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Introduction
The genusArachisencompasses 82 species described based on the integration of varied criteria, such as morphological, cytogenetic, and molecular characterization, geographic distribution, and interspecific cross-compatibility (Gregory et Gregory, 1979; Krapovickas et Gregory, 1994; Valls et Simpson, 2005, 2017; Valls et al.; 2013). It is typified by the common peanut, an allotetraploid cultigen with a genomic constitution AABB (Singh et Moss, 1984), that is well diploidized genetically (Bertioli et al.; 2011).
Further to their relevance for biosystematic studies, interspecific crosses are a first step for gene introgression from wild relatives into the cultigen. However, different ploidy levels impose a barrier for direct gene transfer. While the common peanut has 2n=4x=40 chromosomes, most wild species of the taxonomic sectionArachisare diploid, with 2n=2x=20 or 2n=2x=18 (Fernández et Krapovickas, 1994; Lavia et al.; 2009). The sectionArachisincludes at least six distinct genomes, A (Robledo et al.; 2009), D (Stalker, 1991; Robledo et Seijo, 2008), B, F, K (Robledo et Seijo, 2010), and G (Silvestri et al.; 2015), considering the distribution patterns of both heterochromatic bands and rDNA loci.
Production of synthetic allotetraploids from sterile hybrids between diploid wild species related to the A and B peanut genomes often restores fertility, removing the reproductive barriers, and increasing the possibility of successful construction of pre-breeding lines derived from wild species andA. hypogaea(Simpson, 1991; Fávero et al.; 2006; Foncéka et al.; 2009). Therefore, knowledge on the crossing behavior of wild diploids most closely related to the peanut, those with the A and B genomes, will help to understand species relationships in the genus, and to produce amphidiploid lines of potentially successful use in peanut breeding programs.
Arachis gregoryiis part of a small group of species genetically associated toA. ipaënsis(Burow et al.; 2009; Robledo et Seijo, 2010), the source of the B genome ofA. hypogaea(Kochert et al.; 1996; Seijo et al.; 2004, 2007; Fávero et al.; 2006; Bertioli et al.; 2016).
In the framework of peanut breeding based on the incorporation of related wild species germplasm, research on crossability of wildArachisspecies withA. hypogaeahas been concentrated for decades on species that share the A genome with the crop, and with assorted species then thought to be associated to the B genome (Simpson et Starr, 2001; Singh et Moss, 1984). A review of the genomic characteristics of species in the taxonomic sect.Arachis(Robledo et Seijo, 2010) established a group of true B genome species, gatheringA. ipaënsisandA. williamsii, exclusive to Bolivia, and each represented in genebanks by a single available accession, as well as three species occurring in Brazil,A. magna(also occurring in Bolivia),A. gregoryi, andA. valida. The restricted diversity ofA. ipaënsisdemands further exploration of the closely related species sharing the B genome, for further progress in peanut breeding.
This paper analyses crossing relationships ofA. gregoryiwith members of sectionArachis, including representatives of the A, B, D, F, G, and K genomes, as well as the AB configuration, and withA. paraguariensis, of the taxonomic sectionErectoides.
Material and Methods
Crosses were made during two consecutive spring/summer periods, under screenhouse conditions, from October 2006 to late May 2007, and from September 2007 to April 2008, at Embrapa Genetic Resources and Biotechnology, located in Brasilia, Distrito Federal, Brazil.
The female parent in all crosses wasA. gregoryi, accession number VS 14957 (Table1).
It was collected in the wild, in 2004, at the same original site as VSGr 6389, this one collected in 1981, and is a member of the same natural population already represented in crossing experiments (Valls et Simpson, 2005; Fávero et al.; 2015a, b).
Male parents represent the genomic and cytological variation so far detected in the taxonomic sectionArachisand one species of sectionErectoides(Table 1). Single plants of the female parent were cultivated in rectangular pots, each receiving pollen from a single and specific male donor.
Emasculation of theA. gregoryi14957 flower buds was conducted each day, from 4 to 7 PM. The flower keel was carefully taken out with tweezers, for full anther exposure and removal, and the standard petal was folded again over the exposed stigma, for protection. Pollination of the emasculated flowers was conducted from 7 to 11 AM, the next morning. Pollen grains were transferred from the specific donor with tweezers, which were washed in alcohol between crosses. Emasculated flowers were misted with a fine water spray to facilitate pollen grain adherence to the stigma. Crosses were made during the flowering period of each pair of parents.
Pegs were visible approximately 15 days after pollination and were individually monitored until ripening, or to the end of the life cycle of the individual female parent. Spontaneous seedlings were carefully transferred to smaller, individual plant pots for further development. Harvested fruit segments were dried at room temperature and shelled seeds were later put to germinate under laboratory conditions.
Pollen viability of the parents was estimated both by staining andin vitrogermination. Pollen of F1hybrids was analyzed by staining only. Eight flower buds were collected from each parent at weekly intervals during the crossing period. Pollen grains were set on a slide, stained with 2% aceto-carmine:glycerine, covered with a cover slip, and allowed to stain for 30 minutes in a moist chamber. Counting of well-formed and ill-formed grains was made under the microscope, including five samples of 100 grains each per slide. The analysis byin vitrogermination followed the protocol of Niles et Quesenberry (1992). A basic solution of Medium 11 (10 mg H3BO3, 30 mg de Ca (NO3)4H2O, 20 mg MgSO4+ 7 H2O, 10 ml KNO3, make up to 100 ml of water), with subsequent addition of 1.5 g sucrose to each 10 ml of basic solution at the time of preparation of the slides. The slides with the pollen grains plus a drop of basic solution are stored in humid chamber for 2 h. The number of pollen tubes larger than the pollen grain was counted. Average counts per flower bud were calculated, and the compiled results were expressed in percentage. The percentage of successful pollinations was calculated by division of the number of hybrids obtained by the number of pollinations performed. These results were multiplied by 100 (Table 2).
Results
Pollen viability estimates
Pollen viability estimates by staining, of the parental plants, varied from 46.75% forA. helodesto 99.17% forA. magnaaccession 30097 (Table 2). Values for all additional accessions were over 61.91%, with an average of 87.78%. Pollen viability estimates by germination varied from 41.70% forA. helodesto 88.00% forA. diogoi. Other accessions were over 41.91%, with an average of 75.33%.
