Performing Department
(N/A)
Non Technical Summary
In the United States, soybean diseases result in an estimated average annual yield reduction of 11%. Plant-parasitic nematodes are responsible for annual agricultural losses of $160 billion, severely threatening global food security. Root-knot nematodes (Meloidogynespp.) are considered the most economically important and widely distributed species of plant-parasitic nematode, of which southern root-knot nematode [SRKN,Meloidogyne incognita(Kofold & White) Chitwood] has the most scientific and economic importance. In the United States, SRKN is widely distributed across all 13 Southern states, representing the second most suppressive soybean pathogen in the region.The recent northern distribution of SRKN is highly concerning considering that most soybean varieties grown in this region (maturity groups (MGs) earlier than MG 4) are known to be highly susceptible to SRKN.SRKN is challenging to control due to its short life cycle and high reproductive rates.The observed symptoms of SRKN infection closely resemble those caused by abiotic stressors, including stunted growth, leaf wilting and discoloration, and deformation of the roots. The extent of crop losses relies on historical crop rotation and field usage, environmental factors, initial nematode population density, soil type, and genetic background. Studies with controlled nematode pressure in field conditions have shown yield reduction of up to 97% in susceptible cultivars when exposed to high SRKN pressure. Management based on crop rotation is especially challenging and limited since most flowering plants are hosts to SRKN. Chemical approaches used to be an effective management option to control SRKN, however, fumigants, such as 1,3-dichloropropene are expensive and require special application equipment. Non-fumigants such as seed-applied nematicides are readily available but are ineffective when nematode densities are high due to limited movement from the seed coat to the root system. The use of genetic resistance is the most sustainable approach to efficiently control the damage of SRKN in soybeans.Since most SRKN-resistant cultivars are derived from genetic resources primarily derived from the Forrest cultivar carrying solely the Rmi1 locus (chromosome 10), it raises concerns regarding the long-term sustainability of resistance. As parthenogenic nematodes, minimal diversity and evolution are expected. However, resistance breakdown has been observed in tomatoes against the Mi gene. Understanding the molecular mode of action responsible for the Rmi1 QTL resistance will help better safeguard that resistance once deployed in the field. The impact of a resistance-breaking population in soybeans, although very rare, would be dramatic because of the high concentration and wide distribution of SRKN, the rather narrow base of genetic resistance, as well as the lack of alternative management options. More efforts are necessary to identify and stack novel sources of resistance in developing soybean lines with enhanced and more durable SRKN resistance, particularly in the current scenario where SRKN populations are being detected in northern soybean-producing regions.Therefore,the long-term objective of this proposal is to provide soybean growers with SRKN-resistant cultivars with a broader genetic basis for sustainable security against yield losses caused by this yield-limiting plant-parasitic nematode. More specifically, this proposal is grounded in three main objectives, including i) identification of the major gene on theRmi1locus and molecular characterization of its mechanisms of resistance, ii) expansion of the genetic basis by screening and identifying novel genetic sources and different modes of resistance, and iii) development of soybean breeding populations aiming to stack different genetic sources of resistance.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
Southern root-knot nematode [SRKN,Meloidogyne incognita(Kofold & White) Chitwood] represents one of the yield-limiting plant pathogens with the most scientific and economic importance. In the United States, SRKN was predominantly a problem in the Southern states due to temperature and soil conditions. In this region, SRKN accounted for over $1.54 billion of losses translating to approximately $4,000 cumulative losses per acre over the last two decades. Recently, SRKN has been confirmed in the North Central region across soybean-producing counties in Missouri, Illinois, and Indiana, with Adams County(Illinois)being the northernmost region where SRKN has been confirmed. The use of genetic resistance through SRKN-resistant cultivars is the most sustainable and effective approach to managing the damage of SRKN in soybeans. Most genetic resources for SRKN resistance are within maturity groups (MGs) 5 or later. In Southern and mid-Southern states, soybean growers are switching cultivars from MG 5 to MG 4 or earlier. Therefore, the widespread distribution of SRKN in northern regions and the recent shift toward earlier MGs in the Southern states are highly concerning because most soybean cultivars within these earlier MGs are highly susceptible to SRKN.Furthermore, available SRKN-resistant cultivars are derived from genetic resources majorly derived from Forrest solely carrying the major resistance locus Rmi1 on Chr. 10, which imposes severe selection pressure across environments and growing years. Although SRKNs are parthenogenic nematodes, resistance breakdown has been observed in other crops. In soybeans, the impact of a resistance-breaking population would result in devastating losses because of the high concentration and wide distribution of SRKN, the rather narrow base of genetic resistance, as well as the lack of alternative management options. Therefore, there is a pressing need to develop earlier MG SRKN-resistant soybean cultivars, as well as expand the current genetic basis of resistance to provide long-term protection against this plant-parasitic nematode and support the sustainability of the soybean value chain.The long-term objective of this proposal is to provide soybean growers with SRKN-resistant cultivars with a broader genetic basis for sustainable security against yield losses caused by this yield-limiting plant-parasitic nematode. More specifically, this proposal is grounded in three main objectives, including i) identification of the major gene on the Rmi1 locus and molecular characterization of its mechanisms of resistance, ii) expansion of the genetic basis by screening and identifying novel genetic sources and different modes of resistance, and iii) development of soybean breeding populations aiming to stack different genetic sources of resistance.
