Genetic Projects Lead Vegetables Seeds R&D Syngenta Biotechnology Inc, 3054 Cornwallis Rd. Research Triangle Park, Durham, NC 27709, USA

7 USDA-ARS, Soybean Genomics and Improvement Laboratory, Beltsville Agricultural Research Center, Beltsville, MD 20705, USA

8 USDA-ARS, Soybean/Maize Germplasm, Pathology and Genetics Research Unit, and Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

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from the Rpp4 locus and about 66 cM from the Rpp1 locus (Li et al. 2012). The Japanese cultivar Hyuuga was found to have an Rpp gene, Rpp? (Hyuuga), at or near the Rpp3 locus on chromosome 6 (Monteros et al. 2007, 2010). A second resistance gene from Hyuuga was later mapped to the Rpp5 locus on chromosome 3 (Kendrick et al. 2011). Garcia et al. (2008) discovered recessive resistance genes at different loci in PI 224270 (rpp2 [?]) and in PI 200456 (rpp5). In addition to the six loci thus far reported in the lit-erature, patents have been claimed on at least 10 other loci associated with SBR resistance (Bailey et al. 2014), though the use of proprietary markers to map these genes makes it difficult to determine where the loci are located relative to markers frequently used in the public sector.

Resistance reactions are typically characterized by the development of reddish-brown (RB) lesions with two or fewer uredinia on infected leaf tissue (Bromfield et al. 1980). Immune, or Type 0 reactions are less common, but occur in certain pathotype-Rpp gene interactions, typi-cally involving Rpp1. In contrast, susceptible soybean hosts typically develop tan (TAN) reactions with three or more uredinia per lesion and a tan color resulting from the lesions and/or clumps of urediniospores. The resistance of soybean genotypes to P. pachyrhizi can vary tempo-rally and geographically (Akamatsu et al. 2013; Paul et al. 2013; Twizeyimana and Hartman 2012; Walker et al. 2011, 2014). Durable resistance would provide an economic and environmentally friendly way to protect soybean crops from SBR, but it might likely require pyramiding two or more Rpp genes each effective against a majority of the P. pachyrhizi 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 novel genes that would condition broader or more durable resistance in pyramids with other Rpp genes (Khanh et al. 2013; Miles et al. 2006). Field evaluations of germplasm in several states in the southern USA (Walker et al. 2011, 2014), and in coun-tries in South America (Miles et al. 2008) showed that PI 567104B has high levels of resistance to many populations of P. pachyrhizi. The resistance of accession PI 567104B to P. pachyrhizi populations from North and South Amer-ica is somewhat unusual, since to date many of the acces-sions found to be resistant in South America (Miles et al. 2008) were susceptible to rust populations in the southern USA (Walker et al. 2011, 2014). The SBR resistance of PI 567104B could therefore be of value for the develop-ment of SBR-resistant soybean cultivars. The objectives of this study were to identify the location of a rust-resistance (Rpp) gene or genes in PI 567104B, and to determine if the gene(s) in PI 567104B was different from previously mapped Rpp loci.

Materials and methods

Plant material

A population was developed from the cross of ‘IAR 2001 BSR’ × PI 567104B. IAR 2001 BSR is a high-yielding SBR-susceptible cultivar released by Iowa State Univer-sity-ISURF, Docket # 03542 (Cianzio et al. 2008). PI 567104B is a MG IX accession from Indonesia (Germ-plasm Resources Information Network, http://www.ars-grin.gov/cgi-bin/npgs/acc/). The accession was identified by Miles et al. (2006) to be resistant to a mixture of four P. pachyrhizi isolates from Brazil, Paraguay, Thailand and Zimbabwe. It was also resistant to a local field SBR popu-lation at Capitán Miranda, Paraguay (Miles et al. 2008). In the USA, PI 567104B was highly resistant to field popula-tions in the states of Alabama, Florida, Georgia, Louisiana, and 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 State University research site located at the University of Puerto Rico’s Isabela Sub-station (ISU-PR). It was part of a group of crosses made to transfer P. pachyrhizi resistance to high-yielding elite soybean lines of early maturity groups. For AX20871, six F1 seeds were obtained and planted in June 2006 in Puerto Rico. Each F1 plant was identified and harvested individu-ally in September 2006. The identity of the individual F1 plants was maintained throughout the study even though the hybrid nature of the F1 plants was confirmed using flower and pubescence colors as morphological mark-ers. After additional results confirming PI 567104B field resistance to SBR (Miles et al. 2006, 2008), the F2 seeds of the population which had been maintained in cold stor-age, were planted in Puerto Rico in October 2007, and each individual F2 plant was identified prior to harvest in Janu-ary 2008. A total of 253 F2:3 lines were obtained for the population and were maintained in cold storage until the next planting. One hundred and thirty F2:3 and F4:5 lines were used in the 2008 and 2009 field assays described in the next section.