Of hybrids that produced flowers (Table 2), the highest estimate of 43.6% was obtained for the intraspecific hybridA. gregoryi14957 ×A. gregoryi14753. Estimates for other flowering hybrids varied from 0.3% forA. gregoryi×A. glanduliferato 12% forA. gregoryi×A. hoehnei30006.
Hybridization
A number of 153 confirmed hybrids resulted from the total of 3167 hand pollinations (Table 2). Of those, 1368 were made from October 2006 to late May 2007, and 1799 from September 2007 to April 2008. The number of pollinations in the distinct crossing combinations varied according to the number and quality of flowers produced by the respective female and male parents (Table 2). Some crossing combinations were repeated in the second season, due to low flower production, lack of flower synchrony between parents, or temporary low pollen estimates of the male parent in the first season.
Several seedlings voluntarily produced in the pots of their female parent died at early stages, so that it was impossible to determine whether they were hybrids or just maternal offspring.
According to the percent of successful pollinations, crossing results were classified into four heterogeneous groups (Table 2). Six crossing combinations, which failed or did not perform well in the first crossing season, for reasons beyond our control, were repeated in the second season, this time showing positive results. Such combinations conducted in both seasons are reported individually within the framework of the four groups, and the results of the second attempt obviously better reflect their crossing potential.
The first group encompasses crossing combinations that produced the highest percent of hybrids per pollination events (17.1 to 11.9%), but most of the hybrids produced did not flower. Group 1 involvesA. gregoryi×A. hoehnei13985 (17.1%),A. gregoryi×A. kuhlmannii(15.18%),A. gregoryi×A. williamsii(14.3%),A. gregoryi×A. kempff-mercadoi(14.0%),A. gregoryi×A. schininii(13.3%),A. gregoryi×A. microsperma(12.5%), andA. gregoryi×A. stenosperma, as well asA. gregoryi×A. vallsii(both 11.9%). Five of the male parents are diploid species associated to the A peanut genome, one to the B genome. The genomes ofA. hoehnei13985 and ofA. vallsiiare yet to be determined (Table 2).
Concerning male parents with the A genome, the cross ofA. gregoryiwithA. kuhlmanniiproduced 24 fruit segments, which generated 12 hybrid plants with pollen viability estimates of 4.8%, in average. Hybrids ofA. gregoryi×A. kempff-mercadoiproduced quite normal plants, with a typical main axis and ascending lateral branches, but no flowers. Hybrids withA. microspermaalso were quite normal plants, but did not flower. Showing excellent survival, hybrids withA. schininiiproduced highly proliferated lateral branches, but an abnormally short main axis, and no flowers. Hybrids ofA. gregoryi×A. stenospermashowed slow growth and early loss of the main axis, although the lateral branches remained alive for several months.
Hybrids with the B genome speciesA. williamsii, that stood out as a good result obtained in the first season, showed very intensive vegetative growth, with lateral branches extending longer than 2.5 meters, intensive flowering, and average pollen viability estimate of 2.6%, but no peg formation.
The hybrid ofA. gregoryi×A. hoehnei13985 showed a fast growing rate, erect habit, and intensive lateral branching. However, the plants did not flower, so that pollen viability estimates are not available.
Crosses ofA. gregoryi×A. vallsiiproduced 22 fruit segments, which resulted in ten seedlings. Although their hybrid nature could not be confirmed, due to very slow growth and no flowers, the leaflets of the hybrids showed the paternal parent trait, narrow and lanceolate, not of maternal origin.
The second group of crossing combinations gathers four crosses with a percent of success from 9.6 to 5.4%, involvingA. gregoryi×A. valida(9.6%),A. gregoryi×A. magna30097 (9.5%),A. gregoryi×A. villosa(8.2%) andA. gregoryi×A. diogoi(5.4%), the first two male parents associated to the peanut B genome, and the last two to the A genome.
In hybrids ofA. gregoryi×A. valida, lateral branches were well developed and over 2.5 m long. Flowering was intensive, with an average pollen viability estimate of 5.45%. In spite of this low figure, one peg was produced, showing a fruit segment that, although well formed, did not complete its development and deteriorated in the soil.
The hybrid ofA. gregoryi×A. magna30097 presented average pollen viability estimate of 7.12%.
With a rate of success of 8.2%, crosses ofA. gregoryi×A. villosaproduced four plants, three of which survived well, and were additionally multiplied by cuttings, but never flowered. The hybrid status of the seedlings was easily confirmed by the dense pubescence on the adaxial leaflet surface, a paternal feature.
Hybridization ofA. gregoryi×A. diogoiproduced three small, but vigorous plants, which did not flower.
The third group of hybrids, with a low rate of success between 3.6 and 0.6%, encompasses 15 crossing combinations, involvingA. gregoryi×A. hypogaeasubsp.fastigiatavar. aequatoriana(3.6%),A. gregoryi×A. krapovickasii(3.5%),A. gregoryi×A. ipaënsis(2.8%),A. gregoryi×A. helodes(2.7%),A. gregoryi×A. schininii(2.7%),A. gregoryi×A. simpsonii(2.5%),A. gregoryi×A. cruziana(2.5%),A. gregoryi×A. magna14724 (2.2%),A. gregoryi×A. hypogaeasubsp.hypogaeavar.hirsuta(2.0%),A. gregoryi14957 ×A. gregoryi14753 (1.7%),A. gregoryi×A. glandulifera(1.7%),A. gregoryi×A. benensis(1.4%),A. gregoryi×A. hypogaeasubsp.fastigiatavar.fastigiata(1.4%),A. gregoryi×A. batizocoi(1.2%),A. gregoryi×A. duranensis(1.1%), andA. gregoryi×A. hoehnei30006 (0.6%). Male parents in this group include representatives of three botanical varieties ofA. hypogaea, as well as species either associated to the A or B genomes, besides representatives of the D, F and K genomes, additionally described for species of theArachissection (Stalker, 1991; Robledo et Seijo, 2008, 2010).
In the intraspecific crossA. gregoryi14957 × 14753, 24 fruit segments were formed, but only two hybrid plants survived. Pollen viability estimates varied from 36-55.6%. Although the anthers did not open properly at anthesis, and, consequently, release of pollen-grain was restricted, the two hybrid plants produced many pegs with average thickness of 0.68mm, about half the peg thickness measured on the female parent. This F1plant behaved as typically annual, dying out at the end of the reproductive period. Seeds germinated voluntarily in the pot, generating ten F2plants.
