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. 2026 Sep 24;46(10):97. doi: 10.1007/s11032-026-01719-9

Novel quantitative trait loci conferring resistance to root-knot nematodes in lima bean (Phaseolus lunatus)

Adia M Tajima 1,2, William C Matthews 1, Tra Duong 1, Tran Dang Khanh 1, Anil Baniya 1, R Varma Penmetsa 3, Travis A Parker 3, Andrew Farmer 4, Stephanie English 4, Christine Diepenbrock 3, Paul Gepts 3, Philip A Roberts 1, Bao-Lam Huynh 1,✉
PMCID: PMC13612800  PMID: 42798685

Abstract

Lima bean (Phaseolus lunatus) is a broadly adapted, economically important leguminous crop and a susceptible host of root-knot nematodes (Meloidogyne spp.; RKN), which are a devastating plant pathogen in agricultural systems worldwide. To date, there have been few studies to elucidate the genetic determinants of RKN resistance in lima beans. Understanding the genetic mechanisms underlying resistance is essential for improving resistance traits and incorporating them into lima bean breeding programs. To assist in marker-assisted selection, we aimed to identify and map quantitative trait loci (QTLs) conferring RKN resistance-related traits. Three recombinant inbred line (RIL) populations were used in this study. The RIL populations were derived by crossing two RKN-resistant parents with the same RKN-susceptible parent and with each other. All populations were genotyped using genome-wide single-nucleotide polymorphism (SNP) markers. Each population was screened for root galling (RG) and RKN egg reproduction (ER) in response to M. incognita and M. javanica in greenhouse experiments. Three major QTLs were detected and mapped on chromosome Pl04 (QRk-pl04.1), Pl05 (QRk-pl05.1) and Pl10 (QRk-pl10.1) across populations. Among them, QRk-pl05.1 affected RG and ER in response to both RKN species, while QRk-pl04.1 and QRk-pl10.1 suppressed root galling and/or reproduction responses of M. incognita but not of M. javanica. These chromosomal regions defined by flanking markers will help guide marker-assisted breeding and gene discovery for RKN resistance in lima beans.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s11032-026-01719-9.

Keywords: Lima bean, Genetic resistance, Root-knot nematode, Meloidogyne, Plant Breeding

Introduction

Lima bean (Phaseolus lunatus) is a leguminous crop in the Fabaceae family that is valued for its nutrient-dense and edible seeds. Domesticated separately in Mesoamerica and the Central Andes of Ecuador and northern Peru (Gutiérrez-Salgado et al. 1995; Chacón-Sánchez and Martínez-Castillo 2017; Garcia et al. 2021), lima beans have made their mark in the cultural and historical practices of people across the Americas (Marsh n.d.). Today, lima beans are cultivated in over 80 countries and contribute positively to global food security (Traverso et al. 2024). However, lima bean production and the food security it provides may be threatened due to disease susceptibility. One of the most devastating pathogens for lima beans and agriculture worldwide are the root-knot nematodes (RKN) (Giordani et al. 2021; Huynh et al. 2016). Among them, Meloidogyne javanica and Meloidogyne incognita are prevalent in lima bean production regions (Long et al 2014). These nematodes parasitize plant roots by invading the root vascular tissue and inducing root cell differentiation into giant cells and stimulating surrounding root galling. The RKN juveniles and adult females live and feed within these giant cells. Although rarely lethal, galling and stress caused by RKN can lead to severe root damage, resulting in reduced water and nutrient uptake, stunted growth, and vegetative chlorosis (Giordani et al. 2021). Yield loss from RKN damage in susceptible crops can range anywhere from 20—90% (Garcia et al. 2021). While California’s deep loamy soils are well-suited for lima bean growth, they also foster higher reproduction and penetration rates of RKN in susceptible varieties, resulting in yield losses of up to 70% (Traverso et al. 2024; Long et al. 2014; Peterson 2023).

Current pest management strategies to combat the effects of RKN on crops such as lima beans are not highly effective or feasible on a large scale. Although some chemical nematicides are available, they are expensive, and there are significant concerns surrounding their potential health risks. Due to these concerns, some nematicides are banned from use (Giordani et al. 2021; Abad et al. 2008) and a safe alternative to sustainably manage nematode populations is necessary. Unfortunately, crop rotation approaches to reduce pathogenic effects are not highly effective either, as RKN targets a wide variety of crops and the eggs can persist in the soil for multiple years (Giordani et al. 2021). The most promising method of RKN disease and related crop loss management is the use of cultivars with inherent immunity and genetic resistance (Traverso et al. 2024).