F5:6 recombinant inbred lines (RILs) were developed at the ISU-PR research site by advancing the 253 original F2:3 lines through single-plant selections of each line from 2008 to 2011, when each RIL had reached the F6 generation. The RILs were screened for their reactions to a 2006 P. pachy-rhizi isolate from Mississippi (MS06-1B) in the growth chamber study described below. Genotypic and phenotypic data from 114 of the 130 F4:5 lines phenotyped in the field in 2008 and 2009, and of the 253 F5:6 RILs phenotyped in the growth chamber were 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 from two other crosses with the PI as the SBR-resistant parent were tested for their reactions to the MS06-1B isolate. Three putative F1s from a cross of ‘Osage’(Chen et al. 2007) × PI 567104B and 12 putative F1s from a cross between the University of Geor-gia breeding line G00-3880 and PI 567104B were evalu-ated in 2014 at Stoneville, MS with the same methods used for the growth chamber assay of the RILs.

P. pachyrhizi resistance evaluations

Field evaluations

The 130 F2-derived lines mentioned in the previous section were planted at the University of Florida’s North Florida Research and Education Center (NFREC) in Quincy, FL, during August of 2008 (F2:3 generation) and August of 2009 (F4:5 generation). The remaining 123 lines were not included because the number of seeds available was insuffi-cient to plant replicated experiments. The purpose of the late field planting was to delay flowering until the late summer and early fall, when temperatures and rainfall in north-cen-tral Florida typically become more favorable for pathogen population development. The parents of the cross, as well as PI 200492 (Rpp1), PI 230970 (Rpp2), PI 462312 (Rpp3), and PI 459025B (Rpp4) were also included in the tests.

A randomized complete block design (RCBD) with three replications was used, with single-row plots 1.2 m in length and 90 cm apart. Plots were scored for SBR dis-ease severity and infection type in November 2008 and in November 2009, when plants in the row were at reproduc-tive growth stages R5-R6 (Fehr et al. 1971). A six-point SBR severity scoring scale described by Walker et al. (2011) was modified to a 1–6 scale in which 1 = no vis-ible lesions; 2 = light severity in the bottom of the canopy; 3 = light to moderate lesions in the middle portion of the canopy; 4 = moderate lesions in the middle portion of the canopy; 5 = abundant lesions in the middle of and top of the canopy; and 6 = very high disease severity, equivalent to susceptible checks.

Infection types were classified in the field tests using the criteria described by Bonde et al. (2006) and Bromfield et al. (1980). Resistant genotypes had either an immune (IM) reaction characterized by a lack of obvious symp-toms, or reddish brown (RB) lesions, which are considered indicative of incomplete resistance. The susceptible geno-types had a tan (TAN) infection type, characterized by the presence of abundant urediniospores. Lines with a mixture of TAN and RB plants within the same row were consid-ered to be segregating for the resistance gene. On the basis of the SBR severity scores and infection types observed, plants were rated as resistant or susceptible.

Growth chamber assay

A growth chamber assay using a purified P. pachyrhizi isolate was also conducted to phenotype the 253 F5:6 RIL lines. This assay was conducted in a Conviron Model PGR 15 growth chamber (Conviron Inc., Pembina, ND) at the USDA-ARS facility at Stoneville, MS in January 2013. The parental lines and the same PIs used as controls in the field tests were also included in the growth chamber assay, as well as PI 567102B (Rpp6) (Li et al. 2012), which origi-nated from Indonesia, like PI 567104B.

To prepare the population for screening in the growth chamber, two seeds of each F5:6 RIL were planted in indi-vidual Jiffy Poly-Pak™ pots (Hummert, St. Louis, MO), which were placed in a 27 × 52 cm flat that contained 5 × 10 pots. Autoclaved Metro Mix™ 360 medium (Sun Grow Horticulture Products, Belleview, WA) was used for filling the pots. The seed were planted in the Agronomy Greenhouse at Iowa State University in Ames, IA, on 2 Jan. 2013. Each line was planted in a randomized complete block design (RCBD) with three replications, and plants were watered daily. One week after planting, when the seedlings were at the VE stage (Fehr et al. 1971), each line was thinned to one plant per pot. On 10 Jan. the plants were transported to the USDA laboratory in Stoneville, MS, where they were maintained in a Conviron Model PGR 15 growth chamber (Conviron Inc., Pembina, ND) under a 16 h photoperiod, with a light intensity of 433 µE m−2 s−1 at 25 °C for continuous growth.

Inoculum was prepared using a P. pachyrhizi single-uredinium-derived isolate from Mississippi (MS06-1B) that was originally isolated from field-collected kudzu [Pueraria lobata (Wild.) Ohwi] leaves in 2006 (Li 2009; Li et al. 2007, 2012). The identity of MS06-1B had previously been confirmed by microscopy, enzyme-linked immuno-sorbent assay and polymerase chain reactions as previously described (Li et al. 2007). Urediniospores were increased on the susceptible soybean cultivar Williams 82 (Bernard and Cremeens 1988) at the Stoneville Research Quarantine Facility in Mississippi (Li 2009). Urediniospores of MS06-1B were harvested using a Cyclone Surface Sampler™ (Burkard Manufacturing Co. Ltd. UK). Inoculation was performed on 21-day old seedlings using freshly collected urediniospores from Williams 82 plants. Spore suspensions were made using sterile distilled water containing 0.01 % Tween TM 20 (Sigma-Aldrich, St. Louis, MO). The sus-pensions were mixed and then filtered through a 100 µm cell strainer (BD Biosciences, Bedford, MA) to remove any debris, and clumps of urediniospores. Urediniospore concentrations were quantified using a hemocytometer and were diluted to a final concentration of 40,000 uredinio-spores ml−1. Inoculation was at the rate of 1 ml of spore suspension per plant, and the plants were inoculated using

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a Preval sprayer (Yonkers, NY). After inoculation, plants were placed in a dew chamber in the dark at 22 °C over-night (during 16 h), and then moved to a Conviron Model PGR 15 growth chamber (Conviron Inc., Pembina, ND) in which temperatures were maintained at 23 °C during the day and 20 °C at night. The plants were grown under a 16 h photoperiod and a light intensity of 280 µE m−2 s−1.

