Homoeologous recombination is recurrent in the nascent synthetic allotetraploid Arachis ipaënsis × Arachis correntina4x and its derivatives.

Genome instability in newly synthesized allotetraploids of peanut has breeding implications that have not been fully appreciated. Synthesis of wild species-derived neo-tetraploids offers the opportunity to broaden the gene pool of peanut; however, the dynamics among the newly merged genomes creates predictable and unpredictable variation. Selfed progenies from the neo-tetraploid Arachis ipaënsis × Arachis correntina (A. ipaënsis × A. correntina)4x and F1 hybrids and F2 progenies from crosses between A. hypogaea × [A. ipaënsis × A. correntina]4x were genotyped by the Axiom Arachis 48 K SNP array. Homoeologous recombination between the A. ipaënsis and A. correntina derived subgenomes was observed in the S0 generation. Among the S1 progenies, these recombined segments segregated and new events of homoeologous recombination emerged. The genomic regions undergoing homoeologous recombination segregated mostly disomically in the F2 progenies from A. hypogaea × [A. ipaënsis × A. correntina]4x crosses. New homoeologous recombination events also occurred in the F2 population, mostly found on chromosomes 03, 04, 05, and 06. From the breeding perspective, these phenomena offer both possibilities and perils; recombination between genomes increases genetic diversity, but genome instability could lead to instability of traits or even loss of viability within lineages.

Introduction

Peanut (Arachis hypogaea) is an important crop valued for its high oil and protein content. Originating from South America, peanut is widely grown in the warmer regions of the world yielding a total of 46 million tons in 2018 (http.//www.fao.org/faostat). Cultivated peanut is an allotetraploid with homoeologous subgenomes (AABB; 2n = 4x = 40) sharing greater than 90% DNA sequence similarity (Bertioli ). Its origin was via the formation of a hybrid between two diploid wild species, Arachis ipaënsis (BB; 2n = 2x = 20) and Arachis duranensis (AA; 2n = 2x = 20) followed by a single, or very few, natural polyploidization events less than 10,000 years ago (Bertioli ). This recent polyploid origin created a strong genetic bottleneck and isolated cultivated peanut from its diploid wild relatives (Krapovickas ). The narrow genetic base of cultivated peanut has resulted in limited resistance to many pathogens and diseases. For instance, only moderate levels of resistance to root-knot nematode (Meloidogyne arenaria) and late leaf spot (caused by Nothopassalora personata) were identified among over 1,000 peanut plant introductions in the US (Holbrook and Noe 1992; Holbrook and Anderson 1995). Screening over 10,000 plant introductions for rust (caused by Puccinia arachidis) yielded only 14 moderately rust resistant lines mostly collected from Peru (Subrahmanyam , 1989). In contrast, the wild relatives of peanut harbor strong resistance or immunity to many diseases and pests (Stalker 2017). There are 31 species in the section Arachis with only two of them being tetraploid (2n = 4x = 40), i.e., A. hypogaea and A. monticola. The other 29 species are diploids (2n = 2x = 20 or 2n = 2x = 18). High levels of genetic diversity of the diploid species compared to cultivated peanut is well-known (Kochert ; Moretzsohn ). An early well-documented introgression event resulted in introduction to the peanut crop of near immunity to root-knot nematode from the diploid species Arachis cardenasii (Simpson , 2003; Holbrook ; Nagy ). This introgression used a hybridization scheme known as the tetraploid route (albeit in a complex three-way cross; Simpson 1991). First, an A genome hybrid was made by crossing A. cardenasii with A. diogoi. Then, the B genome (sensu lato) species A. batizocoi was crossed with the A genome hybrid to create a sterile AB hybrid. The AB hybrid was treated with colchicine to double the chromosome number and restore fertility thereby gaining sexual compatibility with cultivated peanut. Subsequently, efforts have focused on the tetraploid route in the simpler form, starting with one A and one B genome species (Favero ; Stalker 2017; Leal-Bertioli ). Recent introgressions from the tetraploid route include a new strong resistance to root-knot nematode from A. stenosperma (Ballen-Taborda ) and improved pod and seed characteristics from A. ipaënsis and A. duranensis (Fonceka , 2012). Besides peanut, introgression from wild relatives brought in significant benefits to other crops such as improved seed yield in Brassica napus (Qian ), fiber quality in cotton (Zhang ), and disease resistance in wheat (Ali ; Rahmatov ).

Following interspecific hybridization and chromosome duplication, active homoeologous recombination can occur between the two distinct types of chromosomes as a consequence of bivalent and tetravalent formation during meiosis (Soltis and Soltis 1999). While bivalent pairing of chromosomes maintains the status of two separate subgenomes, the formation of multivalents leads to the breakdown of their separate identities (Stebbins 1947). The pairing of homoeologous chromosomes can result in replacement of a segment by a copy of the paired homoeologous region through both meiotic crossover and noncrossover (Youds and Boulton 2011). Pioneering cytogenetic studies showed that meiotic chromosomes in peanut consisted of 20 chromosome bivalents in 88–98% of cells; the remainder of cells harbored mostly bivalents together with one or a few univalents, trivalents, or quadrivalents (Husted 1936; Smartt ). This clearly indicated the likelihood of genetic recombination between subgenomes. However, in spite of these findings, almost all genetic studies using DNA markers have assumed only recombination within subgenomes. Only more recently has recombination between subgenomes begun to be quantified, and the effects on genome structure recognized with most of the research focused on neo-tetraploids generated from crosses of cultivated peanut’s putative diploid progenitors (Leal-Bertioli , 2018; Clevenger ).

This study used a neo-tetraploid formed from two wild diploid species. A. ipaënsis Krapov. & W.C. Greg. K30076 (Ipa), and A. correntina (Burkart) Krapov. & W.C. Greg. 9530 (Cor). The former is the B genome progenitor of cultivated peanut. The latter is an A genome, nonprogenitor diploid species that is resistant to late leaf spot, rust, tomato spotted wilt virus, peanut mottle virus, fall armyworm, corn earworm, and aphids (Stalker 2017). New tetraploids [A. ipaënsis K30076 × A. correntina 9530]4x (IpaCor4x) were created from hybrids between Ipa and Cor through colchicine treatment. The first objective of this research was to study the occurrence of homoeologous recombination in the nascent synthetic allotetraploid and the derivative lines from crosses with cultivated peanut. The second objective was to determine the genomic regions and chromosomes predominantly impacted by the homoeologous recombination. The impact of homoeologous recombination was found to increase through generation advancement and was propagated among progenies from A. hypogaea × IpaCor4x crosses, a phenomenon which has been called “the polyploid ratchet” (Gaeta and Pires 2010). The implications of homoeologous recombination events for breeding are discussed.

