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a Embrapa Genetic Resources and Biotechnology, SAIN Parque Estação Biológica, CP 02372, 70.770-900, Brasília, DF, Brazil
b Texas Agric. Exp. Stn., Texas A&M University, 1229 U.S. Hwy. 281 North, Stephenville, TX 76401-0004
c Department of Genetics, Escola Superior de Agricultura "Luiz de Queiroz", ESALQ/USP, C.P. 9, 13418-900, Piracicaba, SP, Brazil. J.F.M.V. received a CNPq Fellowship
* Corresponding author (c-simpson{at}tamu.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Gregory and Gregory (1976) proposed that the peanut could have developed through the crossing between an annual and a perennial species, both of the section Arachis. They suggested that detailed karyotype studies followed by experimental interspecific crossings should be made for parental identification. Re-creation of A. monticola or A. hypogaea was considered by them a real possibility of considerable interest. Smartt et al. (1978) suggested that A. hypogaea probably developed from the hybridization of two diploid wild species with different genomes.
The perennial species of the section Arachis have 20 chromosomes, including Husted's A pair and show more similarity and better crossability to each other than those that do not have the A pair, generally being classified as the A genome species (Stalker, 1989; Stalker et al., 1991). The same applies to the annual A. duranensis and A. stenosperma Krapov. and W.C. Gregory. Section Arachis species without the small pair are all annual and much more heterogeneous, including a group of three with 2n = 18 chromosomes (Lavia, 1998; Peñaloza and Valls, 1997), one with six subtelocentric pairs (Fernández and Krapovickas, 1994), considered quite distant from A. hypogaea and classified by Stalker (1991) as a D genome species, and a third still heterogeneous group, with 20 metacentric or submetacentric chromosomes (or a rare subtelocentric pair), which includes the most probable non-A progenitor of A. hypogaea, A. ipaënsis (Fernández and Krapovickas, 1994). Since 1976, collection has accumulated species and germplasm accessions of section Arachis that cross with the peanut and are not associated to the A genome. Attempts of producing artificial AABB hybrids including A. ipaënsis, apparently the closest species to A. hypogaea on the basis of the karyotype and molecular markers, have consistently failed (Singh and Smartt, 1998). On the other hand, there is growing evidence of strong similarity between A. ipaënsis and A. magna Krapov, W.C. Gregory and C.E. Simpson (Simpson et al., 2001), and other species, such as A. williamsii Krapov. and W.C. Gregory, can also be included in this alliance, which increases the number of accessions available with the possibility of sharing the same B genome of the cultivated peanut.
Gregory and Gregory (1976) initially suggested A. duranensis and A. cardenasii Krapov. and W.C. Gregory as possible parents of the cultivated peanut. Based on cytological characterization and crossability studies, Smartt et al. (1978) suggested that several wild species of Arachis with A chromosomes could be potential donors of the A genome, A. cardenasii being the main candidate, while A. batizocoi Krapov. and W.C. Gregory was considered the possible donor of the B genome. For a long time, A. batizocoi had been the only species of section Arachis with available germplasm, that produced hybrids with A. hypogaea, but did not show the small A chromosome pair. In line with that, Gregory et al. (1980) warned that new collections were being made, so that other species could arise as possible progenitor candidates of A. hypogaea.
Based on seed protein data, Krishna and Mitra (1988) supported the indication of A. batizocoi and A. duranensis or A. cardenasii being the progenitors of A. hypogaea. Singh (1986) suggested that the parents were A. duranensis and A. batizocoi, but in 1988 he accepted the possibility of biphyletic origin, suggesting that A. hypogaea subsp. fastigiata Waldron evolved from a diploid species such as A. batizocoi and A. duranensis while A. hypogaea subsp. hypogaea evolved from diploid species such as A. batizocoi and A. villosa Benth. Hybrids between A. hypogaea and amphidiploids involving A. batizocoi and A. duranensis showed high pollen fertility and good fruit production, as well as a high association in bivalents, however, meiosis was not totally normal.
Contradicting the above information, Stalker and Dalmacio (1986) discarded the hypothesis that A. cardenasii and A. batizocoi could be the ancestors, based on cytological characteristics. Hilu and Stalker (1995), based on RAPD markers, stated that A. duranensis is likely the donor of the A genome of A. hypogaea but also discounted A. batizocoi as the donor of the B genome. Later on, Singh et al. (2002), stated that A. batizocoi could not be a parent of A. hypogaea, based on the relationship of repetitive ribosomal DNA polymorphism units of 77 accessions of wild species and A. hypogaea.