Assessment of infection types was made 14 days after inoculation (DAI). Disease severity was evaluated on the basis of the reaction of the first trifoliate leaf on every plant, as described by Li (2009). A modified version of the five-point lesion density scale described by Miles et al. (2006) was used. The modification involved the inclusion of percentage of infected area as part of the scale, such that 1 = no visible lesions; 2 = few lesions (1–20 % infected area); 3 = moderate lesion density (21–50 % infected area); 4 = heavy lesion density (51–80 % infected area); and 5 = very heavy lesion density over most of the leaf (81–100 % infected area). This rating scale was functionally sim-ilar to the six-point severity scale used for the field assays, but ratings in this case were made on a few leaves that had been manually inoculated, whereas the field ratings took into consideration lesion distribution in the plant canopy as well as lesion density. The infection types on each soybean line were also recorded and classified as previously described by Bonde et al. (2006) and Bromfield (1984). When a mixture of TAN and RB infection types were observed on the same leaf, the reaction was classified as mixed (MIX).

Assay to determine the degree of dominance of the resistance gene in PI 567104B

Putative F1 hybrids from two different crosses were assayed to determine the degree of dominance of the Rpp gene present in PI 567104B. Three putative F1 plants from the cross Osage × PI 567104B and 12 putative F1 plants from a cross between the University of Georgia line G00-3880 and PI 567104B (provided by Z. Li, University of Georgia, Athens, GA) were inoculated with the MS06-1B isolate using the same methods that had been used to inoculate the RIL population. Osage and G00-3880 were included in the assays to confirm that the isolate induced a suscep-tible reaction on those susceptible parents. The Osage × PI 567104B plants were genotyped with the polymorphic SSR marker Satt460 (linked to the Rpp3 locus on Chr 6) to con-firm that they were hybrids. The plants were rated for rust severity and sporulation using scales of 1–5, lesion type (TAN, RB or IM) was also recorded.

DNA isolation and genotyping

At Iowa State University, three seeds of each of the 253 F5:6 RILs, in addition to seeds from the two parents and

from PIs carrying previously reported genes (Rpp1-Rpp4 and Rpp6), were planted in individual Styrofoam cups (240 ml in size) containing 180 ml of Metro Mix™ 360 medium (Sun Grow Horticulture Products, Belleview, WA). The seeds were placed on the surface of the pot-ting mix and covered with an additional 60 ml of the same medium. Each line was planted once, and the planting order of the lines followed the randomization order of a randomized complete block design (RCBD). At plant stage V3 (Fehr et al. 1971), a single trifoliolate leaflet from each of the three plants of each line was collected, frozen in liquid nitrogen, and stored at −80 °C until they were lyo-philized. The sample leaflets from each line were ground by hand into a fine powder using a mortar and pestle after pouring liquid nitrogen on lyophilized leaf tissues. Pul-verized tissue (2–3 ml) from each line was placed into a 15 ml Falcon™ conical centrifuge tube (BD Biosciences, Bedford, MA), and DNA was extracted using the CTAB (hexadecyltrimethy ammonium bromide) protocol of Keim et al. (1988). A similar method was used to extract DNA from the putative F1 plants to confirm the hybrid nature of the plants. Relative concentrations of extracted DNA sam-ples were determined by band intensity following electro-phoresis and were adjusted through dilution prior to PCR amplification.

To determine whether the resistance gene in PI 567104B was at any of the previously reported Rpp gene loci (Rpp1-Rpp6), 33 SSR markers linked to these six loci (Supple-mental Table 1), were used to genotype the two paren-tal lines and also the same 130 F5:6 RILs that had been descended from the F2-derived lines included in the 2008 and 2009 field tests. Of the 33 SSR markers, 12 were poly-morphic between the two parents of the population. Wil-liams 82 was used as positive control for amplification. The 12 polymorphic markers were distributed on Chr 3 (LG N), with two markers flanking the Rpp5 locus; on Chr 6 (LG C2), with two markers flanking the Rpp3 locus; on Chr 16 (LG J), with two markers flanking the Rpp2 locus; and on Chr 18 (LG G), with two markers flanking the Rpp1 locus. In addition, two markers linked to the Rpp4 locus and two markers surrounding the Rpp6 locus were also used, for a total of 12 SSR markers (Supplemental Table 1). The PCR primer sequences for the markers were obtained from Soy-Base (http://soybase.org/resources/ssr.php).

After evidence was found for a statistically signifi-cant association between resistance and the genotype at some markers on Chr 18 (LG G), 19 polymorphic SSR markers from the region between 24.96 cM (Satt235) and 104.01 cM (Sat_372) on Chr 18 (SoyBase http://soybase.org/resources/ssr.php), were used to genotype the 130 F5:6 RILs (Supplemental Table 2). Genotypic data from 19 of the markers were then used to construct a linkage map for that region of Chr 18. Later the SSR marker Sat_315 was

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omitted due to distorted segregation in the populations, leaving 18 SSR markers.

