Update:
Final Report
Iowa Soybean Association
October 13, 2019
Project Title: Evaluation of the Phytophthora resistance Rps12 gene for its utility and identification of tightly linked molecular markers for its selection.
Investigator: Madan K. Bhattacharyya
Agronomy Hall G303
Iowa State University
Ames, IA 50011
515-294-2505
mbhattac@iastate.edu
Iowa State University
Progress report for the period from October 1, 2017 to June 30, 2019
Executive Summary
Here we summarize the overall accomplishment of this one-year project.
The goal of this one-year project was to investigate (i) the usefulness of the Phytophthora resistance Rps12 gene that we mapped earlier under the support of another Iowa Soybean Association grant (Sahoo et al. 2017: https://doi.org/10.1371/journal.pone.0169950), and (ii) developed breeder-friendly markers so that the gene can be rapidly introgressed into soybean cultivars.
To accomplish our goal, we identified two desirable recombinant inbred lines (RILs) that most unlikely carry Rps genes, other than Rps12 and unknown Rps genes next to Rps12. Of the two lines presumably carrying only Rps12, one showed susceptibility to six of the Phytophthora sojae 33 isolates collected from Iowa soybean fields. Molecular analyses of the two lines revealed that the line showing susceptibility to the six isolates inherited a portion of the chromosome, next to Rps12, from the susceptible parent AR12 suggesting that this section of the chromosome from the resistant parent PI399036 contains a new Rps gene that is required for resistance against the six of the 33 P. sojae isolates collected from the soybean fields of Iowa. In our earlier study, resistance encoded by this new gene RpsX was confounded with that of Rps12 and provided broad-spectrum resistance against many isolates of the pathogen because of the complementary Phytophthora resistance functions of Rps12 and a neighboring RpsX gene. Each of the two genes confer resistance against a subset of P. sojae isolates; and together, they confer resistance against many of the P. sojae isolates. Rps12 and RpsX are very closely located on Chromosome 18. As a result, they are inherited as a single gene, which is ideal for transferring to new P. sojae susceptible cultivars.
Our previous study on the Phytophthora resistance Rps1-k gene conferring resistance against a large number of P. sojae isolates also revealed two functional Rps genes (Gao and Bhattacharyya, 2008: https://bmcplantbiol.biomedcentral.com/track/pdf/10.1186/1471-2229-8-29; Gao et al. 2005: https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-18-1035).
Our study discovered two Rps genes that are tightly linked. It is essential that we select both genes using molecular markers to incorporate the broad-spectrum resistance mechanisms against the P. sojae isolates conferred by the two genes. We therefore identified desirable molecular markers to introgress the two genes into new soybean cultivars.
Detailed Final Report
Goals and Objectives: Based on the responses of recombinant inbred lines (RILs) carrying Rps 12 to three P. sojae isolates that can defeat resistance encoded by most of the characterized Rps genes (Sahoo et al. 2017), we hypothesized that Rps12 is a very important Rps gene provideing soybean with broad-spectrum resistance to most, if not all P. sojae isolates. The goals of this project is to establish the utility of this novel gene through infecting lines carrying this gene against a large number of P. sojae isolates and identify molecular markers for pyramiding this gene into commercial cultivars that carry other Rps genes.
We proposed to conduct the following two objectives to reach our goal.
Objective 1. Determine the responses of three recombinant inbred lines carrying Rps12 to a large collection of P. sojae isolates.
Objective 2. Identify molecular markers linked tightly to Rps12.
Progresses made in the last six months are presented under each objective.
Objective 1. Determine the responses of three recombinant inbred lines carrying Rps12 to a large collection of P. sojae isolates.
It was proposed that the Phytophthora resistant PI399036 line contains several Rps genes (Gordon et al. 2007: Phytopathology 97: 113-118). To identify three RILs containing only Rps12, we investigated 42 Phytophthora resistant RILs generated from the cross between PI399036 x AR2 for molecular markers linked to the known Rps regions.
