Results

Phenotyping
The F6 RIL population used here was produced by single seed descent from a cross A. ipaënsis × A. magna. The F1 showed high fertility, reflecting that the parents are very closely related. The population shows large variability for main stem height, length of lateral branches, seed size and number, and resistance to rust. Most individuals show mid-parent values, but the population showed large transgressive segregation. High skewness and kurtosis values showed that four of the 15 traits evaluated were not normally distributed. These were incubation period for 2012 and 2013 (IncPer_2012, IncPer_2013); seed number, average of 3 years; and main stem height for 2011 (MSH_2011). To achieve approximately normal distributions, these four traits were log(x) transformed. All phenotyping information is presented on File S1.

Rust:
The frequency distribution based on the pooled data for TL/LA, number of SL/LA, and incubation period (IncPer) showed strong biased toward resistance, because 41 individuals and the resistant parent presented no lesions in either experiments (Figure 2, A and B). The susceptible parent A. ipaënsis K 30076 showed greater susceptibility than the control, A. hypogaea cv. Runner IAC 886, in all components tested: larger SI, TL/LA and SL/LA, and shorter IncPer than the susceptible control (File S1). Seventeen individuals had greater SI, SL/LA, and TL/LA than the susceptible control. In addition, five had shorter IncPer (File S1). As expected, the number and size of lesions were negatively correlated with Incubation period (Pearson r = – 0.6, P < 0.005). No chlorosis was observed on any accession of the F6/F7 population. On leaves of some of the less-susceptible genotypes, necrotic areas corresponding to colonies aborted at a late developmental stage were observed.
Figure 2  Frequency distribution of rust resistance, peg, and seed characteristics (A−F) in recombinant inbred lines (F6/F7 RILs) derived from a cross of A. ipaënsis K 30076 (Ai) with A. magna K 30097 (Am). For rust tests, A. hypogaea cv. Runner IAC 886 (R) was the susceptible control. With the exception of pod constriction, for all traits the means of the parents are significantly different (P < 0.05). In (A) genotypes without symptoms (therefore without incubation period), this trait was artificially tabulated as 200.

Other agronomic/domestication traits:
Phenotypic evaluations were performed at different generations. Values were normally distributed for most traits for most years. With the exception of PC and PL, the means of the parents are significantly different (P < 0.05). Comparison of the means of the parents and the segregating genotypes reveals that for all traits there is transgressive segregation in the progenies (Figure 2, C−F). On an average for 3 years, A. magna produced more seed than A. ipaënsis, but 10-seed weight was lower (Figures 2, E and F).

Map construction
All markers evaluated in this study were amplified by the use of heterologous primers. With the use of minimum LOD score of 6.0 and a maximum recombination fraction (θ) of 0.35, and after the exclusion of co-segregating markers, we found that 399 markers mapped onto 10 LGs, spanning a total map distance of 678.2 cM. These markers included 378 microsatellites and 21 transposon (miniature inverted-repeat transposable element) markers. LGs were numbered and oriented essentially according to Moretzsohn et al. (2009), but with B05 and B08 reoriented (“flipped”) to ensure forward compatibility with pseudomolecules produced by the Peanut Genome Sequencing Consortium (www.peanutbase.org; Genbank GCA_000816755.1). LGs ranged from 41.5 cM (with 35 markers) to 139.2 cM (55 markers), with an average distance of 1.7 cM between adjacent markers. A total of 91 (22.8%) of the 399 mapped markers deviated from the expected 1:1 ratio at P < 0.05 level. Of these, 79 markers were skewed toward A. ipaënsis and only 12 markers toward A. magna. Most LGs have few distorted markers, and LG B05 and B08 had no distorted markers. In contrast, LG B02 and the upper portion of LG B04 were almost entirely composed of distorted markers. These two LGs and LG B06 were distorted toward A. ipaënsis alleles. Distorted markers at P < 0.05 were identified by # (Figure 3).
Figure 3  A genetic linkage map of the B-genome of Arachis obtained through the analysis of 94 F6 plants, generated from a cross between A. ipaënsis K 30076 and A. magna K 30097. Numbers on the left of each group are Kosambi map distances (cM). Markers that amplified more than one loci have numbers _1 and _2 after the marker name. Quantitative trait loci (QTL) are indicated as colored bars running alongside linkage groups. Colors/textures are according to categories: black, rust resistance [total number of lesions/leaf area (TL/LA), number of sporulated lesions/leaf area (SL/LA), susceptibility index (SI), and Incubation Period (IncPer)]; white, seed characteristics [seed number (SN) and 10-seed weight (10_SW)]; textured, plant architecture [main stem height (MSH)]; and domestication traits [peg length (PL) and pod constriction (PC)]. Distorted alleles (P < 0.05) are indicated by #.