To further narrow down the possible location of the Rpp gene in PI 567104B, 64 SSR markers from the region between Sat_131 (at 31.33 cM in the Song et al. 2004 map) and Satt394 (at 43.38 cM) were tested (Supplemental Table 3). The 12 polymorphic markers identified within the group of 64 were then used to genotype the set of 130 F5:6 RILs and the two parents (Supplemental Table 3). The primer sequences and the genomic locations of the SSR markers were obtained from the BARCSOYSSR_1.0 soybean SSR database (http://soybase.org/BARCSOYSSR/index.php) (Song et al. 2010). Among the polymorphic markers were BARCSOYSSR_18_0331 (abbreviated to “BARC331” in tables and figures) and BARC-SOYSSR_18_0380 (abbreviated to “BARC380” in tables and figures). BARCSOYSSR_18_0331 (r = 0.905, P < 0.001) and BARCSOYSSR_18_0380 (r = 0.892, P < 0.001) were identi-fied as having the highest significant Pearson correlation coef-ficient values with the phenotypic data of the 253 RILs (data not shown).

Polymerase chain reaction (PCR) amplifications were performed in 10 µl reaction volumes on a MyCycle™ Cycler (Biorad, Hercules, CA) using the cycling conditions recommended for specific simple sequence repeat (SSR) primer pairs (Cregan et al. 1994; Narina et al. 2011). The PCR program consisted of an initial denaturation step at 94 °C for 2 min, followed by 40 cycles of 94 °C for 30 s, and a primer annealing step at temperatures between 47 and 57 °C, depending on the optimum annealing tempera-ture for each primer pair, and 30 s of extension at 72 °C. A touchdown protocol was used for some primer pairs. The PCR programs ended with a final 10 min extension at 72 °C. The PCR products were analyzed on a 4 % agarose gel stained with ethidium bromide, as indicated by McMul-len (2003). Molecular biology grade high resolution aga-rose (Low EEO; DNase and RNase free; Fisher Scientific, Pittsburgh, PA) was used for preparing the gels.

Genetic mapping and statistical analysis

Since the experimental error in the growth chamber assay was expected to be lower than that of the field assays, the genotypic and phenotypic data for the F5:6 RILs in the growth chamber experiment were analyzed for genotype-phenotype associations using a two-way contingency table (Gilula 1983). The association between molecular marker genotype and disease severity ratings was tested by Fish-er’s exact test (Sokal and Rohlf 1995) using PROC FREQ procedure of SAS ver. 9.2 (SAS institute, Cary, NC). After significant associations were found with markers on Chr18, a genetic linkage map was created using JoinMap 4.0 (Van Ooijen 2006). A LOD significance threshold of above 3 was used to cluster the markers into a linkage group

following elimination of any marker loci with segregation distortion (P < 0.001). Recombination frequencies were converted to genetic distances using the Kosambi (1944) mapping function.

To construct this genetic map, in addition to the 18 poly-morphic SSR markers described before between the region 24.96 cM (Satt235) and 104.01 cM (Sat_372), two of the highly significant markers, BARCSOYSSR_18_0331 and BARCSOYSSR_18_0380 were also used to genotype the 130 F5:6 RILs. In total 20 SSR markers were used for geno-typing the lines (Supplemental Fig. 1).

Quantitative trait locus (QTL) analysis was conducted to confirm and map the possible location of the Rpp gene on Chr 18 in PI 567104B by using the composite interval map-ping (CIM) function in Windows QTL Cartographer V2.5 (Wang et al. 2007), with a step size of 1 cM. A LOD sig-nificance threshold at the P = 0.01 level was obtained on the basis of 1000 random permutations of the phenotypic and genotypic data. QTL analysis was done using growth chamber assay data from 114 F5:6 RILs that descended from the same F2:3 and F4:5 lines that previously were rated in the field experiments of 2008 and 2009, respectively. Data from 16 of the 130 lines were not used because they had haplotypes that could only be explained by double crossovers between tightly linked markers, an event that rarely may occurs in plants Kosambi (1944). Since inac-curacies in genotyping were a more plausible explanation for the “mosaic” haplotypes (varying fungus population between 2008 and 2009 natural infection), and the 16 lines were omitted from the mapping analysis. The total number of lines included in the CIM analysis was therefore 114.

Statistical analyses were conducted to calculate herit-ability estimates in the broad sense (Chapman et al. 2003; Lewers et al. 1999; SAS 2009, NC). In addition, Skewness and kurtosis values were obtained from the Excel calcula-tions (Microsoft 2003). The heritability estimates were calculated for each field experiment conducted at Quincy, Florida, which had been planted as indicated in a rand-omized complete block design with three replications. Replications and lines were considered random effects. For the heritability estimates in the growth chamber experi-ment conducted at Stoneville, Mississippi, the experimental arrangement was that of a completely randomized design, with three replications. In the growth chamber environ-ment, lines were also considered a random effect.