Materials and methods

Genetic materials

Interspecific hybrids were made between the two diploid species A. ipaënsis K30076 (Ipa; female) and A. correntina 9530 (Cor; male) at North Carolina State University. Multiple plants of each diploid species were used for crossing. Cuttings from the sterile interspecific hybrids were established and shipped to the University of Georgia Tifton Campus for colchicine treatment. Sterility is expected in interspecific hybrids, and the hybrid nature of putative F1s was confirmed by the presence of greater than 80% aborted pollen grains using the Alexander blue staining method (Alexander 1969).

For chromosome doubling, 300 cuttings of the diploid hybrid (∼20 cm long) were immersed in 0.2% colchicine solution for 12–14 hours at room temperature. The treated cuttings were rinsed under continuously running tap water for 1 hour to remove excess colchicine. The cuttings were shortened by 2 cm from the end of the existing cut. The 18 cm long piece was further cut in half yielding two approximately 9 cm long pieces. Fresh cut ends were dipped in Clonex rooting gel (Growth Technology, Western Way, TA, UK) before being planted in Jiffy peat pots (5.7 cm2 sq. × 5.7 cm H) filled with Promix BX growth medium (Premier Tech Horticulture, Quakertown, PA, USA). The cuttings were kept in seed trays enclosed with a clear cover to reach 100% humidity. The colchicine-treated cuttings were grown under 16/8 hours light/dark condition at room temperature for 3–4 weeks to allow root formation before transplanting to a plastic tub (87 × 55 × 20 cm; LxWxH) filled with Promix growth medium in the greenhouse. Cuttings were periodically checked for the formation of pegs and marked with flags. Pods were harvested 40–50 days after peg identification.

S0 plants of IpaCor4x were used as male parents to cross with two advanced peanut breeding lines 13-2113 [(Tifguard × Florida-07) × C725-19-25] and 13-1014 [(Tifguard × Florida-07) × Georgia-06G]. Florida-07 (Gorbet and Tillman 2009), Tifguard (Holbrook ), and Georgia-06G (Branch 2007) are elite peanut cultivars adapted to US southeastern peanut growing regions. C725-19-25 is a high-yielding breeding line with resistance to tomato spotted wilt virus (Holbrook ). Two F2 populations from F1 hybrids (13-1014 × IpaCor4x_S0.2)_F1.4 and (13-1014 × IpaCor4x_S0.5)_F1.4 were grown in the greenhouse for tissue collection and DNA extraction. Each F2 population consisted of 456 individuals.

Genotyping by SNP array

Genetic materials genotyped by the Axiom Arachis 48 K SNP array (Thermofisher Scientific, Waltham, MA, USA) (Clevenger ; Korani ) included DNA from two plants of Ipa and two plants of Cor (Supplementary Table S1). These four plants were sister lines to the original parents used to produce the IpaCor4x neo-tetraploid. The diploid hybrids before and after colchicine treatment were included. Six IpaCor4x_S0 and seven IpaCor4x_S1 neo-tetraploid plants were genotyped. All of the S1 plants were progenies from one mother plant i.e., IpaCor4x_S0.2. Thirty-seven F1s of the A. hypogaea × IpaCor4x_S0 crosses were genotyped together with their A. hypogaea female parents 13-2113 and 13-1014. Two F2 populations (456 lines in each population) descending from (13-1014 × IpaCor4x_S0.2)_F1.4 and (13-1014 × IpaCor4x_S0.5)_F1.4 were genotyped as well. Genomic DNAs were extracted from unexpanded young leaves by Qiagen Plant DNeasy kit (Qiagen, Germantown, MD, USA) and quantified by Quant-iT Picogreen dsDNA assay kit (Thermofisher Scientific, Waltham, MA, USA). Genotyping data were analyzed with the Axiom Analysis Suite (Thermofisher Scientific, Waltham, MA, USA). Based on SNP QC matrix developed by the software, SNP markers were grouped in six categories, i.e., PolyHighResolution, NoMinorHom, MonoHighResolution, CallRateBelowThreshold, OfftargetVariant, and Other. All data analysis in this study was performed with the markers in the PolyHighResolution category since this group of markers has the clearest signal separation.

Curation of homoeologous recombination events in IpaCor4x neo-tetraploid

To study the recombination between the two subgenomes of IpaCor4x, diploid parents, IpaCor2x hybrids, and all of the IpaCor4x S0 and S1 plants were subjected to analysis. To curate the genomic regions hosting homoeologous recombination, we focused on polymorphic markers between Ipa and Cor which would have opposite genotype calls AA versus BB. Without taking into account recombination between subgenomes, we would naively expect IpaCor4x plants would form homoeologous genotyping clusters with AB genotype calls at these loci. In reality, we encountered unexpected genotype calls. All of the genotype calls in four categories AA, BB, AB, and NoCall were output as “call codes” using the Axiom analysis software. The data set was first filtered for polymorphic markers between Ipa and Cor using the “if” argument of Excel. Subsequently, the “countif” function was applied to curate the genotyping of IpaCor4x_S0 and S1 plants deviating from expected homoeologous clusters. All of the markers with this type of sample distribution were visually inspected for clustering patterns. Markers with DNA samples clearly forming a separate cluster between homozygous and heterozygous clusters were assembled. The genotype calls of individuals falling in the separate cluster were adjusted to 75% of the closest homozygous calls. A stretch of at least three consecutive markers demonstrating the same recombination pattern was considered an event of homoeologous recombination.

Curation of new homoeologous recombination events in the F2 populations of A. hypogaea × IpaCor4x_S0 crosses

Manually curating homoeologous recombination events among polymorphic markers of the F2 populations is an arduous task due to the large population size and the abundance of markers. However, monomorphic markers with F2 individuals grouped outside the majority of the population indicated the presence of new homoeologous recombination in these F2 outliers. To curate this type of new homoeologous recombination event, monomorphic markers with F2 individuals demonstrating genotype calls other than the expected AB calls were curated by Excel sorting and countif functions.

Figures of the clustering patterns were exported from the Axiom Analysis Suite software, which carried out clustering in two dimensions (Axiom Analysis Suite User Guide, Affymetrix.com). The X dimension is called “contrast” and the Y dimension is called “size.” They are log-linear combinations of the two allele signal intensities. To illustrate the allele exchange among the groups, sampler nucleotides G and C were used. The actual nucleotides at the targeted loci could be any one of the A, T, C, G. Superscripts next to the nucleotides denoted the sources of alleles. For instance, in the allele combinations such as GacGac, CbiCbi, and GahCah, ac superscript indicated A genome from Cor, bi superscript denoted B genome from Ipa and ah denoted genome composition from A. hypogaea.

To determine the frequency of homoeologous recombination across the genome, the number of lines hosting the recombination was counted for each marker. Genome positions of markers demonstrating homoeologous recombination were referenced to the Ipa genome (Bertioli ).