Of the 30 Rps-linked SSR markers evaluated, 10 SSR markers were found to be polymorphic between the two parents and were applied in evaluating 42 RILs homozygous for Rps12. From the polymerase chain termination reaction (PCR) assays of 420 combinations of 42 RILs and 10 SSR markers, we identified two RILs (RIL12 and RIL14) that carry molecular marker alleles, specific to the Phytophthora susceptible parent AR2. We have obtained 33 P. sojae isolates from the Robertson lab that were collected earlier from the Iowa soybean fields. The isolates were characterized for their pathotypes by inoculating a set of 14 soybean lines that are considered to be differential lines for 14 individual Rps genes. The isolates were used also to infect the two selected RILs (RIL12 and RIL14) and the two parents, PI399036 and AR2, used to develop the RILs. Although RIL12 and RIL14 were shown to contain Rps12 earlier (Sahoo et al. 2017: PLoS ONE 12: e0169950), RIL12 is susceptible to six of the 33 P. sojae isolates collected from the Iowa soybean fields. Based on the genetic make-ups of RIL12 and RIL14 for molecular markers of the Rps12 region and distinct responses of the two RILs to six isolates, it appeared that there could be a functional Rps gene next to Rps12, which we named temporarily RpsX. The preliminary mapping results were reported on October 1, 2018.
We conducted additional mapping experiments to conform the presence of RpsX in PI399036. Six RILs, RILs 6, 9, 12, 42, 49 and 81, containing genetic rearrangements between Rps12 and RpsX due to exchange of chromosomal segments from the two parents were studied to confirm the new RpsX gene. Due to absence of RpsX, the RILs 6, 9, 12, 42, and 49 were susceptible to the P. sojae isolate V13. The five lines however contain Rps12; and therefore, resistant to the mixture of the isolates, R17 and Val 12-11. On the contrary, RIL81 contains RpsX but not the Rps12 gene. Therefore, this RIL is resistant to V13 and susceptible to the mixture of R17 and Val 12-11 isolates.
KPIs/Performance Metrics: In the one-year grant period, under the Objective 1 we expected to accomplish the following:
1. By the end of the one-year grant period, we will complete the evaluation of three selected RILs carrying only Rps12 for their responses to at least 100 P. sojae isolates.
Accomplishment: We were able to identify only two RILs carrying only Rps12 based on molecular analyses of 42 RILs, available to us. The two lines were sufficient enough to test our hypothesis that the Rps12 gene governs broad-spectrum Phytophthora resistance. While testing our hypothesis, we discovered that the broad-spectrum resistance of Rps12 is because of the complementary Phytophthora resistance functions of Rps12 and a neighboring RpsX gene. Each of the two genes confer resistance against a subset of P. sojae isolates; and together, they confer resistance against many of the P. sojae isolates.
The outcome was unexpected. We, therefore, instead of investigating the RILs 12 and 14 against additional 67 P. sojae isolates as proposed, we utilized our resources to map the new Rps gene and identify molecular markers linked to the two Rps genes, Rps12 and RpsX, to facilitate Phytophthora resistance soybean breeding programs for this complex locus.
Objective 2. Identify molecular markers linked tightly to Rps12.
Using the unassembled genome sequences of the two parents of the RILs, PI399036 and AR2, and the reference Williams 82 genome sequence, our collaborator Dr. Anindya Das, identified single nucleotide polymorphic (SNP) loci of the Rps12 region. We developed sequence-based polymorphic (SBP) markers as follows (Sahu et al. 2012: BMC Genomics 13:20). First, we predicted the SNPs between the two parents of the RILs, PI399036 and AR2, by comparing their short-read sequences with sequences of the reference Williams 82 genome sequence. PCR amplified DNA fragments containing this SNPs were then digested with the respective restriction endo nucleases and run on an agarose gel to reveal the polymorphisms. We have mapped 12 molecular markers including four SBP markers. Additional eight SBP markers are detected and will be genetically mapped.