Results

Phenotypic evaluation of resistance to SBR

PI 567104B was highly resistant to the P. pachyrhizi field populations in north-central Florida, USA, in 2008

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and 2009, while IAR 2001 BSR was highly susceptible (Table 1). Accessions with different Rpp genes that were included in the test had variable reactions, depending on the gene and the year of evaluation. All accessions with known genes had higher SBR severity in 2008, indicat-ing that there was more disease pressure in 2008 or that the fungal population included pathotypes that were more virulent or aggressive on plants with resistance genes. PI 200492 (Rpp1) was highly resistant both years, while SBR severity on PI 459025B (Rpp4) was similar to the suscepti-ble parent IAR 2001 BSR in 2008, and more resistant to it in 2009. The Rpp2 and Rpp3 accessions appeared to have been more susceptible to the pathotypes present or most abundant in 2008. The higher disease severity on IAR 2001 BSR in 2008 also suggests that environmental conditions were more favorable for the pathogen in 2008 or that the strains of the pathogen were more aggressive that year. The disease severity ratings of the progeny lines showed a simi-lar pattern to that of the parents and accessions with known genes, having differential interactions between the resist-ance gene(s) and the P. pachyrhizi field populations in each of the 2 years (Table 1; Fig. 1).

The broad sense heritability values were similar in both years, although a higher value was observed in 2008. The observations agree with the information previously men-tioned, in that variations in disease pressure and/or the pathotypes in the fungal populations might be the cause of the variability in disease reactions. The skewness val-ues, which characterize the degree of asymmetry of the frequency distribution of the segregating lines around the mean, indicated that in each of the years the distributions tended to be bimodal (Fig. 1). In 2009 the frequency of resistant lines appeared higher than in 2008, possibly an indication of the variability and/or aggressiveness of the Asian soybean rust fungal populations in each of the

years. The kurtosis values also indicated departure of the observed frequency distributions from normality. In 2009 the departure from normality was more pronounced than in 2008.

Disease severity on most of the F2:3 and F4:5 lines in the field assays was intermediate between rust severity on the two parents (Fig. 1). The progeny lines did not fall into discrete phenotypic classes. The distribution of the quasi-quantitative rating data impeded to carry out a Chi-square test to determine the likely number of segregating Rpp genes. The F4:5 field data from 2009 formed two relatively distinctive peaks close to the mean severities of each the parents of the cross.

The initial concerns about experimental error in the field data prompted the decision to conduct the growth chamber assay with more highly inbred soybean lines, greater envi-ronmental and experimental control, and a single purified (i.e., pathogenically homogeneous) SBR isolate (Table 2; Fig. 2). In the growth chamber assay PI 567104B, as well as PIs containing the Rpp1 through Rpp4 and Rpp6 genes, were classified as resistant to the MS06-1B isolate, while IAR 2001 BSR was classified as susceptible (Table 2). Of the 253 F5:6 RILs in the assay, the three individual plants of 116 RILs were resistant, the plants from 71 of the lines were all susceptible, and 66 lines had a mixture of resistant and susceptible plants (Fig. 2). The results indicate that PI 567104B requires further characterization to understand the nature and durability of the resistance trait in the accession.

The heritability value calculated from the growth chamber experiment was the highest of the three envi-ronments (Table 2). Skewness had a positive value com-pared to field tests (Table 1) indicating that the asym-metry was towards the resistant side of the curve. The kurtosis was negative, and higher in absolute value than for the field experiments. This indicated that the

Table 1 Average soybean rust severity of F2:3 and F4:5 lines evaluated in field experiments

Rust severity of F2:3 and F4:5 lines, parents and resistant controls evaluated in field at Quincy, Florida, during 2008 and 2009 under natural field infection. The soybean rust severity scores are the average of three replications planted in each year of screening. Score range, broad sense herit-ability (H2), skewness and kurtosis values were calculated for each year

Genotypes 2008 2009

Rust severity score/range H2 Skewness/Kurtosis Rust severity score/range H2 Skewness/Kurtosis

PI 567104B (Rpp?) 2.0/2.0–2.0 1.0/1.0–1.0

IAR2001 BSR (S) 6.0/6.0–6.0 4.5/4.0–5.0

F2:3 lines 3.6/1.5–5.5 0.745 −0.43/−0.83

F4:5 lines 2.8/1.0–5.7 0.673 −0.06/−1.40

Known resistance genes

PI 200492 (Rpp1) 1.3/1.0–2.0 1.2/1.0–1.5

PI 230970 (Rpp2) 3.7/3.5–4.0 1.3/1.0–1.5

PI 462312 (Rpp3) 4.0/4.0–4.0 1.3/1.0–2.0

PI 459025B (Rpp4) 4.8/4.5–5.0 2.8/2.0–3.5

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observations under the growth chamber environment had a more pronounced departure from normality than in the field.

The reactions of the confirmed F1 plants challenged in the study showed that the Rpp gene from PI 567104B is dominant. Two of the three putative F1 plants from the cross between PI 567104B and Osage developed a RB-type resistant reaction to the isolate MS06-1B, while the third plant developed a TAN (susceptible) reaction. The SSR marker data confirmed that the susceptible plant was a self, whereas the two resistant plants were confirmed as true F1 hybrids. Of the 12 putative F1 plants from the

G00-3880 × PI 567104B cross, five developed RB lesions, four had a TAN (susceptible) reaction, and three developed tan-colored lesions which had no visible sporulation. The plants with the TAN reactions presumably resulted from self-pollination of G00-3880, and were not true F1 hybrids. The observations that the two confirmed F1s from one cross developed RB reactions and that eight of the putative F1s from an independent cross also developed either RB reac-tions or non-sporulating tan-colored lesions demonstrates the dominance of the Rpp gene from PI 567104B in its interaction with the MS06-1B isolate used in the growth chamber assays.