L-136 is a bright red small seeded variety of lima bean with indeterminate growth habit and late maturity (Helms et al. 2004). The L-136 accession of lima bean (Allard 1954; Helms et al. 2004) was reported to have resistance to Meloidogyne incognita (reduced galling and reproduction) and M. javanica (reduced galling, but normal reproduction), in a previous study by Roberts et al. (2008). In that study, quantitative inheritance analysis of a Henderson x L-136 recombinant inbred line (RIL) population indicated that root galling and reproduction were controlled by different unidentified genes based on phenotypic data from greenhouse and field tests. However, genomic regions and markers for these genes have yet to be reported. The PI 256874 accession of lima bean (PI 256874 GRIN-Global., 2025), known by G code G25467, was selected as a second RKN resistant parent used to develop recombinant inbred lines due to its observed resistance to both Meloidogyne incognita and javanica galling and reproduction in field trials (Roberts & Matthews, personal communication, 2026). PI 256874 is a large seeded white accession of lima bean that exhibits indeterminate growth and is photoperiod sensitive (GRIN-global 2025).

L-136 and PI 256874 were selected as two resistant parents to cross with the susceptible commercial lima bean cultivar Henderson and with each other to develop three F2:9 RIL populations: Henderson x L-136, Henderson x PI 256874, and PI 256874 × L-136. In this study, the three populations were genotyped using Restriction site-Associated DNA sequencing (RAD-Seq), resulting in 739, 2278, and 1759 genome-wide SNP marker bins, respectively. The populations were phenotypically scored for galling and reproduction response to M. incognita and M. javanica in greenhouse pot trials. We report on quantitative trait loci (QTLs) significantly associated with RKN resistance in the three lima bean RIL populations. Flanking markers for these QTL regions can help lead future gene discovery endeavors and marker-assisted backcrossing for the development of RKN-resistant lima bean cultivars.

Materials and methods

Mapping populations

Three segregating lima bean populations were used in genetic mapping. The RIL01 population included 119 F9 RILs derived from a cross between the susceptible cultivar Henderson as a female parent and the resistant accession L-136 as male parent (Roberts et al. 2008). The RIL02 population included 107 F9 RILs derived from a cross between Henderson (female) and another resistant accession PI 256874 (male). The RIL03 population includes 113 F9 RILs derived from a cross between the two resistant parents PI 256874 (female) and L-136 (male). Henderson is a commercial variety of lima bean that is highly susceptible to both Meloidogyne species M. incognita and M. javanica.

Nematode isolates

Two RKN isolates were used to inoculate and phenotype plants for galling and reproduction response. The Meloidogyne incognita isolate “Beltran” was originally isolated from a lima bean field in the San Joaquin Valley in California. The Meloidogyne javanica isolate “811” was originally isolated from a cowpea field in Southern California. Both “Beltran” and “811” isolates were maintained on greenhouse grown tomato plants (cv. Large Red Cherry) susceptible to RKN (Thomason and McKinney 1960). Nematode eggs were extracted from susceptible tomato roots and used to inoculate greenhouse grown lima bean RILs and parents for resistance phenotyping. Each tomato plant yielded approximately 2–3 million nematode eggs or more after extraction. Nematode egg viability was assessed prior to inoculation by conducting a hatch test, where approximately 20–25% of eggs hatched in a healthy sample.

Resistance phenotyping

The three RIL populations were previously developed and phenotypically scored for their response to RKN stress at UCR in the early 2000s. Phenotypic data of the RIL01 population was reported in Roberts et al. (2008). All RIL populations were separately screened in greenhouse pot trials for resistance to M. incognita and M. javanica galling and reproduction response. The trials were conducted in a randomized complete block design. Air temperatures in the greenhouse were maintained between 27 and 35 °C during the day and 24 ± 1 °C at night. Individual plants were grown in plastic pots with steam-sterilized 80:20 sand:peat moss mix. Soil temperatures ranged from 24 to 27 °C during the experiments. Plants were inoculated with 50,000 eggs in 10 mL of water after the first set of trifoliate leaves emerged, approximately 3 weeks after germination. The RKN hatching and infiltration of root systems began within several days of initial inoculation of greenhouse pot tests. Symptoms of RKN infestation, including galling of root systems and development of nematode egg masses visible on the root surface, were used for phenotyping. Roots of RIL lines and parents were washed clean after 2 months of growth and scored for root galling. The RIL01 population was scored differently from the RIL02 and RIL03 populations as specified below.