Results

Homoeologous recombination in the IpaCor4x neo-tetraploid

Out of 300 cuttings of IpaCor2x hybrids treated by colchicine, 21 S0 seeds were harvested. The new IpaCor4x allotetraploid was highly fertile. DNAs from six of the S0 seedlings were genotyped by the SNP array. The occurrence and segregation of chromosome regions demonstrating homoeologous recombination were curated in IpaCor4x_S0.2 and its seven progenies IpaCor4x _S0.2_S1.1 to S1.7 (Table 1). During meiosis of IpaCor4x, genomic regions undergoing homoeologous pairing may result in homoeologous recombination. As one segment of the chromatid is replaced by the opposite subgenome, a set of contiguous homoeologous markers within the replaced chromosome segment should shift as a block from the expected “heterozygote” cluster (alternate SNPs in the two homoeologs are detected) toward the donor cluster. As illustrated at SNP marker AX-147221175 (Figure 1), diploid parents Cor (red triangles, example base call as GacGac; ac denotes A subgenome from Cor) and Ipa (blue triangles, CbiCbi; bi denotes B subgenome from Ipa) were grouped in the genotype clusters AA and BB respectively. Most of the IpaCor4x_S0 plants (S0.5, S0.6, S0.8, S0.10, S0.11, and S0.12) (GacGacCbiCbi, magenta circles) were grouped in the AB cluster as expected indicating their allele composition as GacGacCbiCbi due to the merging of the Ipa and Cor genomes. However, IpaCor4x_S0.2 (two circled magenta triangles) was an outlier since it had a genotype call of AA. This was caused by the replacement of one chromosome segment from Ipa by the homoeologous region from Cor. Consequently, the allele composition became GacGacGacCbi and the hybridization signal was shifted toward the AA cluster where Cor samples were located. Although the software assigned AA genotype calls for the two DNA samples from IpaCor4x_S0.2, the clustering pattern clearly indicated that IpaCor4x_S0.2 and four of her S1 progenies (S1.2, S1.3, S1.4, and S1.7) actually formed a fourth cluster (green oval) between the AB and AA clusters. Their genotype calls were adjusted to 75% of AA (re-coded as 75% of 0 in Table 1) implying one allele from the Ipa subgenome was replaced by Cor. The remaining three S1 progenies S1.1, S1.5, and S1.6 were grouped with Cor. This implied that the allele composition of these individuals became GacGacGacGac i.e., quadriplex for Cor. A set of 19 adjacent markers at the end of chromosome A04/B04 demonstrated the same pattern of homoeologous recombination and segregation among the S1 progenies of IpaCor4x_ S0.2 (Table 1). The size of the chromosome segment was 6.7 Mbp with a marker density of 353 kb/marker. This was the only homoeologous recombination event identified in IpaCor4x_S0.2.

Table 1

Homoeologous recombination between A04 and B04 in IpaCor4x_S0.2 and its segregation among S1 progenies

Probeset_id	Chromosomea	SNP Position (bp)	Ipa	Cor	IpaCor 2x	IpaCor4x _S0.2	IpaCor4x _S0.2_S1.1	IpaCor4x _S0.2_S1.2	IpaCor4x _S0.2_S1.3	IpaCor4x _S0.2_S1.4	IpaCor4x _S0.2_S1.5	IpaCor4x_S0.2 _S1.6	IpaCor4x _S0.2_S1.7	Segment size (bp)	bp/marker
AX-147248448	Araip.B04	124,804,923	0	2	1	75% of 2	2	75% of 2	75% of 2	75% of 2	2	2	75% of 2	6,698,921	352,575
AX-147220907	Araip.B04	124,824,690	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—
AX-147248475	Araip.B04	125,438,618	0	2	1	75% of 2	2	75% of 2	75% of 2	75% of 2	2	2	75% of 2	—	—
AX-147248476	Araip.B04	125,440,304	0	2	1	75% of 2	2	75% of 2	75% of 2	75% of 2	2	2	75% of 2	—	—
AX-147221088	Araip.B04	128,460,377	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—
AX-147248617	Araip.B04	128,469,894	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—
AX-147248627	Araip.B04	128,518,680	0	2	1	75% of 2	2	75% of 2	75% of 2	75% of 2	2	2	75% of 2	—	—
AX-147221124	Araip.B04	129,198,098	0	2	1	75% of 2	2	75% of 2	75% of 2	75% of 2	2	2	75% of 2	—	—
AX-147221160	Araip.B04	129,385,417	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—
AX-147221161	Araip.B04	129,385,840	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—
AX-147248696	Araip.B04	129,600,869	0	2	1	75% of 2	2	75% of 2	75% of 2	75% of 2	2	2	75% of 2	—	—
AX-147221210	Araip.B04	129,796,029	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—
AX-147248730	Araip.B04	129,989,016	0	2	1	75% of 2	2	75% of 2	75% of 2	75% of 2	2	2	75% of 2	—	—
AX-147248735	Araip.B04	130,112,475	0	2	1	75% of 2	2	75% of 2	75% of 2	75% of 2	2	2	75% of 2	—	—
AX-147248739	Araip.B04	130,210,807	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—
AX-176791380	Araip.B04	131,369,085	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—
AX-147221357	Araip.B04	131,423,712	0	2	1	75% of 2	2	75% of 2	75% of 2	75% of 2	2	2	75% of 2	—	—
AX-147221364	Araip.B04	131,478,950	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—
AX-147221371	Araip.B04	131,503,844	2	0	1	75% of 0	0	75% of 0	75% of 0	75% of 0	0	0	75% of 0	—	—

Genotype calls from the SNP array were re-coded as follows, genotype call AA = 0; BB = 2; AB = 1 to avoid confusion with the description of subgenomes. Seventy-five percent of a genotype call indicates the dosage of a subgenome allele was increased by 25% as a result of subgenome recombination.

Chromosome and SNP positions were given relative to Arachis ipaensis genome (peanutbase.org). The recombination events occurred at these loci were actually between the homoeologous chromosomes.

Figure 1

A SNP marker demonstrating a Cor allele replacing an Ipa allele in IpaCor4x_S0.2 and S1 progenies. Cor, red triangles with GacGac as an example of base call, and Ipa (CbiCbi, blue triangles) were grouped in genotype calls of AA and BB respectively. ac superscript indicates A genome from Cor, bi superscript denotes B genome from Ipa. IpaCor2x (GacCbi, green circles), and IpaCor4x_S0.5, 6, 8, 10,11,12(GacGacCbiCi, magenta circles) were clustered in the expected AB group. IpaCor4x_S0.2 (GacGacGacCbi, two circled magenta triangles) and some other samples formed a separate group between cluster AB and AA which was enclosed by a green oval. Four of the S1 progenies from IpaCor4x_S0.2 (GacGacGacCbi, dark purple triangles) fell in this new group. The remaining three S1 progenies (GacGacGacGac, dark purple triangles) grouped with Cor forming a quadriplex locus.