Fig. 1 Frequency distribu-tion of rust severities of 130 AX20871 (‘IAR 2001 BSR’ × PI 567104B) soybean lines from field evaluations at Quincy, Florida, in 2008 (a) and 2009 (b). Arrows indicate the disease severity scores of the parents, PI 567104B and IAR 2001 BSR. The 1–6 rating scale used, was 1 = no visible disease symp-toms to 6 = severely diseased. The values are means of three biological replications. The insets show the disease severi-ties of the parental lines with the standard error bars

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Mapping of an Rpp gene in PI 567104B to Chr 18

Results of the Fisher’s exact test (Sokal and Rohlf 1995) using soybean rust severity data from the growth chamber assay identified the SSR markers Sat_131 (P < 0.001), and Satt394 (P < 0.001), which flank the Rpp6 locus on Chr 18, as having the lowest F values among all of the markers flanking the known Rpp loci (Figs. 3, 4). The results thus indicated that PI 567104B has a P. pachyrhizi resistance gene at or near the Rpp6 locus, previously discovered by Li et al. (2012). The region covered by markers on Chr18

was extended and a linkage map was constructed with 20 SSR markers. The polymorphic markers covered the region between 21.9 cM (Satt235) and 107.7 cM (Sat_372) on the Song et al. (2004) map of Chr 18 (Supplemental Fig. 1). This region includes intervals containing the loci for Rpp6 (33.3–43.4 cM), Rpp4 (76.8–80.4) and Rpp1 (96.6–108.7 cM).

The maximum LOD score occurred in nearly the identi-cal location on the linkage map of Chr 18 when CIM was conducted using the disease severity ratings from the 2009 field assay (Fig. 3), even though the lines were in an ear-lier generation (F4:5) than the F5:6 plants from which DNA was extracted. The average percentage of lines expected to have plants heterozygous at a given locus would have been 12.50 % in the F4 generation and 6.25 % in the F5. This dif-ference did not appear to have much of an effect on shift-ing the position of the maximum LOD score peak when the 2009 field ratings were used.

Omission of the 16 lines with implausible or missing marker data resulted in linkage map interval distances simi-lar to those calculated in the consensus map of Chr 18 (Song et al. 2004) (Fig. 4). The results of CIM using the growth chamber disease severity ratings to test for significant gen-otype-phenotype associations involving SSR markers linked to the Rpp4 and Rpp6 loci (Supplemental Table 3) indicated the presence of a major resistance gene within or adjacent to the interval flanked by markers BARCSOYSSR_18_0331 and BARCSOYSSR_18_0380 (Figs. 3, 4). This corresponds to an 879 kb region in the Williams 82 reference genome (Schmutz et al. 2010), and is in the region of Chr. 18 reported to contain the Rpp6 locus (Li et al. 2012).

Table 2 Average soybean rust severity of F5:6 lines evaluated in growth chamber

Rust severity of F5:6 lines, parents and resistant controls evaluated under controlled growth chamber assay at Stoneville, Mississippi, in 2013 following artificial inoculation with a local isolate of Pha-kopsora pachyrhizi. The soybean rust severity score is the average of three replications planted in growth chamber screening. Score range, broad sense heritability, skewness and kurtosis values are included

Genotypes 2013

Rust severity score/range

H2 Skewness/Kurtosis

PI 567104B (Rpp?) 1.0/1.0–1.0

IAR2001 BSR (S) 4.3/3.0–5.0

F5:6 lines 2.5/1.0–5.0 0.823 0.39/−1.48

Known resistance genes

PI 200492 (Rpp1) 1.0/1.0–1.0

PI 230970 (Rpp2) 1.3/1.0–2.0

PI 462312 (Rpp3) 1.0/1.0–1.0

PI 459025B (Rpp4) 2.0/1.0–3.0

Fig. 2 Frequency distribution of rust severities of 253, F5:6 AX20871 (‘IAR 2001 BSR’ × PI 567104B) soybean lines from a growth chamber assay at Stoneville, Mississippi in 2013. Arrows indicate the disease severity scores of the parents, PI 567104B and IAR 2001 BSR. In the 1–5 rating scale used, 1 = no visible disease symptoms and 5 = severely diseased. The values are means of three biological replications. The inset shows the disease severities of the parental lines with standard error bars

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Fig. 3 Estimated position of the novel Rpp gene for resist-ance to Asian Soybean Rust in Chromosome 18. The possible location of rust-resistance gene identified in PI 567104B through 114 RIL population and 20 SSR markers on Chr. 18 with phenotypes from 2008 field trial (a); 2009 field trial (b) and 2013 growth chamber assay (c). The X-axis shows the genetic distance in cM with SSR markers on the Chr. 18 (LG G). The position was estimated by Windows QTL Cartographer V2.5 (Wang et al. 2007) using composite interval mapping (CIM) function with a step size of 1 cM. The logarithm of odds (LOD) curve is represented on the linkage group and the peak of the LOD lies around the markers Sat_131 and BARC-SOYSSR_18_0380 (solid black arrows). LOD significance threshold at the P = 0.01 level was obtained on the basis of 1000 random permutations of the phenotypic and genotypic data. R2 value is the percentage of phenotypic variation contrib-uted by the novel Rpp locus