Crosses involvingA. gregoryiwithA. ipaënsisproduced 39 fruit segments, that had no spontaneous germination, with the hybrid pollen viability estimate of 5.4%.
Seedlings resulting from two crossing combinations, involvingA. gregoryi×A. batizocoi, showed abnormal growth from the first stages. Fruit segments produced in the respective female plant pots were harvested, some already starting germination. Seedlings and seeds were grownin vitro. The 82 pollinations made in the cross ofA. gregoryi×A. batizocoiresulted in the production of 61 fruit segments, which would imply a very high rate of success, if they developed normal seeds and then plants. However, 46 seeds germinated spontaneously producing abnormal seedlings, lacking the aerial organs above the cotyledons. So, the seeds from the 15 remaining fruit segments were set to germinatein vitro.Only a single hybrid plant developed in a greenhouse, but it did not survive, when later transferred to screenhouse conditions. Embryos rescuedin vitrostill persist but need to be acclimatized.
The crossing ofA. gregoryi×A. cruzianaresulted in 36 fruit segments. Seedlings were vigorous, forming adult plants with lateral branches longer than 2.5 m, and intensive flowering. The average pollen viability estimate was 5.22%.
ConcerningA. gregoryi×A. krapovickasii, 19 fruit segments were formed. The seeds germinated spontaneously, generating vigorous seedlings, but did not continue the development after showing the first leaves. Only one plant survived and flowered, allowing the pollen viability estimate of 0.4%.
The crossing combinationA. gregoryi×A. glandulifera, representing the B and D genomes, produced 27 well-formed fruit segments, but 25 had aborted embryos, and one seed did not reach full maturity. The single well-formed seed produced a normal, flowering plant, with an estimated pollen viability of 0.3%. The hybrid status was easily confirmed by the presence of glands in the abaxial leaflet surface, a typical paternal feature.
In a cross involvingA. gregoryi×A. benensis, which represents the F genome (Robledo et Seijo, 2010), 25 fruit segments were formed, with spontaneous germination of five seeds. Pollen viability estimates were quite low, 0.5% in average.
In a second crossing combination that requiredin vitrosupport, involvingA. gregoryi×A. hoehnei30006, 59 seeds were produced, of which 43 germinated spontaneously. Although under intensive attack of mites, just like the male parent, seedlings started in good shape, but soon died. The remaining 16 seeds were set to germinatein vitro. Only a single plant survived in the greenhouse, and later, under screenhouse conditions. It had profuse lateral branching, with ascending-erect habit, and it flowered. The pollen viability estimate was 12%, with extremes of 9% and 18.2%.
The fourth group involves crosses in which the resulting seeds did not become established as plants, so that the hybrid character could not be confirmed (Table 2). Additionally, the crossesA. gregoryi×A. diogoi,A. gregoryi×A. duranensis,A. gregoryi×A. stenosperma,A. gregoryi×A. ipaënsis, andA. gregoryi × A. vallsii, as performed in the first season, as well asA. gregoryi×A. gregoryi14767,A. gregoryi×A. gregoryi14962,A. gregoryi × A. magna13761,A. gregoryi × A. magna13765,A. gregoryi × A. vallsii,A. gregoryi × A. hypogaea“Xingu” type,A. gregoryi × A. hypogaeasubsp.fastigiatavar.peruviana, andA. gregoryi × A. palustriswere not possible because of low flowering synchrony between parents (LS), or due to low quantity of flowers produced by the parents (LQF), or potential hybrid offspring that died at early stages of seedling development (DES).
In crosses ofA. gregoryi × A. hypogaeasubsp.hypogaeavar.hypogaea,A. gregoryi × A. hypogaeasubsp.fastigiatavar.vulgaris,A. gregoryi × A. monticola2775,A. gregoryi × A. monticola14165,A. gregoryi × A. paraguariensis, embryos aborted (AE) and/or seedlings died at early stages of development (DES).
Factors like low flowering synchrony and low quantity of flowers produced by the male parents, and, most of the time, low vigor of flowers after manipulation for hybridization were significant in the lack of success in the production of hybrid plants.
Table 1. List of theArachisspecies studied, accession codes, accession collectors and numbers, origin and geographic coordinates.
| Arachisspecies 1 | BRA-Code2 | Accession3,4,5 | Origin6 | Municipality | Lat (S) | Long (W) | Alt (m) |
|---|---|---|---|---|---|---|---|
| A. batizocoiKrapov. & W. C.Greg. | 00064972-3 | K 9484 | BOL/SC | Parapeti | 20°05’ | 63°14’ | 700 |