Four new homoeologous recombination events were found in three S1 progenies, IpaCor4x_S0.2 S1.1, S1.5, S1.7 (Table 2, Supplementary Table S2). At the top of chromosome A03/B03, a 5 Mbp chromatid segment had Cor replacing Ipa in IpaCor4x_ S0.2_ S1.1. Most of the chromosome B04 (∼120 Mbp) had homoeologous recombination in both IpaCor4x_ S0.2_ S1.1 and S1.7 yet in opposite directions, i.e., the Cor allele replaced the Ipa allele in S1.1 and vice versa in S1.7. At the top of chromosome B05, a 14 Mbp segment had one copy of alleles from Cor replaced by Ipa in IpaCor4x_ S0.2_ S1.5. These new events among the S1 progenies indicated that active homoeologous recombination continues to occur during generation advancement of the neo-tetraploid.

Table 2

Homoeologous recombination events in IpaCor4x_S0 and S1 plants other than those listed in Table 1

Genotype	Direction of subgenome recombination	Chromosomea	Left border (bp)	Right border (bp)	Segment size (bp)	No. of markers	bp/marker
IpaCor4x_S0.2_S1.1	Cor replaced 50% of Ipa	Araip.B03	2,065,843	7,115,308	5,049,465	19	265,761
IpaCor4x_S0.2_S1.1	Cor replaced 50% of Ipa	Araip.B04	4,420,103	124,301,830	119,881,727	96	1,248,768
IpaCor4x_S0.2_S1.7	Ipa replaced 50% of Cor	Araip.B04	613,773	124,301,830	123,688,057	109	1,134,753
IpaCor4x_S0.2_S1.5	Ipa replaced 50% of Cor	Araip.B05	1,426,280	15,568,257	14,141,977	23	614,869
IpaCor4x_S0.11	Ipa replaced 50% of Cor	Araip.B04	123,807,791	124,301,830	494,039	7	70,577
IpaCor4x_S0.12	Cor replaced 50% of Ipa	Araip.B04	613,773	124,298,527	123,684,754	108	1,145,229
IpaCor4x_S0.6	Cor replaced 50% of Ipa	Araip.B05	1,426,280	148,997,085	147,570,805	91	1,621,657
IpaCor4x_S0.5	Ipa replaced 50% of Cor	Araip.B07	686,538	2,077,018	1,390,480	13	106,960

Chromosome and SNP positions were given relative to A. ipaensis genome (peanutbase.org). The recombination events occurred at these loci were actually between the homoeologous chromosomes.

In addition to the IpaCor4x_S0.2 family, homoeologous recombination was identified in the four other IpaCor4x_S0 plants on chromosomes A04/B04, A05/B05, and A07/B07 (Table 2). The two events on chromosome A04/B04 were opposite in direction of homoeologous recombination and occurred in two separate S0 plants. The event in IpaCor4x_S0.11 spanned a small segment of 0.5 Mbp whereas the event in IpaCor4x_S0.12 almost covered the whole of chromosome A04/B04 (124 Mbp). The event captured on chromosome A05/B05 also encompassed nearly the whole chromosome (148 Mbp) in IpaCor4x_S0.6. The event identified on the top of chromosome A07/B07 was 1.4 Mbp in IpaCor4x_S0.5. Among the nine events found in IpaCor4x_S0 and S1 plants, five of them had Ipa replacing the Cor subgenome and four of them had Cor replacing the Ipa subgenome.

Segregation of genomic regions subjected to recombination among F1 hybrids

Three of the IpaCor4x_S0 plants S0.2, S0.5, and S0.6 were used as males to cross with two elite A. hypogaea breeding lines 13-2113 and 13-1014 and produced 37 F1 progenies. Hybridity of all 37 F1 progenies was confirmed by the expected heterozygous calls from 1024 polymorphic markers between the A. hypogaea and IpaCor4x_S0 parents (Supplementary Table S3). Each of these three IpaCor4x_S0 male parents had a genomic region with chromosomal recombination on chromosomes A04/B04, A07/B07, and A05/B05, respectively (Tables 1 and 2). Segregation of these genomic regions harboring homoeologous recombination among F1 hybrids was observed (Supplementary Table S4; red font genotype calls). Two examples demonstrating segregation of the inherited homoeologous recombination from the IpaCor4x_S0 parents among F1 hybrids are illustrated in Supplementary Figures S1 and S2. In both cases, the IpaCor4x_S0 parent had a genomic region with pre-existing homoeologous recombination and the A. hypogaea female parent presented in either the heterozygote (Supplementary Figure S2) or homozygote (Supplementary Figure S3) cluster.

At the first locus (Supplementary Figure S2A), Cor (GacGac) and Ipa (CbiCbi) had genotype calls of AA and BB, respectively. The two DNA samples from IpaCor4x_S0.6 that showed homoeologous exchange (GacGacGacCbi) grouped in a separate cluster between the AB and AA clusters indicating that one Ipa allele was replaced by Cor. The two A. hypogaea female parents had the genotype call of AB indicating the presence of polymorphism between its own subgenomes (GahGahCbhCbh). Upon hybridization, two F1 genotypes were expected, i.e., GahGacCbhCbi and GahGacGacCbh (Supplementary Figure S2B) and realized; three F1 hybrids had genotype calls of AB (GahGacCbhCbi) and seven F1 hybrids had genotype calls of 75% AA (GahGacGacCbh).

At the second locus (Supplementary Figure S3A), Cor (GacGac) and Ipa (CbiCbi) had genotype calls of AA and BB, respectively. The IpaCor4x_S0.6 formed a separate group between AB and AA indicating that the Ipa allele was replaced by Cor (GacGacGacCbi). The A. hypogaea female parent (CahCahCbhCbh) was monomorphic between its subgenomes and fell in the BB genotype cluster with Ipa and a few other samples. The expected allele compositions of F1 hybrids were CahGacCbhGac and CahGacCbhCbi (Supplementary Figure S3B). Indeed, three of the F1 hybrids had genotype calls of 75% BB indicating their allele composition of CahGacCbhCbi; the remaining seven F1 hybrids had genotype calls of AB indicating their allele composition of CahGacCbhGac.

Segregation of pre-existing homoeologous recombination events was found in most of the F1 hybrids. All of the 10 F1 hybrids from A. hypogaea × IpaCor4x_S0.6 segregated for most of the 91 markers detecting homoeologous recombination on chromosome A05/B05 (Supplementary Table S4). One F1 hybrid 13-1014 × IpaCor4x_S0.6_F1.6 inherited a truncated 7.9 Mbp recombined segment at the top of chromosome A05/B05 suggesting an additional round of recombination occurred in this F1 hybrid. Among the 16 F1 hybrids from A. hypogaea × IpaCor4x_S0.5 crosses, five of them inherited the ∼1.4 Mbp segment that showed homoeologous recombination from the male parent and the remaining 11 F1 hybrids had the same genotype call as the A. hypogaea parents at the top of chromosome B07. Expected segregation was found in most of the F1 hybrids from the A. hypogaea × IpaCor4x_S0.2 crosses. All of the 18 markers within the 6.7 Mbp segment at the bottom of A04/B04 showed segregation, in which six F1 hybrids shared the genotype call of the male parent and the remaining four shared the genotype call of the female parent (Supplementary Table S4, Table 1). Only one F1 hybrid from this cross, 13-1014 × IpaCor4x_S0.2_F1.4 was an exception. This individual experienced a new round of homoeologous recombination during hybridization. Consequently, it had a homoeologous recombination opposite to the other 10 F1 siblings, that is, it had one chromosome segment of Ipa replacing that of Cor in this region. The expected segregation patterns among the F1 hybrids of the homoeologous recombination events from neo-tetraploid parents confirmed the presence and inheritance of these events.