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Discussion

The research was conducted to identify the location of a rust-resistance (Rpp) gene(s) in the accession PI 567104B, and to compare its genomic location with previously mapped Rpp loci. A resistance gene locus was mapped to a region also containing the Rpp6 locus on chromosome 18, the resistance gene in PI 567104B appears to be dominant. The interval contain-ing the gene(s) is flanked by the SSR markers Satt131 and Satt394, and includes the SSR markers BARC-SOYSSR_18_0331 and BARCSOYSSR_18_0380. The resistance gene from PI 567104B is different from the known genes Rpp1–Rpp4 previously identified. Addi-tional research will be required to establish the relation of the resistance gene in PI 567104B with that of the resistance gene Rpp6.

Field assessment of PI 567104B

The field assays confirmed that PI 567104B was resistant. These and the previous data collected for PI 567104B (Har-ris et al. 2015; Miles et al. 2006; Walker et al. 2011, 2014) indicate that resistance conditioned by the Rpp gene from PI 567104B may be of value for breeding programs. Some caution, however, needs to be exerted in the interpretation of the field data. The natural development, reproduction and spread of P. pachyrhizi in the field are highly influenced by environmental conditions, among them temperature and the amount and duration of moisture on the surfaces of leaves on the hosts. Additionally, the pathotype composition of the pathogen field populations and the variation in the virulence of P. pachyrhizi populations observed remains unknown. Even though PI 567104B has been resistant to most field populations of P. pachyrhizi in the United States since 2006,

Fig. 4 Genomic location of the novel Asian soybean rust resistance gene in PI 567104B. The genetic linkage map was created using JoinMap 4.0 software (Van Ooijen 2006). The possible location of the Rpp gene identified on Chromosome 18 (LG G) using data from a 114 F5:6 RIL population, shown with a solid black arrow (a). The consensus map (b) (Song et al. 2004) and the physical map (c) of Chr 18 are included as reference. All of the marker information was obtained from http://www.soybase.org. The estimated locations of other Rpp genes identified earlier are also shown on the map (broken-black arrows)

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low levels of sporulation have sometimes been observed on field infected leaves (Harris et al. 2015; Miles et al. 2006; Walker et al. 2011, 2014). These observations indicate that resistance to some endemic P. pachyrhizi pathotypes is incomplete. These factors might have complicated the efforts to classify some reactions into discrete Mendelian segregation ratios.

An additional problem with the field early generations tested is that although the gene from PI 567104B was found dominant in the F1 progeny of crosses between PI 567104B and two other susceptible elite lines, the degree of domi-nance over the allele from IAR 2001 BSR is not known and might not have been complete. Garcia et al. (2011) found that the degree of dominance of some Rpp genes can vary considerably depending on the allele or genetic back-ground of the susceptible parent used in the cross. In the assay conducted to determine whether the Rpp gene from PI 567104B was dominant, PI 567104B plants from two different seed sources exhibited both IM and RB reactions. Plants from one source were predominantly immune, with a single plant developing RB lesions, while plants from the second source were predominantly RB, with a single plant lacking any visible lesions. The results obtained with a single-spore isolate under the growth chamber conditions demonstrate that even the reactions of homozygous PI 567104B plants to a purified P. pachyrhizi isolate can vary to some degree. Additionally, all of the putative F1 plants that had a resistance reaction developed RB lesions, and none were immune.

Incomplete dominance could in part account for the continuous distribution of the reaction that was observed in the field assays, particularly if PI 567104B was itself incompletely resistant to the field populations of the fun-gus. A high percentage of the progeny lines with a major-ity of individuals heterozygous at the resistance gene locus potentially interacting with more than one pathotype in the field populations, might have resulted in the large number of intermediate reactions observed in the progeny lines. Possible explanations to these reactions include heterozy-gosity of lines or among plants within the line, heteroge-neity in the fungal populations combined with differential host reactions, incomplete dominance of the Rpp gene(s) against some pathotypes, and even possible epistatic or genetic background effects. Additionally, the plants in the field would have had lesions of varying ages, whereas the lesions of the plants in the growth chamber assay were evaluated exactly 2 weeks after inoculation.

Rpp gene from PI 567104B located at or near the Rpp6 locus

The mapping of the Rpp gene suggests future research leads. One question is whether PI 567104B has a second

rust resistance gene at a different Rpp locus. In the present study, a resistance gene in addition to the Rpp6 gene might be possibly located in the same region. The bulked seg-regant analysis conducted by Harris et al. (2015), however, implicated the possible presence of resistance genes at both the Rpp4 and Rpp6 loci in PI 567104B. Two Rpp genes were previously identified in the Japanese cultivar Hyu-uga (Kendrick et al. 2011). The Rpp? (Hyuuga) gene was mapped to Chr 6, at or near the Rpp3 locus (Monteros et al. 2007, 2010), and it was initially thought to be a different allele than Rpp3 because of differences in the reactions of Hyuuga and PI 462312 to certain SBR populations and iso-lates. When the mapping population was phenotyped using a different rust isolate, a second gene was later detected that mapped to Chr 3 (LG-N), at or near the Rpp5 locus (Kendrick et al. 2011). In the present study, after applying CIM to the field data and to the growth chamber data, there were no indications of a resistance gene at the Rpp4 locus inherited from PI 567104B. Although the lines were chal-lenged with different sources of P. pachyrhizi inoculum in each assay, the actual Rpp4 gene from PI 459025B has not conditioned high levels of resistance to isolates and field populations from the USA. It is therefore conceivable that the gene or another resistance allele at the same locus could have gone undetected in assays with P. pachyrhizi isolates or strains that were able to defeat the resistance conditioned by the second gene. Further research might clarify this issue.