Kochert et al. (1991) using RFLP markers, suggested that the parents of A. hypogaea are A. ipaënsis and A. duranensis. Krapovickas and Gregory (1994), Fernández and Krapovickas (1994), and Seijo et al. (2004) also support this possibility. Fernández and Krapovickas (1994) demonstrate that the so-called B chromosome pair is present in every diploid Arachis species, including those also showing the small A pair. Therefore, the presence alone of a chromosome with a secondary constriction in a section Arachis species will not automatically qualify such species as a potential B genome donor. Fernández and Krapovickas (1994) suggest that the presence of a single pair with a secondary constriction in A. hypogaea is a consequence of the amphiplastic inhibition of one of such pairs brought in by both diploid parent species. Kochert et al. (1996) considered that A. duranensis was the female parent of the original hybridization event, while stressing a large amount of RFLP variability was found among the accessions of A. duranensis. Accessions they considered the most similar to the A genome of A. hypogaea were identified as accessions mostly concentrated in the Salta Province of Argentina.
Paik-Ro et al. (1992), using RFLP markers in 14 accessions of A. hypogaea, seven of A. monticola, four of A. batizocoi, four of A. cardenasii, five of A. duranensis, and four of A. glandulifera, suggested that A. duranensis is the closest diploid species to A. hypogaea, mainly the accession PI 468201 (also from Salta), that had similarity in more than 11 RFLP probes from a total of 13 probes. However, A. ipaënsis was not considered in the analysis. A closer affinity of A. ipaënsis than that of A. duranensis to the A. hypogaea/A. monticola accessions or introgression lines has been documented by Gimenes et al. (2002), using AFLP and RFLP markers. Most probably, each of the two species is showing its closest relationship to a different genome of the tetraploid A. hypogaea, and there is an appreciable agreement in the literature as concerns their complementary importance to the origin of the cultivated peanut.
Krapovickas and Gregory (1994) state that A. monticola, a wild tetraploid species that crosses with A. hypogaea, generating fertile descendents, could be a parent of the cultivated peanut or a derivative. However, they believe that the most likely parental species of A. hypogaea would be A. ipaënsis and A. duranensis, and they do not discard the possibility of a polyphyletic origin. A possible area for the origin of the peanut would be in the southeast of Bolivia and northwest of Argentina, where natural populations of A. ipaënsis, A. duranensis, A. batizocoi, and A. monticola could come together, corroborating the hypothesis that two wild sympatric species, carrying the A and B genomes, were crossed by bee pollination, generating a sterile hybrid that would be naturally chromosome doubled. Those fertile hybrids would have been domesticated by the native people of the area.
Later on, using RAPD and ISSR markers in a study of genetic diversity, Raina et al. (2001) observed that A. villosa, A. ipaënsis, A. monticola, and A. hypogaea form a group in the dendrogram, while A. duranensis formed a distinct group with A. cardenasii. Raina and Mukai (1999a, 1999b) considered A. ipaënsis and A. villosa Benth. to be the most probable progenitors of A. hypogaea, based on the observation of 18S-5,8S-26S, and 5S ribosomal loci for fluorescent in situ hybridization (FISH) and for genomic in situ hybridization (GISH). It is remarkable that their findings would discard the possibility of A. duranensis being one of the parents of the cultivated peanut. However, a more comprehensive FISH study by Seijo et al. (2004) reestablishes A. duranensis as the most probable donor of the A genome, although A. villosa is only discarded on geographic and morphological grounds.
Stalker et al. (1991) tried to hybridize A. ipaënsis and A. hypogaea, without success. Singh and Smartt (1998) took this as an indication that perhaps A. ipaënsis would not be the donor of the B genome of A. hypogaea. They suggest that one should apply more probes to enhance the covering of the genome before defending this hypothesis.
Singh and Smartt (1998) suggested that until a fertile synthetic amphidiploid is produced between A. duranensis and A. ipaënsis and it is crossed with A. hypogaea to produce a fertile hybrid, hypotheses on the probable parents of A. hypogaea would not be confirmed. They suggested that A. batizocoi would continue being the probable donor of the B genome, because of the cytogenetic similarity with A. hypogaea and stated that the re-creation of A. hypogaea could not be made in an exact way, due to the long time between the present and the origin of the species. It is important to note the proven usefulness of A. batizocoi in breeding programs, as a component of amphidiploids that produce fertile hybrids with A. hypogaea, therefore allowing the introgression of resistance genes from wild relatives into the cultigen (Simpson and Starr, 2001). The status of A. batizocoi as a putative progenitor of A. hypogaea is denied by the results obtained by Seijo et al. (2004), based on the physical mapping of the 5S and 18S-25S rRNA genes by FISH.