New homoeologous recombination events captured in F1 hybrids

In addition to the inherited recombination events, 27 new recombination events on chromosomes A02/B02, A03/B03, A04/B04, A05/B05, A06/B06, and A07/B07 were captured among the F1 hybrids (Table 3, Supplementary Table S4). There were four events on chromosome 2, where the segment sizes ranged from 2 to 106 Mbp. One event was identified on chromosome 3 with a segment size of 5 Mbp. There were seven events on chromosome 4, where the segment sizes ranged from 0.5 to 130 Mbp. The 0.5 Mbp recombination event was identified in two F1 hybrids, i.e., 13-1014 × IpaCor4x_S0.2_F1.4 and 13-2113 × IpaCor4x_S0.2_F1.6. The inheritance of this 0.5 Mbp region in the F2 population descending from 13-1014 × IpaCor4x_S0.2_F1.4 was demonstrated (Table 3). Three events were found on A05/B05 with the segment sizes ranging from 3 to 17 Mbp. Seven events were identified on A06/B06 with segment sizes ranging from 2 to 111 Mbp. Four events were found on A07/B07 with segment sizes ranging from 0.3 to 124 Mbp. As for the direction of genome recombination, there were 16 events where Cor alleles replaced the Ipa alleles and 11 events that demonstrated the opposite direction of recombination. Most of the recombination break points occurred closer to the ends of the chromosome arms. Most of the events were in different regions except for two consecutive recombination events in opposite directions on A06/B06 in 13-2113 × IpaCor4x_S0.2_F1.6 (Supplementary Table S4).

Table 3

New homoeologous recombination detected among F1 progenies of A. hypogaea × IpaCor4x_S0 crosses

Genotype	Direction of subgenome recombination	Chromosomea	Left border (bp)	Right border (bp)	Segment size (bp)	No. of markers	bp/marker
13-2113 × IpaCor4x_S0.6_F1.1	Cor replace 50% of Ipa	Araip.B02	102,655,016	104,599,332	1,944,316	9	216,03
13-2113 × IpaCor4x_S0.5_F1.1	Cor replace 50% of Ipa	Araip.B02	61,718,684	106,235,603	44,516,919	25	1,780,677
13-1014 × IpaCor4x_S0.5_F1.7	Cor replace 50% of Ipa	Araip.B02	295,929	95,321,834	95,025,905	73	1,301,725
13-1014 × IpaCor4x_S0.6_F1.7	Cor replace 50% of Ipa	Araip.B02	295,929	106,273,695	105,977,766	103	1,028,910
13-2113 × IpaCor4x_S0.6_F1.1	Cor replace 50% of Ipa	Araip.B03	2,029,212	6,739,039	4,709,827	25	188,39
13-1014 × IpaCor4x_S0.2_F1.4	Ipa replace 50% of Cor	Araip.B04	123,807,791	124,298,527	490,736	4	122,684
13-2113 × IpaCor4x_S0.2_F1.6	Ipa replace 50% of Cor	Araip.B04	123,807,791	124,298,527	490,736	4	122,684
13-2113 × IpaCor4x_S0.5_F1.3	Ipa replace 50% of Cor	Araip.B04	129,385,417	130,961,045	1,575,628	8	196,954
13-2113 × IpaCor4x_S0.5_F1.7	Cor replace 50% of Ipa	Araip.B04	3,490,627	6,128,842	2,638,215	15	175,881
13-1014 × IpaCor4x_S0.6_F1.1	Cor replace 50% of Ipa	Araip.B04	204,623	6,097,596	5,892,973	44	133,931
13-1014 × IpaCor4x_S0.2_F1.1	Ipa replace 50% of Cor	Araip.B04	140,971	23,049,636	22,908,665	70	327,267
13-2113 × IpaCor4x_S0.6_F1.2	Ipa replace 50% of Cor	Araip.B04	647,417	46,100,243	45,452,826	63	721,473
13-1014 × IpaCor4x_S0.6_F1.4	Ipa replace 50% of Cor	Araip.B04	605,143	130,961,045	130,355,902	132	987,545
13-2113 × IpaCor4x_S0.5_F1.2	Cor replace 50% of Ipa	Araip.B05	145,358,753	148,997,085	3,638,332	9	404,259
13-1014 × IpaCor4x_S0.2_F1.3	Cor replace 50% of Ipa	Araip.B05	6,425,776	16,814,071	10,388,295	23	451,665
13-2113 × IpaCor4x_S0.5_F1.2	Cor replace 50% of Ipa	Araip.B05	7,141,413	24,364,583	17,223,170	23	748,833
13-1014 × IpaCor4x_S0.6_F1.4	Cor replace 50% of Ipa	Araip.B06	133,505,937	135,831,090	2,325,153	33	70,459
13-2113 × IpaCor4x_S0.2_F1.6	Cor replace 50% of Ipa	Araip.B06	129,731,047	135,639,285	5,908,238	90	65,647
13-2113 × IpaCor4x_S0.2_F1.6	Ipa replace 50% of Cor	Araip.B06	123,590,343	129,526,826	5,936,483	19	312,446
13-2113 × IpaCor4x_S0.6_F1.1	Ipa replace 50% of Cor	Araip.B06	129,740,694	135,831,090	6,090,396	85	71,652
13-2113 × IpaCor4x_S0.6_F1.2	Cor replace 50% of Ipa	Araip.B06	127,136,710	135,831,090	8,694,380	91	95,543
13-2113 × IpaCor4x_S0.5_F1.3	Cor replace 50% of Ipa	Araip.B06	121,832,857	134,519,804	12,686,947	92	137,90
13-1014 × IpaCor4x_S0.5_F1.1	Ipa replace 50% of Cor	Araip.B06	249,894	110,859,588	110,609,694	74	1,494,726
13-2113 × IpaCor4x_S0.2_F1.2	Ipa replace 50% of Cor	Araip.B07	763,999	1,029,451	265,452	3	88,484
13-1014 × IpaCor4x_S0.6_F1.2	Cor replace 50% of Ipa	Araip.B07	686,538	1,394,692	708,154	8	88,519
13-1014 × IpaCor4x_S0.2_F1.2	Ipa replace 50% of Cor	Araip.B07	686,538	5,579,063	4,892,525	41	119,330
13-2113 × IpaCor4x_S0.5_F1.7	Cor replace 50% of Ipa	Araip.B07	2,276,988	125,807,926	123,530,938	96	1,286,781

Chromosome and SNP positions were given relative to A. ipaensis genome (peanutbase.org). The recombination events occurred at these loci were actually between the homoeologous chromosomes.