The RIL01 population’s root systems screened in greenhouse pot trials were scored for M. incognita and M. javanica galling and reproduction (Roberts et al. 2008) using a rating convention of absence (resistant, no gall/egg mass) and presence (susceptible, galls/egg masses present across the root system). For QTL analysis, the absence and presence data were converted to 0 and 4, respectively, because the gall/egg mass distribution on the root systems of susceptible plants resembled the score of 4 based on the 0–10 rating chart (Bridge and Page 1980). The egg mass distribution on roots was visualized by staining egg masses with an erioglaucine solution (Omwega et al. 1988). Four biological replicates (plants) of each line were planted in separate pots and measured at the same time, and the mean score was used in QTL analysis. In some cases, one plant of the four replicates died, or had a different score from most of the samples (a 4 instead of a 0, or vice versa), indicating that the line was still segregating. The average score was taken from the four replicates, leading to a slight deviation from the mean value.

The RIL02 and RIL03 populations, with PI 256874 as a parent, had more varied galling and egg mass production than the RIL01 population. This difference led to the use of a numerical scale for gall scoring and using egg counts instead of presence or absence of galls and egg masses. The RIL02 and RIL03 populations were visually scored for galling on a scale of 1–10 (Supplemental Fig. 2), based on Bridge and Page (1980). Scores of 1 to 3 indicated that the tap or primary root was free of damage, though small galls on secondary roots could be seen. Roots from > 3 to 6 were considered intermediate, with damage observed on the primary root ranging from roughness (score = 4) to a few small uncoalesced galls (score = 5) to increasing amount of galls (score = 6), plus increasing amounts of galling on secondary roots. Scores of > 6 were considered fully susceptible, with primary roots of 8 score fully galled (lack of any ungalled tissue on the primary root). A root score of 9 had lost most of the secondary roots to rot, with a score of 10 indicating complete root rot due to plant death. Egg production was assessed by extracting egg masses from root systems using a 0.5% NaOCl solution and vigorous shaking in an industrial paint shaker. Eggs per root system (ERS) for the RIL02 and RIL03 populations were calculated by counting extracted eggs using a tabletop Zeiss light microscope and cell counting slides. The egg : root mass ratio trait was determined by weighing root systems and dividing the ERS value by their root weight (in grams), to provide an eggs/gram root (EGR) estimate.

Fig. 2.

Fig. 2

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Chromosomal regions associated with (a) M. incognita galling, (b) M. incognita reproduction, and (c) M. javanica galling in the RIL01 (Henderson x L-136) population. Markers flanking the QTL peak are colorized. Chr04, Chr05, Chr10: Chromosomes Pl04, Pl05, and Pl10, respectively. Numbers on the left of each marker represent chromosomal location in centimorgans

RIL02 had 6 plant replicates for M. javanica and M. incognita galling and 2 plant replicates for M. javanica and M. incognita ERS. RIL03 had 3 plant replicates for M. incognita and 2 replicates for M. javanica (galling and ERS). The ERS and EGR values from the replicates were log-adjusted by calculating the log10(n + 1) score and used in phenotype histograms and QTL analysis. Log10(n + 1) transformation of the ERS and EGR scores normalized the data so that the variance did not increase proportionally with its means. Broad sense heritability (H2) was calculated for all traits based on the variance component attributable to variation among lines (VG) and residual variation (VE) [H2 = VG/(VG + VE)] using the lme4 package (Bates et al. 2015) in RStudio Server (Posit Team 2025).

Marker genotyping

Leaf tissue of all three RIL populations was collected at UC Riverside from greenhouse-grown plants. DNA of the RIL01 population was extracted using the Qiagen DNEasy Plant Minikit according to the manufacturer's protocol. Leaf tissue of individual RIL02 and RIL03 plants were prepared in a two-step protocol, with an initial DNA extraction done using the Hu and Lagarias method (Hu and Lagarias 2020) that was followed by a column purification with a Zymo Research ZR-96 DNA clean up kit according to manufacturer’s protocol. Purified DNA for all three RILs were quantified by PicoGreen fluorometry, normalized to equal concentrations and SNP genotyped at the UC Davis Genome Center’s core genotyping facility using reduced representation Restriction site-Associated DNA sequencing (RAD-Seq) using the single enzyme Nla-III whose isoschizomer CviAII has been previously documented to provide genomewide coverage in lima bean (Ariani et al. 2016). Adapters were trimmed from reads using cutadapt (Martin 2011) and subsequently aligned to the reference genome with Minimap2 (Li 2018). Sample variants were identified using GATK HaplotypeCaller (McKenna et al. 2010). The R package mpr.genotyping (adf-ncgr 2025) was used to reduce noise in genotypes by minimizing the number of recombination events locally in each sample. Markers with minor allele frequencies greater than 0.2 were retained for linkage analysis.