QTL identification:
The framework map, containing 399 markers, was used for QTL analysis. LOD significance threshold estimated for each trait ranged from 2.5 to 3.2, and only QTL with LOD values exceeding these values were included. At least one QTL was detected for each of the 15 traits analyzed, with a total of 38 QTL mapped. A summary of QTL is provided in Table 1. Detailed information about markers and QTL are presented in File S1.

QTL for rust resistance:
For rust resistance, 13 QTLs were identified on the two bioassays. A major QTL for the four components of rust resistance (SI, TL/LA, SL/LA, and IncPer) was consistently identified in both bioassays and mapped at the same marker interval on map position 35.1−42.9 cM on LG B08, with LOD scores between 2.9 and 8.2. Its closest marker is Ah-280. This QTL explained 5.8–59.3% of the phenotypic variance of the four different traits (SI, TL/LA, SL/LA, and IncPer). Another QTL for SI and TL/LA evaluated in 2012 and for IncPer_2013 was found at 25.4−33.1 cM on the same LG B08 (Table 1, Figure 3), explaining 13.2–34.8% of the total variance in 2012 and 2013, respectively. The closest markers are AHGS1350 and AHGS2541. In addition to these two QTL, two minor QTL were identified in LGs B04 and B07 for SI_2012. For all QTL, resistance was derived from A. magna. The contribution of A. magna alleles for the rust resistance-related traits was evaluated by calculating the average phenotypes of the homozygote plants for the A. magna allele vs. the average phenotype of those homozygote for the A. ipaënsis allele (Figure 4). Arachis magna alleles contributed significantly for the reduction of SI, TL/LA, and SL/LA. The effect of A. magna alleles was less pronounced for IncPer (Figure 4).
Figure 4  Bar graph of contribution of A. ipaënsis K 30076 (blue bars) vs. A. magna K 30097 (orange bars) alleles to the rust resistance-related traits: susceptibility index (SI), number of sporulated lesions/leaf area (SL/LA), total number of lesions/leaf area (TL/LA). and incubation period (days/20) (IncPer/20).

QTL for other agronomic/domestication traits:
Agronomic and domestication traits were evaluated in three different years. QTL were consistent between years; therefore, for final QTL analyses, data from different years were pooled and considered different replicates of the same experiment. The exception was MSH, which was not pooled, because measures were performed in different stages of plant growth in each year. In total, 25 QTL were identified. Interestingly, the QTL for two domestication traits (PC and PL) were placed in the same map positions: 37.1−40.8 cM in LG B01 and 64.3−64.9 cM in LG B04 (Table 1, Figure 3). LG B04 also harbors a cluster of QTL for MSH at 64.3−64.9 cM, explaining 10.7–30.4% of the total phenotypic variance. For all these traits, the main markers associated were AHGS1917 and AHGS2155. Alleles increasing values of domestication-related traits originated from both parents. However, alleles increasing seed number are all derived from A. ipaënsis. No QTL for domestication/productivity traits co-localized with the rust resistance QTL.

KASP primer design and validation on a tetraploid background
Of a total of 24 assays designed, 22 worked well with the samples tested. Nineteen of them successfully distinguished A. ipaënsis and A. hypogaea from A. magna and A. batizocoi. Four cluster configurations were observed: (1) with nine assays, two clusters were present: one with A. ipaënsis plus the six cultivars of A. hypogaea and another with A. magna, A. batizocoi, and their derived induced allotetraploids MagSten and BatSten1 (noted in File S1 as Ah = Ai≠(Ab = Am = MagSten = BatSten)). An example is shown in Figure 5A; (2) with eight assays, one extra cluster was observed: A. hypogaea formed a different cluster intermediate in position (noted in File S1 as Ah≠Ai≠(Ab = Am = MagSten = BatSten))(Figure 5B); (3) with two assays, a different extra cluster was observed: MagSten was distinguished from all other genotypes (noted as (Ah = Ai)≠(Ab = Am = BatSten)≠MagSten)); (4) and finally, in three assays, A. ipaënsis formed an isolated cluster, being the other cluster formed by all the other genotypes (noted as Ai≠(Ah = Ab = Am = MagSten = BatSten)).
Figure 5  Screenshots of the two most common examples of Arachis B-genome single-nucleotide polymorphism genotyping using Kompetitive allele-specific polymerase chain reaction assays. Both patterns show differentiation between A. ipaënsis K 30076 and the B-genome of A. hypogaea from the wild species A. magna K 30097 and A. batizocoi K 9484 and the induced allotetraploids MagSten and BatSten1. In (A), two clusters are present: one with A. ipaënsis and all A. hypogaea cultivars, and another with the wild species and induced allotetraploids (noted in File S1 as Ah = Ai≠(Ab = Am = MagSten = BatSten)). In (B), three clusters are present. In these cases, A. hypogaea forms a different cluster, intermediate in position [noted in File S1 as Ah≠Ai≠(Ab = Am = MagSten = BatSten)]. All genotypes with A. batizocoi derived rust-resistant cluster in different groups to the susceptible genotypes.