Our study discovered two Rps genes that are tightly linked. It is essential that we select both genes using molecular markers to incorporate the broad-spectrum resistance mechanisms against the P. sojae isolates conferred by the two genes. We therefore identified desirable molecular markers to introgress the two genes into new soybean cultivars. The Figure 4 shows the molecular markers that can be used in marker assisted selection of the Rps12 and RpsX genes in breeding Phytophthora resistant soybean cultivars,
KPIs/Performance Metrics: In the one-year grant period, under the Objective 2 we expected to accomplish the followings:
1. We would map at least 20 molecular markers of the Rps12 region by September 30, 2018.
2. We would identify the candidate Rps12 genes by September 30, 2018.
Accomplishment: In our proposal, we proposed to identify 20 molecular markers to saturate the Rps12 region. We have mapped 12 molecular markers in the region containing Rps12 and RpsX genes. Eight additional molecular markers physically mapped to the region. Genetic mapping of these markers will be completed shortly. The genetically mapped molecular markers are sufficient to breed the two genes into soybean cultivars.
We have not been able to identify the candidate Rps12 genes for the following reason. The Rps12 is most likely comprised of simple sequences making it difficult to assemble the sequences. Our collaborators Drs. Anindya Das and Xiaoqiu Huang failed to generate the continuous sequence for the Rps12 region. The resistance gene regions are novel and cannot be predicted from the genome sequence of Williams 82, which has been sequenced. Our collaborators requested to sequence the PI399036 genome for additional coverages to facilitate the de novo assembly of the genome sequence.
Here it is worth noting that although genome of Williams 82 was sequenced in 2010 and again recently, the Rps1-k region is missing in the assembled genome sequence. We cloned the gene using conventional approaches involving cloning and isolation of bacterial artificial chromosomes and subsequently published the sequence of Rps1 region (Gao and Bhattacharyya, 2008: https://bmcplantbiol.biomedcentral.com/track/pdf/10.1186/1471-2229-8-29; Gao et al. 2005: https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-18-1035). Thus, our failure to identify the candidate Rps12 genes was not unexpected.
Although identification of the candidate Rps12 and/or RpsX genes was not possible due to lack of sufficient sequences, the outcomes of this project however provided adequate information for conducting marker-assisted selection of the two novel Rps genes for developing Phytophthora resistant soybean cultivars.
View uploaded report 
The goal of this one-year project was to investigate (i) the usefulness of the Phytophthora resistance Rps12 gene that we mapped earlier under the support of another Iowa Soybean Association grant (Sahoo et al. 2017: https://doi.org/10.1371/journal.pone.0169950), and (ii) developed breeder-friendly markers so that the gene can be rapidly introgressed into soybean cultivars.
To accomplish our goal, we identified two desirable recombinant inbred lines (RILs) that most unlikely carry Rps genes, other than Rps12 and unknown Rps genes next to Rps12. Of the two lines presumably carrying only Rps12, one showed susceptibility to six of the Phytophthora sojae 33 isolates collected from Iowa soybean fields. Molecular analyses of the two lines revealed that the line showing susceptibility to the six isolates inherited a portion of the chromosome, next to Rps12, from the susceptible parent AR12 suggesting that this section of the chromosome from the resistant parent PI399036 contains a new Rps gene that is required for resistance against the six of the 33 P. sojae isolates collected from the soybean fields of Iowa. In our earlier study, resistance encoded by this new gene RpsX was confounded with that of Rps12 and provided broad-spectrum resistance against many isolates of the pathogen because of the complementary Phytophthora resistance functions of Rps12 and a neighboring RpsX gene. Each of the two genes confer resistance against a subset of P. sojae isolates; and together, they confer resistance against many of the P. sojae isolates. Rps12 and RpsX are very closely located on Chromosome 18. As a result, they are inherited as a single gene, which is ideal for transferring to new P. sojae susceptible cultivars.
Our previous study on the Phytophthora resistance Rps1-k gene conferring resistance against a large number of P. sojae isolates also revealed two functional Rps genes (Gao and Bhattacharyya, 2008: https://bmcplantbiol.biomedcentral.com/track/pdf/10.1186/1471-2229-8-29; Gao et al. 2005: https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-18-1035).
Our study discovered two Rps genes that are tightly linked. It is essential that we select both genes using molecular markers to incorporate the broad-spectrum resistance mechanisms against the P. sojae isolates conferred by the two genes. We therefore identified desirable molecular markers to introgress the two genes into new soybean cultivars.