| A. benensisKrapov.; W. C. Greg. & C. E.Simpson | 00065872-4 | KGSPSc 35005 | BOL/BE | Trinidad | 14°47’ | 64°55’ | 155 |
| A. cruzianaKrapov.; W. C. Greg. & C. E. Simpson | 00065840-1 | WiSVg 1302-2 | BOL/SC | San José de Chiquitos | 18°50’ | 60°53’ | 285 |
| A. diogoiHoehne | 00065993-8 | Vp 5000 | BRA/MS | Corumbá | 17°50’ | 57°33’ | 92 |
| A. duranensisKrapov. & W. C. Greg. | 00065723-9 | VNvEv 14167 | ARG/SA | Salta | 24°50’ | 65°27’ | 1206 |
| A. glanduliferaStalker | 00065512-6 | VSPmSv 13738 | BRA/MT | Porto Esperidião | 16°13’ | 59°07’ | 160 |
| A. gregoryiC. E. Simpson, Krapov. & Valls | 00065978-9 | VOfSv 14753 | BRA/MT | Pontes e Lacerda | 15°59’ | 59°33’ | 275 |
| 00065982-1 | VOfSv 14767 | BRA/MT | Vila Bela da S. Trindade | 16°05’ | 59°58’ | 247 | |
| 00066017-5 | VS 14957 | BRA/MT | Vila Bela da S. Trindade | 15°22’ | 60°14’ | 230 | |
| 00066020-9 | VS 14962 | BRA/MT | Vila Bela da S. Trindade | 15°23’ | 60°13’ | 220 | |
| A. helodesMart. ex Krapov. & Rigoni | 00064934-3 | VSGr 6325 | BRA/MT | Santo Antônio do Leverger | 15°52’ | 56°04’ | 150 |
| A. hoehneiKrapov. & W. C. Greg. | 00065832-8 | KG 30006 | BRA/MS | Corumbá | 18°15’ | 57°28’ | 100 |
| 00065620-7 | VMPzW 13985 | BRA/MS | Corumbá | 19°31’ | 57°25’ | 85 | |
| A. hypogaeaL. “Xingu” type | 00063839-5 | VGaRoSv 12549 | BRA/MT | São José do Xingu | 10°49’ | 50°41’ | 345 |
| A. hypogaeasubsp.hypogaeavar.hypogaea | 00063838-7 | VGaRoSv 12548 | BRA/MT | São José do Xingu | 10°49’ | 50°41’ | 345 |
| A. hypogaeasubsp. hypogaeavar.hirsutaH. A. Köhler | 00063836-1 | Mf 1538 [ex ARG] | ECU/PI | Quito | 0°02’ | 78°26’ | 2560 |
| A. hypogaeasubsp.fastigiataWaldron var.fastigiata | 00064542-4 | IAC ‘Tatu’ | BRA/SP | Campinas | 22°53’ | 47°03’ | 665 |
| A. hypogaeasubsp.fastigiatavar. peruvianaKrapov. & W. C. Greg. | 00063833-8 | Mf 1560 [ex ARG] | ECU/ES | Quinindé | 0°07’ | 79°25’ | 260 |
| A. hypogaeasubsp.fastigiatavar.vulgarisHarz | 00223807-9 | IAC ‘Tatuí’ | BRA/SP | Campinas | 22°53’ | 47°03’ | 665 |
| A. hypogaeasubsp.fastigiatavar.aequatorianaKrapov. & W. C. Greg. | 00063831-2 | Mf 1678 [ex ARG] | ECU/SU | Shushufindi | 0°22’ | 76°39’ | 390 |
| A. ipaënsisKrapov. & W. C. Greg. | 00065831-0 | KGBPScS 30076 | BOL/TA | Ipa | 21°00’ | 63°25’ | 650 |
| A. kempff-mercadoiKrapov.; W. C. Greg. & C. E. Simpson | 00064968-1 | V 13250 | BOL/SC | Santa Cruz de la Sierra | 17°41’ | 63°08’ | 420 |
| A. krapovickasiiC. E. Simpson, D. E. Williams, Valls & I. G. Vargas | 00065839-3 | WiSVg 1291 | BOL/SC | San José de Chiquitos | 18°14’ | 60°51’ | 314 |
| A. kuhlmanniiKrapov. & W. C. Greg. | 00065544-9 | VSPmSv 13779 | BRA/MT | Cáceres | 16°13’ | 57°23’ | 190 |
| A. magnaKrapov.; W. C. Greg. & C. E. Simpson | 00065836-9 | KGSSc 30097-o | BOL/SC | San Ignácio de Velasco | 16°22’ | 60°58’ | 370 |
| 00065540-7 | VSPmSv 13761 | BRA/MT | Vila Bela da S. Trindade | 15°21’ | 60°04’ | 210 | |
| 00065671-0 | VSPmSv 13765 | BRA/MT | Glória d’Oeste | 15°48’ | 58°23’ | 150 | |
| 00065516-7 | VOfSv 14724 | BRA/MT | Vila Bela da S. Trindade | 15°19’ | 60°03’ | 204 | |
| A. microspermaKrapov.; W. C. Greg. & Valls | 00065646-2 | VMPzW 14042 | BRA/MS | Porto Murtinho | 22°05’ | 57°34’ | 102 |
| A. monticolaKrapov. & Rigoni | 00219752-3 | SeSnHoCh 2775 | ARG/JU | Lozano | 24°04’ | 65°24’ | 1546 |
| 00065721-3 | VOa 14165 | ARG/JU | Yala | 24°07’ | 65°23’ | 1436 | |
| A. palustrisKrapov.; W. C. Greg. & Valls | 00065019-2 | VPmSv 13023 | BRA/TO | Filadélfia | 07°25’ | 47°37’ | 192 |
| A. paraguariensisChodat & Hassl. subsp.paraguariensis | 00065159-6 | VRGeSv 7677 | BRA/MS | Bela Vista | 22°08’ | 56°44’ | 168 |
| A. schininiiKrapov.; Valls & C. E. Simpson | 00065028-3 | VSPmSv 9923 | PRY/AM | Bella Vista | 22°20’ | 56°19’ | 246 |
| A. simpsoniiKrapov. & W. C. Greg. | 00065536-5 | VSPmSv 13728 | BOL/SC | San Matías | 16°19’ | 58°26’ | 120 |
| A. stenospermaKrapov. & W. C. Greg. | 00064932-7 | VSv 10309 | BRA/MT | Rondonópolis | 16°28’ | 54°39’ | 235 |
| A. validaKrapov. & W. C. Greg. | 00065464-0 | VPzRcSgSv 13514 | BRA/MS | Corumbá | 19°07’ | 57°32’ | 90 |
| A. vallsiiKrapov. & W. C. Greg. | 00065027-5 | VRGeSv 7635 | BRA/MS | Miranda | 20°07’ | 56°42’ | 100 |
| A. villosaBenth. | 00065862-5 | VMiIrLbGvAn 14309 | BRA/RS | Uruguaiana | 29°47’ | 57°13’ | 50 |
| A. williamsiiKrapov. & W. C. Greg. | 00065838-5 | WiDc 1118 | BOL/BE | Trinidad | 14°48’ | 64°53’ | 150 |
1Species additionally cited in the text:A. linearifoliaValls, Krapov. & C. E. Simpson.
2Germplasm accession code in the Embrapa Alelo Portal.