Genetic mapping

Linkage analysis and QTL mapping were performed using the QTL IciMapping 4.2 software (Meng et al. 2015). Markers that were inherited together without recombination were grouped into bins, from which one marker per bin was used in map construction and QTL analysis. Linkage maps were constructed using the Kosambi function, with reference to the SNP physical locations from the UC92 x UC Haskell reference genome (Garcia et al. 2021). A QTL analysis was performed using the Inclusive Composite Interval Mapping (ICIM) method (Wang 2009) run with the QTL IciMapping software using the BIP method with a window size of 1 cM and significance thresholds determined by 1000 permutations. In brief, the ICIM involved three consecutive steps: (1) Single marker analysis was used to select significant markers (P < 0.001) associated with phenotypes, (2) phenotypic values were adjusted for the selected markers except for the two markers flanking the current mapping interval, and (3) the adjusted phenotypic values were used in composite interval mapping, which involves testing QTL additive effects. Linkage maps and QTLs were visualized using the MapChart software (Voorrips 2002).

Results

Phenotypic variation

Bimodal distributions were observed for root galling response in the RIL01 population tested with M. incognita and M. javanica, with the resistant parent L-136 showing a lower galling than the susceptible parent Henderson (Fig. 1a,c). The M. incognita egg masses assayed in the RIL01 population also show a bimodal distribution (Fig. 1b) but were not significantly correlated (r = 0.135; P > 0.05, n = 119) with the root galling response. The M. javanica egg masses were produced in all RILs and parents of this population and were evenly distributed among RIL lines. The broad sense heritabilities of the M. incognita galling and egg mass phenotypes for RIL01 were 0.9956 and 0.9868, respectively. The broad sense heritability of M. javanica galling for RIL01 was 0.9929.

Fig. 1.

Fig. 1

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Variation in root-knot nematode galling and reproduction response (ERS or EGR) of the RIL01, RIL02 and RIL03 populations. ERS: Eggs per root system, EGR: number of eggs/g of roots

The galling phenotype in the RIL02 population showed a bimodal distribution in response to M. incognita and M. javanica (Fig. 1d,g). For M. javanica ERS and EGR (Fig. 1e,f), the distribution of phenotypes was skewed towards the Henderson phenotype, with PI 256874 and Henderson both having a relatively lower reproduction response compared to the average distribution of the population. There was a significant correlation between ERS and EGR (r = 0.663, P < 0.001, n = 107) in M. javanica, however, their correlation with root galling response was not significant (r = 0.323 and 0.158, respectively; P < 0.05, n = 107). The broad sense heritabilities of the M. javanica galling, ERS and EGR phenotypes were 0.85, 0.58 and 0.48, respectively. For M. incognita ERS and EGR (Fig. 1h,i), the population was more evenly distributed with the PI 256874 line showing less galling and reproduction response overall compared to Henderson. There was a significant correlation between ERS and galling response in M. incognita (r = 0.62, P < 0.001, n = 107), as well as ERS and EGR (r = 0.737, P < 0.001, n = 107). The broad sense heritabilities of the M. incognita galling, ERS and EGR phenotypes were 0.87, 0.79 and 0.74, respectively.

The RIL03 population showed wide variation in reproductive and total EGR responses to M. incognita and M. javanica, while both parents had similar values (Fig. 1k,l,n,o). The reproduction data were generally skewed towards the higher value whereas the parental lines were centered. In the RIL03 population, there was a negligible correlation between M. javanica reproduction and galling, whereas there was a strong correlation between M. incognita galling and reproduction response (r = 0.717, P < 0.001, n = 113). Galling data showed that L-136 was more susceptible to M. javanica galling, while PI 256874 exhibited higher susceptibility to M. incognita galling (Fig. 1j,m). Galling data for RIL03 did not follow a bimodal distribution like those seen in the RIL01 and RIL02 populations. Significant correlations between M. incognita reproduction and galling response were observed in both the RIL02 and RIL03 populations (r = 0.62 and 0.72, respectively; P < 0.001, n = 107, 113) In RIL03, the broad sense heritabilities of the M. incognita galling, ERS and EGR phenotypes were 0.98, 0.81 and 0.746, respectively. For M. javanica, broad sense heritabilities for galling, ERS and EGR phenotypes were 0.77, 0.76 and 0.72, respectively.

QTL identification

Three significant QTL peaks were identified on chromosomes Pl04, Pl05, and Pl10 in the RIL01 population (Fig. 2a,b,c) with a significance LOD threshold of 2.95 determined by 1000 permutations. A major QTL on Pl04 corresponded to the M. incognita galling response, explaining some 90% of total phenotypic variance (PVE) (LOD = 125.8, PVE = 89.6%). Another major QTL region mapped to Pl05 and was associated with M. incognita egg mass and reproduction response (LOD = 78.5, PVE = 92.0%). The M. javanica galling response phenotype was associated with a major-effect QTL region on chromosome Pl10 (LOD = 110.1, PVE = 92%) (Fig. 2c). The favourable (low galling and reproduction) allele for each QTL was contributed by the resistant parent L-136.