The objective of the present work is to report the crossability between A. ipaënsis and A. duranensis, and the accomplishment of tetraploidization through colchicine use, followed by successful crosses of the synthetic amphidiploid with several accessions of A. hypogaea. This work was conducted to study the evolution of the cultivated peanut through interspecific crossings, assuming the principal candidates to parents of A. hypogaea to be A. ipaënsis and A. duranensis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The hybridization technique consisted of emasculation of flowers of the female parents in bud phase between 1600 and 1900 h. In the morning of the following day, emasculated flowers were pollinated from 0700 to 0800 h, using pollen of the male parent.
Between August and October 2001, hybrids were identified by molecular analysis. Microsatellite marker technique was used for the separation of hybrid and possible self-pollinated individuals. This technique was chosen due to its codominant nature, easiness, and speed in providing results.
Molecular Characterization of the Hybrid between A. ipaënsis and A. duranensis
Leaves of each plant of the progeny were collected individually and DNA was extracted from leaves according to the adapted protocol of Murray and Thompson (1980). This stage of the research was done in the Laboratory of Plant Cellular and Molecular Biology of the Department of Genetics of the Faculty of Agriculture Luiz de Queiroz, ESALQ/USP. The amount of DNA was quantified by the use of agarose gels (1.2%) with 80 V for 1 h and diluted to the new concentration (2.5 ng mL–1). DNA amplification reaction (polymerase chain reaction [PCR]) had a final volume of 13 mL, and the reagents were mixed in the cocktail form, separately from DNA. Each reaction contained PCR buffer (10mM Tris-HCl pH 8.3 and 50 mM KCl), 1.5 mM MgCl2, 2.5 mM dNTPs, 5 pmol primers pair, 5 U mL–1 Taq DNA polymerase, 10 mg mL–1 BSA (bovine serum albumine), and 2.5 ng mL–1 DNA. Sterile Milli-Q water was added to complete the volume to 13 mL in the reaction. Mineral oil (50 mL) was applied to avoid evaporation of the cocktail. The PCR program consisted of the reaction: (i) 5 min at 94°C; (ii) 29 cycles with three stages (1 min at 94°C, 1 min at 56°C, and 1 min at 72°C); (iii) 7 min at 72°C. Amplified products were separated in 4% (w/v) agarose gel, using TBE buffer pH 8.0 (0.09 of Tris, 0.09 M boric acid, and 2 mM EDTA), at a constant 90 V cm–1. The gels were stained with 10 mL of ethidium bromide (10 mg mL–1) diluted in 100 mL of TBE and documented under ultraviolet light (GelDoc 2000, Biorad, Hercules, CA). The primers used were A1–041, A1–558, and LEC-1.
Pollen Viability
Estimation of pollen viability was made in eight flowers per hybrid combination; anthers were macerated on a slide and pollen staining was done with 2% acetic carmine. Stained pollen grains were counted to estimate pollen viability.
Amphidiploid Production
Cuttings approximately 20 cm long were taken from growing plants, transferred to assay tubes with the growing tips submerged in 0.2% colchicine, closed with PVC plastic film, and submitted to controlled conditions of fluorescent white light and controlled temperature from 28 to 30°C for 8 h. After treatment, cuttings were washed in running water for 20 min, cut at an angle in the oldest internode. Rooting hormone, indole butyric acid, was applied and cuttings were transferred to the screenhouse in plastic cups with vegetable substratum. Cups were conditioned for approximately 20 d in trays covered with transparent plastic bags to maintain high humidity.
Chromosome Counting of Synthetic Amphidiploid Cuttings
In January of 2002, the detached leaf protocol of Moraes and Salgado (1984) was adapted for rooting petioles (Fávero et al., 2004). New, totally expanded leaves of the colchicine-treated cuttings were cut and the petioles were treated with indole butyric acid and immediately transferred to cups with vegetable substratum. After, two or more weeks, roots grew and were collected between 1000 and 1400 h.