In the present study, the Chr 18 haplotypes of 10 random resistant and 10 random susceptible F5:6 RILs at 20 SSR loci on Chr 18 showed that the resistant lines had marker alleles from PI 567104B at the SSR loci around the Rpp6 locus, e.g., Sat_131, BARCSOYSSR_18_0331 and BARC-SOYSSR_18_0380, and Satt394. The susceptible RILs typically had susceptible parent alleles at those loci. In this research, the haplotypes further support the hypothesis that a gene or genes inherited from PI 567104B are located in the interval containing the Rpp6 locus between 32.88 cM (Sat_131) and 43.27 cM (Satt394) of the Song et al. (2010) consensus linkage map. Significant marker-phenotype associations in the remnant 123 F6 RILs also confirmed the location of the gene(s).

The estimation of the degree of similarity between the Rpp gene(s) at or near the Rpp6 locus in PI 567104B and the actual Rpp6 gene from PI 567102B was not possible in this study. The Rpp6 gene was reported by Li et al. (2012) after this research had been conducted. Additional research, however, informed that the genetic dissimilarity between PI 567104B and PI 567102B based on the allelic differences between 34,142 SNPs genotyped with SoySNP50 K Bead-chip was 0.23, suggesting that the genetic background of the two accessions was different (Song et al. 2015). The similarity and/or dissimilarities between the two plant

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introductions, however, will require further research, including the development of a mapping population derived from the cross of the two PIs, and the corresponding pheno-typing under controlled conditions, along with the genomic characterization of the segregating progeny. It is impor-tant to note that the two accessions were obtained from the same research station in Indonesia, and they resemble one another in morphological traits. These facts indicate that it is important to elucidate the similarity/dissimilarity nature of the genetic resistance to SBR of each of the two accessions.

The research communicated and conducted on PI 567104B, both in field and growth chamber experiments indicate that PI 567104B is an important source of SBR resistance. Already PI 567104B has been crossed to sev-eral elite lines adapted to the southern and northern USA, and agronomical promising rust-resistant lines selected from the populations could become useful sources of SBR resistance as a better alternative than the original plant introduction.

Author contribution statement M. L. and S. S. con-ducted molecular, statistical and linkage analyses of phe-notypic data. S. L. conducted phenotypic screenings (field and Conviron growth chamber), provided guidance to molecular analysis, wrote the sections on growth chamber screening, preparation of inoculum, inoculation descrip-tion, designed the severity rating scale and contributed to data summary. B. B. S. provided initial guidance to map of resistance in PI 567104B. Q. S. analyzed the data to pro-duce the final linkage and LOD score maps. D. R. W., L. F. L. and A. C. planted and conducted field screenings. D. R. W. supplied F1 seeds. M. K. B. provided guidance on molecular analyses and marker used. S. R. C. planned the research, developed the populations, and supervised the project. M. L., D. R. W. and S. R. C. prepared the manuscript.

Acknowledgments The study was funded by grants from the Iowa Soybean Association (ISA), ISU Project 4403 and the United Soybean Board (USB), Grant # 528, ISU Project Number 4403. The study was also partially supported by the USDA-ARS Projects 6402-21220-012-00D, Crop Genetics Research Unit, Stoneville, Mississippi, and by the Home Economic and Agricultural Experi-ment Station, College of Agriculture, Iowa State University. Appre-ciation is extended to the following collaborators of Iowa State University: Dr. Michelle Graham (ARS-USDA), and to graduate students Chantal Liepold, Alexander Luckew, Jordan Baumbach and to Assistant Researchers Peter Lundeen for their help, advice and support during the conduct of this work, to Gregory G. Gebhart for driving the plants to the USDA lab in Stoneville, Mississippi for SBR resistance screening, and to Nieves Rivera-Velez, for the scoring of the plants in the field tests conducted at Quincy FL. We also appreciate the help that David Wright and Jim Marois of the University of Florida provided in planting and managing the plots

in Quincy, FL., and to Dr. Zenglu Li, Dr. Donna Harris, of the Uni-versity of Georgia for providing molecular markers, and F1 hybrid seed obtained with PI 567104B. Appreciation is also extended to Drs. James Buck, Dan Phillips, and Roger Boerma of the Univer-sity of Georgia for providing suggestions and reviews of the manu-script. Our gratitude also goes out to Drs. Perry Cregan and David Hyten, who were both at the USDA-ARS Soybean Genomics and Improvement Laboratory in Beltsville, MD at the time this research was conducted, and provided support in the molecular analysis of the RILs.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

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