In the RIL02 population, a major QTL region was identified on chromosome Pl05 in significant association with M. incognita and M. javanica galling, reproduction, and EGR traits (Fig. 3a,b). The calculated significance LOD threshold was 3.1. The LOD and PVE values varied within these traits, with the highest LOD and PVE scores being associated with M. incognita ERS and EGR (LOD = 20.6 and 16.4, PVE = 60.1% and 45%, respectively), followed by M. javanica ERS and EGR (LOD = 8.4 and 8.1, PVE = 34.1% and 33.4%, respectively). The QTL on Pl05, associated with M. incognita galling, ERS, and EGR and M. javanica galling overlapped with the QTL on Pl05 associated with M. incognita egg mass from the RIL01 population. The favourable (low galling and reproduction) allele was contributed by the resistant parent PI 256874.

Fig. 3.

Fig. 3

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Chromosomal regions associated with galling, egg count and the egg count : root mass ratio response to (a) Meloidogyne incognita and (b) Meloidogyne javanica in the RIL02 (Henderson x PI 256874) population. Markers flanking the QTL peak are colorized. Chr05: Chromosome Pl05; ERS: Eggs per root system, EGR: eggs/g of roots

The RIL03 population exhibited a major QTL on chromosome Pl04 in significant association with M. incognita galling (LOD = 22.9; PVE = 58.9%), ERS (LOD = 12; PVE = 31.4%) and EGR (LOD = 22.2; PVE = 17.4%), with the favorable (low galling and reproduction) allele being contributed by the parent L-136. (Fig. 4a; Table 1). There was also a minor QTL on Pl05 associated with M. javanica and M. incognita EGR (LOD = 4.4 and 5.8; PVE = 3.1% and 3.2%, respectively), which overlaps with the QTLs identified in the RIL01 and RIL02 populations (Fig. 4b). Another QTL mapped to Pl10 in significant association with M. incognita EGR (LOD = 31.9, PVE = 33.2%), with the favorable (low galling and reproduction) allele being contributed by the parent PI 256874 (Fig. 4c; Table 1). Finally, Pl08 harbored two QTLs associated with M. javanica EGR (LOD = 12.4 and 20.4, PVE = 10.5 and 21.1%), with the favourable (low galling and reproduction) allele contributed by parents L-136 and PI 256874, respectively (Fig. 4d and Table 1). The significance LOD threshold for RIL03 QTLs was 2.92.

Fig. 4.

Fig. 4

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Chromosomal regions associated with root-knot nematode resistance in the RIL03 (PI 256874 × L-136) population. a depicts LOD peaks associated with M. incognita egg count (green), M. incognita EGR (pink) and M. incognita galling (red) on chromosome 4. b depicts LOD peaks associated with M. incognita ERS (green), M. incognita EGR (pink) and M. javanica EGR (red) on chromosome 5. c depicts LOD peaks associated with M. incognita EGR on chromosome 10. d depicts LOD peaks associated with M. javanica EGR on chromosome 8. Markers flanking the QTL peak are colorized. Chr04, Chr05, Chr08, Chr10: Chromosomes Pl04, Pl05, Pl08, and Pl10, respectively. ERS: Eggs per root system, EGR: eggs/g of roots

Table 1.

Quantitative trait loci associated with root galling and reproduction response to different root-knot nematode species assessed in three RIL populations: Henderson x L-136 (RIL01), Henderson x PI 256874 (RIL02), and PI 256874 x L-136 (RIL03)