Segregation of the F2 population descending from 13-1014 × IpaCor4x_S0.2_F1.4 at the bottom of chromosome 4

Of the two F2 populations descending from 13-1014 × IpaCor4x_S0.2_F1.4 and 13-1014 × IpaCor4x_S0.5_F1.4 that were genotyped by the SNP array, there was only one pre-existing homoeologous genomic region at the bottom of A04/B04 of 13-1014 × IpaCor4x_S0.2_F1.4 identified in this study (Supplementary Table S4). In the genotyping profile of the parental lines and F1 hybrids (Figure 2A), Cor and Ipa had genotype calls of BB (GacGac) and AA (CbiCbi), respectively, at marker AX-147221124 (one of the markers at the end of B04). The A. hypogaea female parent was polymorphic between its subgenomes (GahGahCbhCbh). IpaCor4x_S0.2 and six F1 hybrids from A. hypogaea × IpaCor4x_S0.2 had one allele from Cor replacing that of Ipa (CbiGacGacGac) forming a cluster in between genotypes AB and BB. Four other F1 hybrids fell in the AB cluster as expected. However, IpaCor4x_S0.2_F1.4 and a few other samples formed a cluster between AA and AB (GahCbiCbCbi) indicating that a new round of homoeologous recombination during hybridization led to the opposite direction of exchange in this individual, i.e., Ipa replaced Cor.

Figure 2

Segregation of the F2 population derived from 13-1014 × IpaCor4x_S0:2_F1:4 when the female parent was genotyped as AB at this locus. (A) Genotyping profile from the SNP array of the parental lines and F1 hybrids. Homoeologs at this locus were detected in the A. hypogaea parent (GahGahCbhCbh; golden circles), and the IpaCor4x_S0.2 parent (CbiGacGacGac; two circled magenta circles) had one allele from Ipa replaced by Cor. It was expected to produce two types of F1 hybrids (panel C, black font), i.e., GahGacCahGac (genotype call 75% BB) and GahCbiCbhGac (genotype call AB). Indeed, six of the F1 hybrids (circled white circles in the green oval) were in the 75% BB group and four F1 hybrids (circled white triangles in the yellow oval) were in the AB group. The 11th F1 hybrid, 13-1014 × IpaCor4x_S0.2_F1.4 (green font), experienced a new round of homoeologous recombination with the Ipa allele replacing Cor resulting in GahCbiCbiCb (genotype call 75% AA). (B) The F2 population derived from this hybrid segregated disomically in three clusters following a ratio close to the expected 1:2:1. Although most F2 individuals fall in the expected clusters, there were three F2s that clustered in the 75% BB group indicating that new homoeologous recombination occurred in these three individuals during selfing. The ac superscript indicates A subgenome from A. correntina (red filled samples), bi superscript denotes B subgenome from A. ipaensis (blue filled samples). The ancestral origin of the A. hypogaea parents is unclear at this locus, therefore, ah is used to denote their genome composition.

Segregation of the F2 population descended from 13-1014 × IpaCor4x_S0.2_F1.4 confirmed the genome composition of this hybrid. The F2 population consisted of 456 individuals (Figure 2B). The majority of the population segregated into three clusters following a ratio of 109:219:121 among three genotypes GGCC: GCCC: CCCC as expected from the genotype composition of GahCbiCbCbi of the F1 hybrid (Figure 2C). This ratio was close to 1:2:1 (Chi-square = 0.91; P = 0.63) indicating that disomic segregation was predominant. In addition, there were three F2 individuals that clustered with IpaCor4x_S0.2 (CGGG) indicating a new homoeologous recombination event in these three individuals resulting in 75% allele composition from Cor and 25% from Ipa. A stretch of 19 adjacent markers including 15 markers within the region inherited from IpaCor4x_S0.2 and four neighboring markers came from a new homoeologous recombination event in the F1 hybrid (Table 4).

Table 4

Segregation of the F2 population from 13-1014 × IpaCor4x_S0.2_F1.4 at the end of chromosome B04a

								Genotype distribution of the F2 population
Event history	probeset_id	Start position (bp)a	Ipa	Cor	IpaCor4x _S0.2	13- 1014	13-1014 × IpaCor4x _S0.2_F1.4	Genotype call 0	Genotype call 75% of 0	Genotype call 1	Genotype call 75% of 2	Genotype call 2	No Call
New event	AX-147248422	123,807,791	0	2	75% of 2	1	75% of 0	103	228	120	2	0	3
in F1 hybrid	AX-147248424	124,023,584	0	2	75% of 2	1	75% of 0	103	226	123	2	0	2
	AX-147220849	124,269,424	2	0	75% of 0	1	75% of 2	0	2	124	224	103	5
	AX-147248402	124,298,527	2	0	75% of 0	1	75% of 2	0	2	123	225	103	5
Inherited from	AX-147248448	124,804,923	0	2	75% of 2	1	75% of 0	105	224	121	2	0	4
IpaCor4x_S0.2	AX-147220907	124,824,690	2	0	75% of 0	1	75% of 2	0	2	127	226	103	0
	AX-147248475	125,438,618	0	2	75% of 2	1	75% of 0	102	226	120	2	0	6
	AX-147248476	125,440,304	0	2	75% of 2	1	75% of 0	104	225	125	2	0	0
	AX-147221088	128,460,377	2	0	75% of 0	1	75% of 2	0	3	123	222	107	4
	AX-147248617	128,469,894	2	0	75% of 0	1	75% of 2	0	2	116	220	108	10
	AX-147248627	128,518,680	0	2	75% of 2	1	75% of 0	105	219	121	2	0	9
	AX-147221124	129,198,098	0	2	75% of 2	1	75% of 0	109	219	119	3	0	7
	AX-147221160	129,385,417	2	0	75% of 0	1	75% of 2	0	2	121	215	110	7
	AX-147221161	129,385,840	2	0	75% of 0	1	75% of 2	0	2	119	217	111	7
	AX-147248696	129,600,869	0	2	75% of 2	1	75% of 0	110	219	119	2	0	6
	AX-147221210	129,796,029	2	0	75% of 0	1	75% of 2	0	3	113	218	106	16
	AX-147248730	129,989,016	0	2	75% of 2	1	75% of 0	110	218	117	2	0	9
	AX-147248735	130,112,475	0	2	75% of 2	1	75% of 0	110	218	120	2	0	6
	AX-147248739	130,210,807	2	0	75% of 0	1	75% of 2	0	2	123	216	115	0
	AX-176791380	131,369,085	2	0	75% of 0	0	1	31	101	191	101	32	0
	AX-147221357	131,423,712	0	2	75% of 2	2	1	32	103	191	100	30	0
	AX-147221364	131,478,950	2	0	75% of 0	0	1	30	104	183	107	32	0
	AX-147221371	131,503,844	2	0	75% of 0	0	1	30	103	188	104	31	0

Genotype calls from the SNP array were re-coded as follows: genotype call AA = 0; BB = 2; AB = 1 to avoid confusion with the description of subgenomes. Seventy-five percent of a genotype call indicates the dosage of a subgenome allele was increased by 25% as a result of subgenome recombination.