3Collector’s abbreviations: An= A. Carneiro; B= D. J. Banks; Ch= J. Chalian; Dc= D. Claure; Ev= A. V. Etcheverry; G= W. C. Gregory; Ga= M. L. Galgaro; Ge= M. A. N. Gerin; Gr= A. Gripp; Gv= F. R. Galvani; Ir=B. E. Irgang; Ho= D. Hojsgaard; K= A. Krapovickas; Lb= L. R. M.Baptista; M= J. P. Moss; Mi= S. T. S. Miotto; Nv= L. J. Novara; Oa= O. Ahumada; Of= F. O. Freitas; P= J. R. Pietrarelli; Pm= R. N. Pittman; Pz= E. A. Pizarro; R= V. R. Rao; Rc= R. C. Oliveira; Ro= D. M. S. Rocha; S= C. E. Simpson; Sc= A. Schinini; Se= J. G. Seijo; Sg= A. K. Singh; Sn= V. G. Solís Neffa; Sv= G. P. Silva; V= J. F. M.Valls; Vg= I. G. Vargas; Vp= V. J. Pott; W= W. L. Werneck; Wi= D. E. Williams.
4Mf= Estación Experimental de Manfredi, Córdoba, Argentina. Accession numbers assigned for the 1997-1998 regeneration season (All three accessions originally collected in Ecuador).
5IAC= Instituto Agronômico de Campinas, São Paulo, Brazil. IAC cultivars.
6Countries/Departments, provinces or states: ARG= Argentina/JU= Jujuy; SA= Salta; BOL= Bolivia/BE= Beni; SC= Santa Cruz; TA=Tarija; BRA= Brazil/MT= Mato Grosso; MS= Mato Grosso do Sul; TO= Tocantins; RS= Rio Grande do Sul; SP= São Paulo; ECU= Ecuador/ES= Esmeraldas; PI= Pichincha; SU= Sucumbíos; PRY= Paraguay/AM= Amambay.
Table 2. Classification of male parentArachisspecies and accessions used, with respective genome. Female parent alwaysArachis gregoryiV 14957.
| Group | Male parents | Accession | Genome3 | %SH4 | %PVS5a | %PVG5b | NFS6 | NH7 | %PVH8 | HB/BH9 |
| 1 | A. hoehnei 2 | V 13985 | ? | 17.1 | 93.85 | 82.1 | 34 | 19 | NF | GSD |
| A. kuhlmannii 2 | V 13779 | Apn | 15.1 | 74.97 | 67.25 | 24 | 12 | 4.8 | GSD | |
| A. williamsii1 | Wi 1118 | B | 14.3 | 97.65 | 79.35 | 28 | 10 | 2.6 | GSD | |
| A. kempff-mercadoi 2 | V 13250 | Ach | 14,0 | 61.92 | 41.9 | 17 | 7 | NF | GSD | |
| A. schininii 2 | V 9923 | Apl | 13.3 | 92.25 | 77.9 | 14 | 11 | NF | GSD | |
| A. microsperma 2 | V 14042 | A | 12.5 | 95.4 | 86.1 | 8 | 8 | NF | GSD | |
| A. stenosperma 2 | V 10309 | Apn | 11.9 | 84.22 | 78.9 | 27 | 16 | NF | DES | |
| A. vallsii 2 | V 7635 | ? | 11.9 | 94.3 | 81.15 | 22 | 10 | NF | GSD | |
| 2 | A. valida 1 | V 13514 | B | 9.6 | 71.92 | 60.05 | 36 | 11 | 5.45 | GSD |
| A. magna 1 | K 30097 | B | 9.5 | 99.17 | 82.45 | 11 | 4 | 7.12 | GSD | |
| A. villosa 1 | V 14309 | A | 8.2 | 93.97 | 84.2 | 4 | 4 | NF | GSD | |
| A. diogoi 2 | Vp 5000 | Apn | 5.4 | 95.45 | 88.00 | 20 | 3 | NF | GSD | |
| 3 | A. hypogaeavar.aequatoriana 2 | Mf 1678 | AB | 3.6 | 76.82 | 69.25 | 46 | 4 | NF | AE/DES |
| A. krapovickasii 1 | Wi 1291 | K | 3.5 | 93.7 | 86.15 | 19 | 2 | 0.4 | DES | |
| A. ipaënsis 2 | K 30076 | B | 2.8 | 94.37 | 83.75 | 39 | 3 | 5.4 | GSD | |
| A. helodes 2 | V 6325 | Apn | 2.7 | 46.75 | 41.7 | 22 | 3 | NF | GSD | |
| A. schininii 1 | V 9923 | Apl | 2.7 | 97.17 | 87.45 | 5 | 1 | NF | GSD | |
| A. simpsonii 2 | V 13728 | Apn | 2.5 | 81.42 | 66.95 | 18 | 2 | NF | GSD | |
| A. cruziana 1 | Wi 1302-2 | K | 2.5 | 98.37 | 87.4 | 36 | 3 | 5.22 | GSD | |
| A. magna 1 | V 14724 | B | 2.2 | 98.62 | 72.15 | 4 | 2 | 10.45 | GSD | |
| A. hypogaeavar.hirsuta 2 | Mf 1538 | AB | 2.0 | 90.95 | 72.85 | 13 | 1 | NF | AE/DES | |
| A. gregoryi 1 | V 14753 | B | 1.7 | 93.9 | 75.75 | 24 | 2 | 43.6 | DES | |
| A. glandulifera 1 | V 13738 | D | 1.7 | 95.85 | 60.65 | 27 | 1 | 0.3 | AE/GSD | |
| A. benensis 2 | K 35005 | F | 1.4 | 93.8 | 86.55 | 25 | 1 | 0.6 | GSD | |
| A. hypogaeavar.fastigiata 2 | IAC ‘Tatu’ | AB | 1.4 | 69.57 | 57.75 | 31 | 1 | NF | AE/DES | |
| A. batizocoi 1 | K 9484 | K | 1.2 | 89.6 | 86.2 | 61 | 1 | NF | IV/DES | |
| A. duranensis 2 | V 14167 | Apelo | 1.1 | 91.55 | 82.35 | 32 | 1 | NF | DES | |
| A. hoehnei 1 | K 30006 | ? | 0.6 | 90.2 | 78.85 | 59 | 1 | 12 | IV/DES | |
| 4 | A. diogoi 1 | Vp 5000 | Apn | 0.0 | 90.27 | 68.5 | 4 | 0 | - | LS/DES |
| A. duranensis 1 | V 14167 | Apl | 0.0 | 87.95 | 82.2 | 0 | 0 | - | LS/LQF/DES | |
| A. stenosperma 1 | V 10309 | Apn | 0.0 | 92.75 | 84.95 | 0 | 0 | - | LS/LQF/DES | |
| A. ipaënsis 1 | K 30076 | B | 0.0 | 94.55 | 78.95 | 10 | 0 | - | DES | |
| A. gregoryi 1 | V 14767 | B | 0.0 | 95.25 | 81.3 | 30 | 0 | - | DES | |
| A. gregoryi 1 | V 14962 | B | 0.0 | 84.57 | 73.8 | 6 | 0 | - | LS/DES | |
| A. magna 1 | V 13761 | B | 0.0 | 94.17 | 84.1 | 0 | 0 | - | LS/LQF | |
| A. magna 1 | V 13765 | B | 0.0 | 96.62 | 72.15 | 8 | 0 | - | DES | |
| A. vallsii 1 | V 7635 | ? | 0.0 | 88.6 | 73.1 | 0 | 0 | - | LS/LQF | |
| A. hypogaea“Xingu type”2 | V 12549 | AB | 0.0 | 62.8 | 50.25 | 3 | 0 | - | LS | |
| A. hypogaeavar.hypogaea 2 | V 12548 | AB | 0.0 | 83.27 | 72.65 | 11 | 0 | - | AE/DES | |
| A. hypogaeavar.peruviana 2 | Mf 1560 | AB | 0.0 | 88.12 | 74.95 | 1 | 0 | - | LS/LQF/DES | |
| A. hypogaeavar.vulgaris 2 | IAC ‘Tatuí’ | AB | 0.0 | 88.15 | 72.25 | 39 | 0 | - | AE/DES | |
| A. monticola 2 | Sj 2775 | AB | 0.0 | 90.9 | 82.3 | 10 | 0 | - | AE/DES | |
| A. monticola 2 | V 14165 | AB | 0.0 | 81.1 | 78.3 | 8 | 0 | - | AE/DES | |