QTL RIL population Trait name Chromosome (Pl) Position (cM) leftmarker rightmarker LOD PVE (%) Add
QRk_pl04.1 RIL01 incognita gall 4 43 Chr04_46033436 Chr04_47051812 125.8 89.6 1.98
RIL03 incognita gall 4 30 Chr04_46059040 Chr04_46692671 22.9 58.9 1.6
RIL03 incognita ERS 4 30 Chr04_46059040 Chr04_46692671 12 31.4 0.37
RIL03 incognita EGR 4 30 Chr04_46059040 Chr04_46692671 22.2 17.4 0.41
QRk_pl05.1 RIL03 incognita ERS 5 33 Chr05_37475031 Chr05_37629354 3.4 7.38 −0.15
RIL01 incognita EM 5 37 Chr05_38095727 Chr05_38145597 78.5 92.0 1.93
RIL03 javanica EGR 5 39 Chr05_38009108 Chr05_38209596 4.4 3.1 −0.19
RIL03 incognita EGR 5 40 Chr05_38198047 Chr05_38209596 5.8 3.2 −0.15
RIL02 javanica ERS 5 47 Chr05_38009108 Chr05_38068926 8.4 34.1 0.31
RIL02 javanica EGR 5 47 Chr05_38009108 Chr05_38040209 8.1 33.4 0.29
RIL02 javanica gall 5 48 Chr05_38080061 Chr05_38270149 4.3 15.7 0.39
RIL02 incognita gall 5 48 Chr05_38080061 Chr05_38270149 19.7 54.8 0.69
RIL02 incognita ERS 5 48 Chr05_38080061 Chr05_38270149 20.6 60.1 0.36
RIL02 incognita EGR 5 48 Chr05_38080061 Chr05_38270149 16.4 45 0.31
QRk_pl08.1 RIL03 javanica EGR 8 39 Chr08_30191213 Chr08_45533429 12.4 10.5 0.35
RIL03 javanica EGR 8 41 Chr08_49235566 Chr08_50917130 20.4 21.1 −0.49
QRk_pl10.1 RIL02 incognita EGR 10 35 Chr10_45103536 Chr10_47644799 3.2 6.3 0.12
RIL03 incognita EGR 10 36 Chr10_44019455 Chr10_50732730 31.9 33.2 −0.56
QRk_pl10.2 RIL01 javanica gall 10 56 Chr10_54670417 Chr10_54858682 110.1 92.0 2.00
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Add Additive effects: In RIL01, positive effects indicate the high-value allele comes from Henderson. In RIL02, positive and negative effects indicate that the high-value allele comes from Henderson and PI 256874, respectively. In RIL03, positive and negative effects indicate that the high-value allele is contributed by PI 256874 and L-136, respectively. PVE Percentage of variance explained

EGR Eggs/gram of root, ERS Eggs per root system, EM Egg mass

Discussion

The bimodal distribution of the root galling and reproduction phenotypes in the RIL01 and RIL02 populations (Fig. 1a, b, c, d, g) supports the presence of a major QTL in each RIL population. The QTL peak on chromosome Pl04 associated with M. incognita galling and reproduction response in the RIL03 population spans approximately 633 kb, which falls within the 1000-kb QTL (~ 2 cM) on the same chromosome associated with M. incognita galling in the RIL01 population (Figs. 2 and 4). Hereafter, this QTL region is referred to as QRk-pl04.1. There was also overlap among QTLs on chromosome Pl05 mapped in different RIL populations. These include the QTL for M. incognita egg mass in the RIL01 population (Fig. 2), the QTL for M. incognita and M. javanica galling and reproduction in the RIL02 population (Fig. 3), and the QTL for M. javanica and M. incognita EGR in the RIL03 population (Fig. 4). Hereafter, this QTL region is referred to as QRk-pl05.1, which spans around 200 kb based on consensus flanking markers (Chr05_38009108 and Chr05_38209596) across the three overlapping QTL. Identification of QTL(s) in the RIL01 population (chromosome 4) and the RIL01 and RIL02 populations (chromosome 5) that overlap with the QTLs on chromosome 4 and 5 from the RIL03 population provide cross-population corroboration of these QTLs. The other major QTL region on Pl10, hereafter QRk-pl10.1, was associated with M. incognita EGR in both the RIL02 and RIL03 populations (Figs. 3 and 4). The QTL in the RIL02 population, which spans approximately 2.5 Mb (Chr10_45103536..Chr10_47644799) falls within the QTL in the RIL03 population on chromosome 10, spanning 6.7 Mb (Chr10_44019455..Chr10_50732730). The RIL01 population also has a separate QTL on chromosome 10, hereafter QRk-pl10.2, about 4 Mb beyond the end of the RIL03 interval associated with M. javanica galling (Fig. 2). The significant parental effects at QRk-pl04.1, QRk-pl05.1, and QRk-pl10.1 in the RIL03 population indicate that these QTLs are likely multi-allelic (i.e., L-136 and PI 256874 may carry different resistance alleles at each QTL) and/or are subject to epistatic interactions involving the respective genetic backgrounds of the resistance sources. The presence of common QTLs across populations confirms their conserved effects that could be valuable for marker-assisted breeding. Additional minor QTL peaks detected on chromosomes Pl01, Pl02, Pl03, Pl08, and Pl09 in either the RIL02 or RIL03 populations (Supplemental Fig. 1) indicate the presence of minor resistance genes and their possible interactions.

A previous quantitative genetic analysis by Roberts et al. (2008) suggested the presence of three nuclear genes with independent control in the RIL01 cross of Henderson x L-136. These genes are Mir-1, a recessive gene that controls M. incognita egg production, Mig-1, a dominant gene that controls M. incognita galling, and Mjg-1, a dominant gene that controls M. javanica galling. Based on F1 data (Supplementary Table 3), the L-136 resistance to galling by M. incognita and M. javanica is dominant in both cases, while the L-136 resistance to M. incognita reproduction is recessive. The QTLs associated with the RIL01 population correspond genotypically to these previously described genes. QRk-pl04.1, associated with M. incognita galling response, is consistent with the dominant Mig-1 gene. QRk-pl05.1, associated with M. incognita egg mass and reproduction response, corresponds to the recessive Mir-1 gene. Finally, QRk-pl10.1 is associated with M. javanica galling and is consistent with the dominant Mjg-1 gene. The QTLs identified in this study offer a genomic localization for these previously described genes and confirm their independent genetic control.