Chromosome and SNP positions were given relative to A. ipaensis genome (peanutbase.org). The recombination events occurred at these loci were actually between the homoeologous chromosomes.

The last four markers at the end of chromosome A04/B04 (Table 4) shared a different segregation pattern due to the homozygous genotype call of the female parent (Figure 3). At marker AX-147221375, Cor and Ipa had genotype calls of BB (GacGac) and AA (CbiCbi), respectively. The female parent was monomorphic between its two subgenomes (Figure 3A) and positioned in the genotype BB cluster along with Cor. The male parent IpaCor4x_S0.2 had Cor alleles replacing Ipa (GacGacGacCbi) due to homoeologous recombination. Two types of F1 hybrids were expected to be produced with genotype compositions of GahGacGahGac and GahGacGbhCbi (Figure 3C). Indeed, six of the F1 hybrids fell in the 75% BB (GahGacGbhCbi) genotype group; four of the F1 hybrids fell in the BB (GahGacGahGac) genotype group. 13-1014 × IpaCor4x_S0.2_F1.4 (GahCbiGbCbi) shifted right from the other F1 hybrids and grouped in the AB genotype due to a new round of homoeologous recombination. As expected, the F2 population from this hybrid segregated into five clusters, i.e., GGGG, GGGC, GGCC, CCCG, CCCC (Figure 3C) at this locus in a ratio of 30:107:191:102:38 or close to the expectation for a disomic segregation ratio of 1:4:6:4:1 (Chi-square = 6.78; P  = 0.14) (Table 3). The remaining three markers at the bottom of B04 shared similar segregation ratios. Therefore, disomic segregation of the F2 population was observed for all of the markers at the bottom of A04/B04 where the subgenome exchange from the male parent was inherited.

Figure 3

Segregation of the F2 population derived from 13-1014 × IpaCor4x_S0.2_F1.4 when the female parent was genotyped as BB at this locus. (A) Genotyping profile from the SNP array for the parents and F1 hybrids. The A. hypogaea parents (GahGahGbhGbh; golden triangles) were monomorphic at this locus and IpaCor4x_S0.2 parent (GacGacGacCbi; two circled magenta circles) had one allele from Ipa replaced by Cor. It was expected to produce two types of F1 hybrids (panel C black font), i.e., GahGacGahGac (genotype call BB) and GahGacGbhCbi(genotype call 75% BB). Indeed, six of the F1 hybrids (circled white circles) were in the 75% BB group and four (circled white triangles) were in the BB group. The 11th F1 hybrid, 13-1014 × IpaCor4x_S0.2_F1.4 (green font), experienced a new round of homoeologous recombination in which the Ipa allele replaced Cor resulting in the genome composition of GahCbiGbCbi (genotype call AB). (B) The F2 population from this hybrid was expected to segregate into five clusters. (C) The distribution of the F2 population followed a ratio close to the disomic segregation, i.e., 1:4:6:4:1.

New recombination events captured in the two F2 populations descending from A. hypogaea × IpaCor4x crosses

New homoeologous recombination events were captured from the F2 populations by curating monomorphic markers harbored by F2 individuals and deviating from the parental calls. At marker AX-176801822 (Figure 4), there were five genotype clusters formed among the parents and the F2 population. Cor (GacGac) and two F2 individuals (GGGG) had a genotype call of BB whereas Ipa (CbiCbi) had a genotype call of AA. The male parent IpaCor 4x_S0.2 (GacGacCbiCbi) had an AB genotype call indicating there was no pre-existing homoeologous recombination in the male parent at this locus. The female parent 13-1014 (GahGahCbhCbh), 13-1014xIpaCor 4x_S0.2_ F1.4 (GacGahCbiCbh), and most of the F2 individuals were grouped in the AB cluster as well. However, there were 18 F2 individuals that shifted right from the AB cluster and formed a separate cluster (GCCC) between the AB and AA genotype calls. This suggested that an Ipa allele replaced a Cor allele in these individuals as a result of homoeologous recombination. There were another 29 F2 individuals that shifted left from the AB cluster and formed a separate cluster (GGGC) between AB and BB genotype calls. These F2 individuals were evidence of homoeologous recombination with a Cor allele replacing an Ipa allele at this locus. Two F2 individuals (GGGG) had become quadriplex at this locus with all alleles from Cor. Therefore, there were 49 F2 progenies that deviated from the expected monomorphic AB calls and demonstrated homoeologous recombination in both directions at this locus. New homoeologous recombination events captured in the two populations were listed (Supplementary Table S5) and presented as a zoomed-out image for global view (Supplementary Figure S4). The two populations shared a similar distribution of recombination events across the chromosomes (Supplementary Figure S4). A03/B03, A04/B04, A05/B05, and A06/B06 were densely populated with homoeologous recombination events and accounted for 95% of the total number of events. The ratio of markers showing Ipa replacing the Cor subgenome versus those with Cor replacing the Ipa subgenome was 0.995 across both populations. However, there was a preference for one direction of homoeologous recombination over the other on individual chromosomes (Figure 5). The frequency of Cor replacing Ipa was higher than the frequency of Ipa replacing Cor on chromosome A03/B03 and lower on chromosomes A04/B04, A05/B05, and A06/B06. The incidence of homoeologous recombination events was higher at distal regions of chromosomes and lower near the centromeres. Among the 907 F2 lines, 416 (46%) hosted at least one event of homoeologous recombination. Tetrasomic recombination was identified in four F2 individuals on chromosome A03/B03 (Supplementary Table S5 dark blue and dark pink highlighted events).

Figure 4

A monomorphic marker between IpaCor4x_S0.2 and 13-1014 parents demonstrated new homoeologous recombination among the F2 population. There were five genotype clusters formed among the parents and the population. Cor (red triangles, GacGac) and two F2 individuals (nonfilled squares, GGGG) grouped in the purple oval with genotype calls of BB. Ipa (blue triangles and squares, CbiCbi) formed a group in the brown oval with genotype call of AA. The female parent 13-1014 (orange circle; GahGahCbhCbh), male parent IpaCor 4x_S0.2 (magenta circles, GacGacCbiCbi) and 13-1014xIpaCor 4x_S0.2_ F1.4 (yellow circle; GacGahCbiCbh) were grouped in the yellow oval with AB genotype call indicating there was no pre-existing homoelogous exchange in the male parent and the F1 hybrid. Most of the F2 individuals (nonfilled circles) were grouped in the AB cluster as expected from this type of monomorphic marker. However, there were 18 F2 individuals (upward clear triangles; GCCC) shifted right from the AB genotype cluster and formed a separate cluster (red oval) between the AB and AA genotype calls. This suggests that an Ipa replaced a Cor allele in these individuals as a result of homeologous recombination. There were another 29 F2 individuals (downward triangles; GGGC) shifted left from the AB genotype cluster that formed a separate cluster (blue oval) between AB and BB clusters. These F2 individuals were subjected to homeologous recombination with a Cor allele replacing an Ipa allele at this locus. Two F2 individuals (nonfilled squares in the purple circle; GGGG) derived from fusion of recombinant gametes resulted in quadriplex alleles from Cor at this locus.