| A. palustris 2 | V 13023 | G | 0.0 | 86.1 | 75.85 | 18 | 0 | - | DES | |
| A. paraguariensis 2 | V 7677 | E | 0.0 | 87.5 | 79.45 | 8 | 0 | - | AE/DES |
1First cross season 2006/2007.
2Second cross season 2007/2008.
3A genome subgroups (Robledo et al. 2009): Ach= Chiquitano; Apn= Pantanal; Apl=La Plata River Basin.
4Percentage of success for obtaining hybrids (% SH)
5a, b Percentage of viability of the pollen-grain of the parents by staining (% PVS) and by germination (% PVG)
6Number of fruit segments produced by crossing combination (NFS)
7Number of confirmed hybrids (NH)
8Percentage of estimated viability of pollen-grain of hybrids by staining (% VPH)
9Hybrids behavoir/bariers for hybridization (HB/BH): good seedling development (GSD), death at the early stages of seedling development (DES), abortion of the embryo (AE), material cultivated in vitro (IV), low synchrony of flowering between parents (LS), low quantity of flowers of the parents (LQF), no flowering (NF).
Discussion
Pollen viability estimates
No method for estimation of pollen viability is fully satisfactory, as the stains used in chemical tests react with chemical constituents or structures of the grains, and may not reflect the real germinating capacity (Stanley et Linskens, 1974). In spite of this, estimates of pollen viability are important to ensure that the plant materials involved present, at least in theory, the ability to trigger the complex reproductive process that involves morphological, physiological and genetic aspects.
Comparing the pollen staining and germination estimates, the difference of 10 to 20% is considered a common fact, most possibly due to overestimation with the staining technique and underestimation with germination, as concerns the viable pollen grains (Galetta, 1983). Even though some authors do not consider pollen staining as a viable estimate of plant fertility, the present authors are not as skeptical as to the positive aspects of this technique. Pollen staining has been reported in the evaluation ofArachiscrossing programs by several authors (Gregory et Gregory, 1979; Simpson, 1991; Krapovickas et Gregory, 1994; Wondracek-Lüdke et al.; 2015; Fávero et al.; 2015a, b) and is generally accepted byArachisresearchers as a useful estimate of fertility.
Although a few accessions have shown lower pollen estimates for some time along the crossing season, all others consistently presented high estimates, so that all male parents were considered fit to fertilize the flowers of the female parent. It is not uncommon for a species such asA. helodesto have a low pollen count when some strenuous environmental conditions occur. If daytime temperature exceeds 42ºC, fertile plants of manyArachisspecies may have a pollen stain of < 50% for two or three consecutive days (Simpson CE, unpublished results).
Hybridization
The female parentA. gregoryi14957, from the same site of 6389, has shown a broad crossing plasticity, as it was successfully pollinated and developed fruit segments in most crossing combinations, involving distinct genomes, ploidy levels, and taxonomic sections. Although these two accessions were assigned distinct collection numbers and germoplasm codes (BRA), due to the long time elapsed between field collections, previous data and the results of the present research confirm the potential value of this species and of the wild population represented by both the 6389 and 14957 accessions for hybridization studies.
The possibility of polyploidization of the hybridA. gregoryi6389 ×A. linearifolia, producing an amphidiploid, which was then successfully crossed withA. hypogaea, has been demonstrated (Fávero et al.; 2015a). So, it is estimated that additional accessions ofA. gregoryi, with a B genome somewhat similar to that ofA. ipaënsis, may be used as efficient bridge species for the introgression of genes from wildArachisspecies into the cultivated peanut.
Group 1
Crossing combinations in this group were the most successful, although they showed differentiated potential. The five hybrid combinations involving B × A genome holders (A. gregoryi×A. kempff-mercadoi,A. kuhlmannii,A. microsperma,A. schininii, orA. stenosperma, the last two with worse results in the first season), all showing low pollen counts, are good candidates for production of colchicine induced highly fertile amphidiploids. Diploid hybrids ofA. gregoryiwith species showing the A chromosome pair are of special interest to peanut breeding, once their chromosome doubling will produce lines with the AABB genome constitution, for direct crossing withA. hypogaea. Furthermore, several of the A genome species of which hybrids where obtained show variable degrees of resistance/tolerance to foliar diseases caused byCercospora arachidicolaHori (early leaf spot),Cercosporidium personatum(Berk. & M.A. Curtis) Deighton (late leaf spot), andPuccinia arachidisSpeg. (rust) (Fávero et al.; 2009, 2015a).