The identification of multiple QTLs for RKN resistance in lima beans is also consistent with previous reports on the multigenic control of RKN resistance in other crops. For example, in common bean (Phaseolus vulgaris), the minimum number of genes involved in RKN resistance was around 2–3 (Pesqueira 2025, Orsi 2025). The concentration of QTLs on chromosome Pl05 in the RIL02 population indicates that a gene, or more likely a complex or cluster of genes, is involved in much of the resistance to M. incognita and M. javanica in the PI 256874 line. For example, a single gene or complex of linked genes acting as a single gene on Pl05 likely explains the prior observation that 13 lines exhibited resistance to M. incognita galling, M. incognita reproduction (low egg counts), as well as somewhat lower M. javanica galling, but no resistance to M. javanica reproduction in the phenotypic Class 4 (Supplementary Table 1). Perhaps accordingly, the QTL (flanked by Chr05_38009108 and Chr05_38068926) in the RIL02 population associated with resistance to M. javanica reproduction did not overlap with the resistance to M. incognita (galling and reproduction) and the M. javanica galling QTL (flanked by Chr05_38080061 and Chr05_38270149), indicating a possible separate but linked gene control of the M. javanica reproduction response. The 17 lines observed in phenotypic Class 3 (Supplementary Table 1) with low M. javanica galling but weakened resistance to M. javanica reproduction is likely due to the above QTL (flanked by Chr05_38080061 and Chr05_38270149) combined with the resistance associated with the QTL on chromosome Pl09. The M. incognita galling trait exhibited a negative additive effect for one of the QTL peaks (Pl03) with which the trait was associated, indicating PI 256874 as contributing to the unfavorable, or high value, allele. For the rest of the trait-associated peaks, the positive additive effects indicate that Henderson contributes to the high-value allele and that the gene(s) on Pl05 from PI 256874 contributes a majority of the resistance seen in the RIL02 population.

The RIL03 population is somewhat more complex in differentiating between the resistance effects of each parent line. Based on additive effects, the resistance towards M. incognita and M. javanica was contributed by both L-136 and PI 256874 parents. The QRk-pl04.1 locus was associated with resistance contributed by the L-136 parent, while the QRk-pl05.1 and QRk-pl10.1 loci were associated with resistance contributed by the PI 256874 parent. It is of interest that L-136 and PI 256874 have different geographic origins. L-136 originated from Puerto Rico (of Mesoamerican origin) and PI 256874 originated from Peru (of Andean origin) (Allard 1954, Helms 2004, PI 256874 GRIN-Global, 2025). The small and large seed size of L-136 and PI 256874, respectively, provides some evidence to support their difference in place of origin. This difference raises the possibility that the observed QTLs may not be allelic in nature.

Two major resistance QTL—QRk‑pl04.1 (from L‑136) and QRk‑pl05.1 (from PI 256874) were identified in the RIL01 (Henderson × L‑136) and RIL02 (Henderson × PI 256874) populations, respectively. In the RIL03 population (PI 256874 × L‑136; RIL03), the same genomic regions were present, but the QTL signals differed in strength, trait association, and PVE. Interestingly, other resistance QTLs were also detected. For example, in the case of M. javanica, QTLs on chromosomes Pl05 and Pl09 were involved in resistance response in RIL02, while QTLs on chromosome 1 and 2 contributed to M. javanica resistance response in RIL03 (Supplemental Fig. 1). Differential signal strength, trait association and PVE could be due to the effects of other minor QTLs and genes and their interactions involved in the resistance response.

Supplementary Table 2 demonstrates the galling, ERS and EGR scores associated with lines possessing the resistance QTL haplotypes on chromosomes Pl04, Pl05 and Pl10 in RIL03. For example, lines that possess the L-136 haplotype at QRk-pl04.1 and the PI 256874 haplotype QRk-pl05.1 and QRk-pl10.1 on average had enhanced resistance to M. incognita and M. javanica (lines 47, 63, 70, 72, 81, 83). There are also examples of lines that did not carry any alleles of the resistant parent at each of the loci and were susceptible to M. incognita and M. javanica (lines 54, 71, 87). In general, lines with resistance QTL haplotypes had lower galling and egg production scores than those with the susceptible QTL haplotype. The RIL03 population also had lines with different combinations of genes resulting in highly resistant lines (more resistant than either resistant parent, e.g., lines 47 & 81, resistance to M. incognita reproduction), as well as lines that had no resistance (susceptible recombinants, e.g., line 71, susceptible to M. javanica reproduction). These lines demonstrate the transgressive segregation resulting from the presence or absence of multiple resistance QTLs in the RIL03 population.