Project Methods
This research proposal consists of a multi-disciplinary effort including expertise in plant pathology, molecular genetics, and plant breeding. The proposal is divided into three main objectives including the i) identification and molecular characterization of the major gene on the Rmi1 locus (Objective 1); ii) screening and identification of novel genetic resources utilizing quantitative resistance metrics (Objective 2); and iii) development of soybean breeding populations aiming to stack different genetic sources of resistance (Objective 3).Objective 1. Identification and molecular characterization of the major gene on the Rmi1 locusa) Identification of the major gene on the Rmi1 locus: The Rmi1 locus will be narrowed down to a reasonably small genomic region so that a few promising candidate genes can be identified for further functional analysis. A recombinant mapping population from an advanced RIL (F7) heterozygous at the Rmi1 locus defined by two flanking markers (P1 and P2) was developed. Seven new recombinants have been identified. The homozygous lines will be obtained from these recombinants for assays with SRKN in the greenhouse. The galling response will be used to grade the degree of resistance. Based on the past screenings, the differences between resistant and susceptible plants were clear and easy to distinguish. These recombinants will also be genotyped with more molecular markers designed from the genome sequence information to estimate their recombination breakpoints. More recombinants will be identified by screening the remaining seeds derived from the heterozygous plants (F7) at the Rmi1 locus.b) Targeted long-read and transcriptome sequencing of PI 438489B and Magellan: To reveal any substantial sequence differences (particularly long insertions or deletions) between PI 438489B and Magellan in the Rmi1 locus, both PI 438489B and Magellan will be sequenced with long-reads using the PacBio HiFi sequencing platform through Novogene. The obtained sequences will be compared between the two parents as well with the Williams 82 reference genome to reveal any sequence differences in the Rmi1 locus, especially long insertions that may contain additional genes.c) Functional analysis of the candidate genes in composite transgenic plants through overexpression and CRISPR knockouts: To validate the correct gene for Rmi1, the identified candidate genes (possibly 3 to 4) will first be evaluated using hairy root transgenic composite plants. Most promising resistant genes evaluated from composite transgenic system followed by SRKN phenotyping will be used to develop stable transgenic plants. The selected candidate genes (their coding sequences, or CDS) will first be cloned from the resistant parent PI 438489B into the donor vector pDONR/Neocin (Invitrogen). The cloned CDS will then be cloned into the binary vector pCAMGFP-CvMV:GWox via the Gateway cloning technology to be driven by the strong CvMV promoter for over-expression.d) Characterization of Rmi1-mediated resistance: Precise cellular localization of proteins controlling disease resistance and their interaction with other cell signaling components are crucial for the proper activation of plant immunity. We will first determine the cellular compartments to which Rmi1 candidate proteins are targeted, utilizing previously generated binary constructs in the pCAMGFP-CvMV:GWox vector.Objective 2. Screening and identification of novel genetic resources utilizing quantitative resistance metrics and microscopic observationsa) Genetic and phenotypic screening to identify novel genetic resources by using quantitative resistance metrics:An inoculated greenhouse pot assay will be used to evaluate the susceptibility of soybean entries based on SRKN reproduction and galling. Two seeds will be planted in 656 cm3 cone-trainers containing pasteurized sand. Seedlings will be thinned to one seedling per pot 2 days after emergence. Each seedling will be inoculated with 5,000 eggs of M. incognita in 2 ml of water. An isolate of M. incognita was collected from soybean in Arkansas and maintained on tomato at the Lonoke Extension Center. Each root system will be rated for galling 60 days after inoculation and eggs extracted from an 8 g subsample.b) Microscopic identification of spatiotemporal resistant root responses at the plant-nematode interface:To shed light on the spatiotemporal aspects of defense responses, newly identified genetic resources (8 to 10 accessions) exhibiting strong SRKN resistance will be utilized to conduct microscopic observations to identify differences in cellular responses of susceptible and resistant roots using a Leica Stellaris DMI8 Confocal Microscope.To achieve this goal, 2-week-old soybean lines [both resistant and susceptible (Magellan)] grown in microscopy rhizosphere chambers will be infested with 500 hatched M. incognita J2s per plant, and infected roots will be harvested for microscopic observations at 2-, 4-, 7-, and 14-days post-inoculation.Objective 3. Development of soybean breeding populations aiming to stack different genetic sources of resistance:Between 15 to 20 bi-parental breeding populations will be developed in Arkansas including a high-yielding elite modern cultivar and a resistant genetic resource (PI 438489B, PI 567516C, PI567305, and more based on the results from Objective 2). An off-season nursery in Ponce, Puerto Ricowill be used to conduct off-season hybridization schemes and generation advancement (three growing seasons in a year). Between 100 and 150 F4 single plants per bi-parental population will be selected and threshed separately in Puerto Rico, and F4:5 seeds from selected F4 plants will be grown in non-replicated progeny rows. Selected rowswill be further evaluated in multi-environment trials for yield and agronomic traits. Breeding lines with superior yield potential and combined modes of resistance will be tested in controlled environments for SRKN resistance and may serve as parental lines to develop additional breeding populations.