It must be noted that hybrids ofA. gregoryi×A. stenospermahave already been reported, but they involve different accessions of each parental species (Fávero, 2015b; Valls et Simpson, 2005; Leal-Bertioli et al.; 2017). These previous hybrids flowered in both experiments cited, showing low pollen viability estimates, from 0.4 to 4.9%. Besides peculiarities of the distinct accessions involved, environmental differences at the distinct experimental facilities may not be ruled out as responsible for the lack of flowering in the present study.
The easy B × B crossing ofA. gregoryi×A. williamsii, associated to the very low pollen viability estimates of the hybrid produced, suggests a potential for successful restoration of fertility through chromosome doubling. Such tetraploid line, gathering characteristics of two B genome species, may be useful for gene pyramidation in the B side.
The easiest crossing, that ofA. gregoryi×A. hoehnei13985, the most prolific male parent in this crossing program, may be of high interest for peanut breeding, but further study of theA. hoehneigenome relationships is still on demand.
Seven accessions ofA. hoehneiare available in the Embrapa WildArachisGenebank, all from the state of Mato Grosso do Sul, Brazil. But they are not uniform, showing differences in their reproductive cycle and crossing behavior, and doubts still persist about the presence or absence of the A chromosome pair in distinct accessions of the species (Fernández et Krapovickas, 1994; Custodio et al.; 2013).
Easy crossing ofA. gregoryi×A. vallsii, attained in the second season, but by no means in the first, is not only of genetic, but also of taxonomic importance.Arachis vallsiiwas originally described as a member of the taxonomic sectionProcumbentes(Krapovickas et Gregory, 1994). Cytogenetic evidence has confirmed its closer relationship to theArachissection, although it does not show the A chromosome pair (Lavia et al.; 2009) nor a typical B genome configuration (Robledo et Seijo, 2010). Confirmation of the crossability ofA. gregoryi×A. vallsiisuggests a possibility of incorporating some peculiar features of the latter into peanut breeding programs, especially those related to the ecological preferences ofA. vallsii, which thrives in periodically flooded areas, producing its underground fruits in muddy soils (Simpson et al.; 2018).
Group 2
As well asA. gregoryi, associated to the B genome (Robledo et Seijo, 2010),A. validahas a potential value for peanut breeding, as it shows resistance to leaf spots (Fávero, 2009), and possibly also to flooding, considering the area of occurrence of its natural populations (Krapovickas et Gregory, 1994). In a parallel study (Wondracek-Lüdke et al.; 2015),A. validahas shown a similar potential to that ofA. gregoryifor producing hybrids with all representatives of the B genome.
Concerning the hybrid ofA. gregoryi×A. magna30097, similar low values for pollen viability estimates were obtained in Texas (Simpson et Faries, 2001), involving the same male parent and the 6389 accession ofA. gregoryi, already mentioned as a member of the same natural population of our female parent. This means that, althoughA. gregoryiandA. magnaseem to be closely related species, even showing individual plants somewhat difficult to discriminate on morphological grounds, and have several sympatric populations, they must have developed genetic barriers, which maintain their reproductive isolation and allow for their persistence as distinct species.
As to theA. gregoryi×A. villosahybridization, the latter, collected in Brazil on the edge of the Uruguay river, is cytogenetically, as well as geographically considered a member of the La Plata River Basin subgroup of A genome species, therefore narrowly related toA. duranensis(Robledo et al.; 2009). Due to this close cytological affinity to the A genome donor ofA. hypogaea(Seijo et al.; 2004), this AB diploid hybrid is of utmost interest for chromosome duplication and incorporation to peanut breeding programs.
AnotherA. gregoryi×A. villosahybrid, obtained by using, respectively, theA. gregoryiaccession 6389 and the Uruguayan accession ofA. villosaVGoMrOv 12812, not tested here, had a much lower percent of success (0.92%), but flowered, with estimated pollen viability of 7.67% (Fávero, 2015b).
TheA. gregoryi×A. diogoidiploid hybrid is another interesting line for chromosome duplication, once the paternal species, then represented by the distinct Paraguayan accession GKP 10602, has been used to produce the amphidiploid line that resulted in the release of the cultivar ‘COAN’ (Simpson et Starr, 2001), which has near immunity to the root-knot nematode speciesMeloidogyne arenaria(Neal) Chitwood andM. javanica(Treub) Chitwood. Natural populations ofA. diogoioccur along the Paraguay river basin and its tributaries, in areas subject to periodic flooding (Krapovickas et Gregory, 1994), and have shown resistance to early and late leaf spots, thrips, and jassids (Fávero, 2009).
Group 3
In spite of the distinct ploidy levels involved, crosses ofA. gregoryi×A. hypogaeadid not require special techniques. However, embryo abortion at early stages of seed formation was frequent. Mature seeds were produced in crosses with three varieties (aequatoriana,hirsuta, andfastigiata). None has shown dormancy, and they generally germinated, but most seedlings died at early stages. All four surviving plants ofA. gregoryi×A. hypogaeasubsp.fastigiatavar.aequatorianawere triploid (2n=3x=30) and highly sterile, while the single surviving plant ofA. gregoryi×A. hypogaeasubsp.hypogaeavar.hirsutawas too weak, and roots were not adequate for chromosome counting, but its morphology, quite different from the female parent, led to the assumption of the hybrid state. Also, a single slow growing, confirmed cytologically as a triploid plant was rescued from the combinationA. gregoryi×A. hypogaeasubsp.fastigiatavar.fastigiata, which, although healthy, never flowered.
Manipulation of triploid interspecific hybrids ofArachisto produce useful breeding lines is laborious. It is first necessary to duplicate chromosomes to produce a fertile hexaploid line, which is then backcrossed toA. hypogaea, until it stabilizes at 2n=40, due to chromosome loss (Simpson, 1991, 2001), but with no guarantee of a balanced AABB genome structure. In spite of the more complicated pathway, triploid hybrids produced in this program have a potential use in peanut breeding. Different behavior presented by crossing combinations ofA. gregoryiwith these three botanical varieties ofA. hypogaeawould recommend additional trials, involving the remaining varieties, as well as accessions of the “Xingu” type, morphologically divergent, so far unclassified at the subspecific level, and more closely related toA. monticola(Nascimento et al.; 2020), of which no hybrids were obtained in this study.