QRk-pl04.1 confers resistance exclusively to M. incognita, while QRk-pl05.1 confers resistance to both M. incognita and M. javanica. This may be due to the fact that different nematodes excrete specific effector proteins to suppress immunity in host cells (Yadav et al. 2025). QRk-pl04.1’s exclusive resistance to M. incognita could also be due to a difference in specificity of the host recognition pathway in the RIL population to effectors from this species (Yadav et al. 2025; Chen et al. 2024). Meloidogyne incognita and M. javanica both thrive in warm, tropical and subtropical environments with a wide range of host species, however M. incognita has a wider range of up to 3,000 host plants, while M. javanica has a range of around 800 host plants and tends to occur in areas with a warmer climate in comparison to M. incognita (Nemaplex 2026). Hosts and parasites co-evolve and QTLs that provide resistance are often specialized towards specific parasites. The QTL shared in the RIL01 and RIL03 populations on chromosome Pl04 that mediates resistance to M. incognita could be the result of a species-specific defense response, as opposed to the more broad spectrum resistance offered by the QTL on chromosome Pl05 that is shared in all three RIL populations.

There are multiple avenues for further analysis based on these QTL discoveries. Backcrossing analyses can be performed in order to further refine the genic region underlying galling resistance in L-136, as well as to investigate the recessive resistance to galling and reproduction observed in PI 256874. RNA-sequencing is another important avenue to narrow down candidate genes within the QTL regions, as reported for cowpea (Santos et al. 2018) and common bean (Orsi et al. 2025). A potential future experiment could be to select one of the RIL populations to analyze gene expression timelines using RNA sequencing after exposure to nematode stress. Another future avenue could be to utilize transgressively resistant lines that carry a full complement of the resistance traits from the RIL03 population for marker-assisted selection in lima bean breeding. Overall, our report has helped to elucidate major QTLs associated with RKN resistance, paving the way for marker-assisted backcrossing or further candidate resistance gene characterization.

Conclusion

This study has identified QTLs of significance across three RIL populations that can be used for marker-assisted breeding of resistance to root-knot nematodes M. javanica and M. incognita in lima beans. By integrating genotypic data with phenotypic analyses of root galling and nematode reproduction response, we were able to confirm that the M. incognita-induced galling and reproduction response, and M. javanica galling in the RIL01 population are under separate genetic control. Analyses of the RIL02 and RIL03 populations identified QTLs on chromosomes Pl04, Pl05, and Pl1010 associated with RKN resistance in all three populations. Analyses of F1 populations established that resistance to galling in L-136 is dominant, while resistance to reproduction in L-136 and resistance to galling and reproduction in PI 256874 is recessive. This is important for marker-assisted breeding to select for recessive resistance alleles, which is a challenge when using phenotypic selection alone. Further research should focus on refining the QTL regions of interest through backcrossing and gene expression experiments to identify genes in resistant parental lines and to subsequently develop gene-based molecular markers for use in resistance breeding.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 2596 KB) (2.5MB, docx)

Acknowledgements

This work was supported by grant No. 2022-51181-38323 (to P.D. Paul Gepts) of the Specialty Crop Research Initiative (SCRI) Program at the United States Department of Agriculture (USDA) and AES Hatch Project (to P.A.R. Philip A. Roberts). The authors wish to thank Peggy Mauk and team at the Agricultural Operations facility on UC Riverside campus for cooperation and trial management. The authors also would like to thank Sarah Dohle for thoughtful conversation regarding parental accessions.

Author contributions

P.A.R. and W.C.M. conceived the experiment. W.C.M. made the crosses, developed the RIL lines and collected data. T.D., A.V.P., T.P., A.F., and S.E. contributed to genotyping efforts. B.L.H. constructed the genetic maps and guided the genetic discovery effort. A.M.T. performed data analysis, QTL mapping, manuscript writing, and editing. B.L.H., P.A.R., W.C.M., C.D. and P.G. contributed to manuscript editing. P.A.R. and P.G. obtained funding. All authors read and approved the final manuscript.

Funding

This work was supported in part by the USDA-NIFA-SCRI grant no. 2022–51181-38323.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors have no competing interests to disclose.

Footnotes

Publisher's Note

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Associated Data

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Supplementary Materials

Supplementary file1 (DOCX 2596 KB) (2.5MB, docx)

Data Availability Statement

No datasets were generated or analysed during the current study.

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