Figure 5

Frequency of new recombination events captured in the F2 populations descended from 13-1014 × IpaCor4x_S0.2_F1.4 and 13-1014 × IpaCor4x_S0.5_F1.4. Pink dots stand for Cor subgenome replacing that of Ipa. Blue dots stand for Ipa subgenome replacing that of Cor. The four chromosomes plotted accounted for 95% of the total number of recombination events.

Discussion

In an effort to increase the genetic diversity of cultivated peanut, crosses were made between two peanut diploid relatives Ipa and Cor. The resulting IpaCor2x hybrids were highly sterile, consistent with the well-documented sterility of diploid hybrids from A and B genome species (Krapovickas ). Formation of seeds from the colchicine-treated hybrids indicated the success of chromosome doubling. In newly formed allotetraploids, the two distinct genomes are expected to function in one cytoplasm and to be inherited through regular meiotic divisions (Lukens ). However, it is known that while the majority of the genome of many allotetraploids does pair bivalently within each subgenome during meiosis, meiotic recombination between the subgenomes can occur when multivalent associations form. In addition, homoeologous chromosomes may be used as templates to repair double-stranded breaks. Both of these mechanisms, homoeologous meiotic recombination and homoeologous repair, can result in the duplication or elimination of corresponding homoeologous regions and alter gene dosage (Szadkowski ; Youds and Boulton 2011; Leal-Bertioli ). Our genotyping data from IpaCor4x S0 plants revealed five independent homoeologous recombination events in each of the five S0 plants. All had one block of alleles on a chromosome segment replaced by its homoeologous alleles. This observation suggests that during meiosis of the tetraploid cells, multivalent association of homoeologous chromosomes and homoeologous recombination occurred as early as the formation of S0 neo-tetraploids. Our finding is consistent with previous cytological studies with Arachis interspecific F1 hybrids (Stalker 1991) and F1 hybrids derived from A. hypogaea × allotetraploids (Gardner and Stalker 1983). Both studies presented evidence of multivalent formation in meiotic cells of the hybrids although at low frequencies.

The homoeologous event in the neoallotetraploid was heritable, as evidenced by the segregation of the recombined region among the S1 progenies. In our study, three S1 from IpaCor4x S0.2 became quadriplex and the other four shared the genotype of the mother plant. This suggests normal segregation of the established homoeologous recombination event during generation advancement. In addition to the inherited recombinant subgenome, four new homoeologous recombination events were found in three of the S1 progenies indicating that recombination between subgenomes continued throughout generation advancement. Therefore, it is apparent that homoeologous recombination occurred at the nascent and early generations of the neo-tetraploids similar to reports on synthetic polyploids of Brassica (Song ; Lukens ; Szadkowski ).

Crossing the neo-tetraploids with cultivated breeding lines initiates introgression of chromosomal segments from wild genomes. The inheritance of the established subgenome exchange from IpaCor4x S0 parents was observed in the F1 hybrids from crosses between A. hypogaea and IpaCor4x. In the meantime, new events of homoeologous exchange were identified in the F1 hybrids suggesting active homoeologous recombinants were captured in the first zygotes of crossing. The established homoeologous recombination event on chromosome 04 captured in the IpaCor4x male parent was found to segregate disomically in most of the F2 population. The impact of homoeologous recombination events on allele dosage is potentially large given their accumulation within the F2 population with nearly half of the population possessing at least one event. Interestingly, the new events were preferentially distributed on chromosomes A03/B03, A04/B04, A05/B05, and A06/B06. These chromosomes hosted over 95% of the homoeologous recombination events. The A. hypogaea genome contains historical quadriplex loci on these chromosomes derived from the homoeologous recombination between the ancestral A. duranensis and Ipa (A. ipaënsis) subgenomes (Leal-Bertioli ; Bertioli , 2019). It is possible that the existing tetrasomic regions in cultivated peanut increase the frequency of homoeologous recombination among progeny derived from crosses between A. hypogaea and interspecific materials. Most recently, hot spots of homoeologous recombination were found pre-dominantly located within genic regions in wheat and other polyploids including peanut (Zhang ). Consequently, novel gene transcripts and proteins produced would contribute to neo- and sub-functionalization of genes and phenotypic changes. For instance, spontaneous flower color change from yellow to orange was reported in the new synthetic allotetraploid A. ipaënsis × A. duranensis4x, which was ascribed to homoeologous recombination (Bertioli ). Yield increase due to intersubgeomic heterosis was reported in B. napus upon introgression from Brassica rapa (Qian ).

The frequent homoeologous recombination in these genetic materials derived from the neo-tetraploid poses a challenge in genetic mapping. Most often, genetic markers linked to the homoeologous recombination were excluded from genetic map construction since they appear to be present/absent or cause severe segregation distortion from the disomic inheritance model. However, QTL analysis with genetic markers associated with presence and absence (PAV) variations was able to identify major effect QTL for seed quality and flowering pattern in B. napus (Stein ). Disease resistance QTL against Sclerotinia stem rot and blackleg disease for oilseed rape were identified in the PAV regions (Gabur ). Homoeologous recombination was found to be the underlying cause of these trait variations. More recently, mixed inheritance following both disomic and tetrasomic patterns of inheritance among the population from a cross between A. hypogaea and (A. duranensis × A. batizocoi)4x supported the occurrence of homoeologous recombination in these progenies (Nguepjop ). Therefore, genetic analysis of peanut populations with wild introgression needs to consider the markers associated with homoeologous recombination.

The high frequency of genome instability of the neo-tetraploid and its derivative lines may offer both possibilities and perils from the breeding perspective. On the one hand, the instability offers an unprecedented opportunity to introduce new phenotypes and variations. Conversely, the desired traits may be unstable and can be lost as generations advance. Backcross and generation advancement may stabilize the genome and trait expression. To this end, we are advancing a BC1F1 population by single seed descent with A. hypogaea as the recurrent parent. The population, being on average 75% of the domesticated genome should predominantly express cultivated phenotypes. The other 25% of wild genome composition may be stabilized through generations of selfing. This type of genetic material will provide the peanut breeding community with valuable genetic diversity to improve disease resistance and other agronomic traits in cultivated peanuts.