Considering B × B hybrids, it is noteworthy that only one intraspecific combination (A. gregoryi14957 × 14753) has been successful, out of three attempted. The accessions tested, 14957 and 14753, are from adjacent counties in Mato Grosso State, but were collected in nature 99 km apart. Although pollen viability estimates of this hybrid reached 43.6%, the highest figure for all hybrids obtained in the present study, it is far below estimates of over 90% mentioned for other intraspecific and even some interspecific crosses involving otherArachisspecies (Krapovickas et Gregory 1994, Simpson et Faries 2001; Fávero et al.; 2015 a, b; Wondracek-Lüdke et al.; 2015). So, crossing data obtained in this study for the specific accession used as the female parent should be taken carefully as concerns otherA. gregoryimaterials, except in comparisons with the 6389 accession, recognized as a member of the same natural population.
Contrary to the general trend, seeds resulting from theA. gregoryi×A. ipaënsiscrossing did not present spontaneous germination. Lack of spontaneous germination is a useful trait, allowing for timely use of the hybrid seed in breeding programs. Due to the relevance ofA. ipaënsisin the origin ofA. hypogaea(Kochert et al.; 1996; Seijo et al.; 2004, 2007; Fávero et al.; 2006; Bertioli et al.; 2016), and the fact that there is only one accession of the former species available in genebanks, hybrids withA. ipaënsisare important for potentially expanding the possibilities of introgression of favorable characteristics associated to the B genome, for the improvement of peanut cultivars.
Similar to the hybrids withA. ipaënsis, and contrary to the behavior of theA. gregoryi×A. magna30097, seeds ofA. gregoryi×A. magna14724 did not show spontaneous germination. This emphasizes the diversity of the available A. magnaaccessions, already put in evidence by molecular cytogenetics (Custodio et al.; 2013) and molecular marker analyses (Moretzsohn et al.; 2013).
Although at lower rates of success, once again B × A genome crosses produced interesting candidates for induced chromosome doubling for direct crossing withA. hypogaeaat the tetraploid level, including the one involvingA. duranensis, the A genome donor ofA. hypogaea(Kochert et al.; 1996; Seijo et al.; 2004, 2007; Fávero et al.; 2006; Bertioli et al.; 2016), besidesA. helodes,A. schininii, andA. simpsonii.
Crosses of the B genomeA. gregoryiwith representatives of the D, F and K genomes, though important to the comprehension of genetic and evolutionary aspects of the genus, are not likely to produce amphidiploids as close toA. hypogaea, but that does not exclude their interest for peanut breeding, once such species eventually present favorable attributes, related to biotic and/or abiotic constraints. For instance, the K genomeA. batizocoihas been used in the construction of TxAG-6, the breeding line that gave rise to the cultivar ‘COAN’ (Simpson et Starr, 2001). While, in the present study, hybrid seedlings ofA. gregoryi×A. batizocoiproduced mostly abnormal seedlings, lacking the aerial organs above the cotyledons, a reverse hybrid involving the same accession ofA. batizocoiandA. gregoryi6389 has shown normal development and flowering, with a pollen viability estimate of 4.5% (Simpson et Faries, 2001).
The low pollen viability estimate of theA. gregoryi×A. glanduliferahybrid possibly reflects a wide genetic distance between the parents involved. Intraspecific hybrids involving distinct accessions ofA. glandulifera, all from Bolivia, and interspecific hybrids ofA. glandulifera×A. batizocoiandA. duranensis, have been obtained elsewhere (Stalker, 1991). However, despite 835 pollinations in both ways, involvingA. glanduliferaand two cultivars ofA. hypogaearepresenting subspecieshypogaeaandfastigiata, not a single triploid hybrid was obtained (Stalker, 1991). Successful crosses ofA. gregoryi×A. glanduliferamay open a pathway to bringing desirable genes of the highly prolificA. glanduliferainto pre-breeding lines similar to the peanut B genome, eventually conditioned to chromosome duplication.
It is interesting to notice the distinct results shown by the crossing combinationA. gregoryi×A. hoehnei. The most successful crossing in this study has been obtained usingA. hoehnei13985 as the male parent, while that withA. hoehnei30006 lies in the third group. The founder seed of the male parentA. hoehnei30006 has been collected in the field at the same natural site of the type collection (Krapovickas et Gregory, 1994), so this accession is the most typical ofA. hoehnei, while other accessions listed at the moment under the same name, such as 13985, may belong to a distinct taxon, yet to be described (Custodio et al.; 2013).
Group 4
Although this group encompasses unsuccessful crosses or crosses in which the hybrid nature of seed produced could not be confirmed, this cannot be taken as a definitive indication of incompatibility. For example, the unsuccesfull hybrization ofA. gregoryiwithA. diogoi,A. duranensis, andA. stenosperma(three A genome species) andA. vallsii(undetermined genome) in the first season was superseded by successful crosses in the second season. Such differences, which we cannot easily account for, may be under the influence of several factors, such as environmental factors, vigor of plants, attack of pests, and even the manipulation of the flowers of each parent during emasculation. But it must be considered that they did not affect crosses in the first season so intensely, when involvingA. williamsii,A. validaandA. magna30097 (B genome species), andA. villosa(A genome).
The genetic diversity of accessions of a same species that show different crossability results needs to be evaluated. This refers to the distinct varieties and off-types ofA. hypogaea, as well as to species such asA. gregoryi,A. magna, andA. hoehnei.
In summary, it has been shown thatA. gregoryiis a promising diploid wild species to be incorporated into peanut breeding programs. It has shown plasticity for crosses with species of several genome types present in the taxonomic sectionArachis. Its inclusion in breeding programs will expand the diversity of species and breeding lines associated to the B genome, with special attention to the actual availability of a single accession ofA. ipaënsis, involved in the origin ofA. hypogaea.
Similarly positive crossing results with distinct botanical varieties ofA. hypogaeaindicate that the use ofA. gregoryiin pre-breeding efforts is a possibility, also through the more laborious triploid/hexaploid/backcrossing strategy.
Finally, crossability ofA. gregoryiwith several representatives of the A genome group, and the general sterility of the AB hybrids, suggests a good potential use ofA. gregoryiin pre-breeding efforts through the amphidiploid strategy, which has already produced relevant results in the genetic breeding of the cultivated peanut.