Chromosome counting was done at the Laboratory of Cytology of the Department of Genetics of ESALQ/USP. Methodologies of root tips treatment and cytological preparations were adapted from Aguiar-Perecin and Vosa (1985) and Silvarolla and Aguiar-Perecin (1994).
After observation good preparations were selected and cover slips were removed in 45% acetic acid and mounted with Canadian balsam. Photomicrographs of the chromosomes were obtained with a Zeiss photomicroscope (Thornwood, NY), using Kodak Technical Pan (ISO 25) film (Rochester, NY).
Morphological Characterization
Diploid and tetraploid flower structures were measured for evaluation of morphological differences. A digital caliper was used to measure standard length and width, wing length and width, lower and upper lip length, and hypanthium length; t test was used for average comparisons (Table 2).
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Molecular Characterization of Progenies of A. hypogaea and Synthetic Amphidiploid Hybridization
Molecular marker studies were done in the Laboratory of Plant Genetic Characterization of Embrapa Genetic Resources and Biotechnology. In March 2003, leaves were removed from the individual progenies originating from crosses between A. hypogaea and the synthetic amphidiploids for DNA extraction and identification of hybrids using microsatellite markers. Protocol used was adapted from Ferreira and Grattapaglia (1995). Quantification stages, PCR reactions and electrophoresis conditions were the same as before. Primer used was Lec-1. Polyacrylamide gel electrophoresis with silver staining was used for distinction among individuals based on microsatellite marker polymorphism. (Bassam et al., 1991).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Microsatellite markers proved to be efficient in the identification of hybrids. All individuals of the progenies could be identified without doubt as to the hybrid or self-pollinated condition.
Twenty-four pollinations were made, resulting in five hybrid plants, with a percentage of success of 20.83%. A diploid cell of the hybrid with a chromosome A is shown in Fig. 1. From experience of evaluating Arachis chromosomes, it is known that the other small bodies shown in Fig. 1 are chromosome satellites. Stained pollen was 0.98%.
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Measurement data of morphological structures of diploid and tetraploid flowers are shown in Table 2. There were significant differences for all the floral structures measured, except for the upper lip length (Table 2).
Arachis hypogaea and Synthetic Amphidiploid Hybridizations
Figure 2 shows it is possible to distinguish with microsatellite markers between hybrid individuals and self-pollinated progeny. Polyacrilamide gel was used for progeny distinction of a family of A. hypogaea cv. BR-1 x [A. ipaënsis (KGBPScS 30076) x A. duranensis (VNvEv 14167)]4x. Individuals of the progeny indicated by the black arrows were considered as hybrid because they had all bands of both parents. A total of 13 microsatellites were used, but only three were polymorphic in the diploids and just one, Lec-1, was polymorphic in the amphiploids (Fig. 2). It is not clear why all three were not polymorphic.
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An important diagnostic morphological marker is the yellow flower color in the tetraploid hybrids. All A. hypogaea accessions used in the work had orange flowers as did A. ipaënsis. Arachis duranensis (VNvEv 14167) with yellow flowers was always used as male parent, and the diploid hybrids consistently showed yellow flowers. Another remarkable morphological characteristic is the significant increase in the number of trichomes on the edges of leaves and on stems of hybrids.
The hybrids obtained from the cross between A. hypogaea and the amphidiploid A. ipaënsis x A. duranensis indicate that these species are closely related to A. hypogaea. In evolutionary studies of the cultivated peanut, the possibility of successful hybrids involving A. hypogaea, A. ipaënsis, and A. duranensis was a goal of primary importance to the validation of several studies in molecular, morphological, and cytogenetic characterization previously published (Kochert et al., 1991, 1996; Krapovickas and Gregory, 1994; Fernández and Krapovickas, 1994; Gimenes et al., 2002; Seijo et al., 2004), where different authors agreed that A. ipaënsis and A. duranensis would be the most probable progenitors of the cultivated peanut.
Singh and Smartt (1998) stated that, since a fertile hybrid had not been obtained from A. ipaënsis x A. duranensis and crossed to the cultivated peanut, it was not possible to confirm this hypothesis of the origin of A. hypogaea. As the result of our work, hybrids were obtained from crosses between six different botanical varieties of A. hypogaea and a fertile synthetic amphidiploid A. ipaënsis x A. duranensis. All tetraploid hybrid F1 plants produced pegs and seeds, documenting the fertility of the F1 interspecific hybrids (Table 3).
Further studies of chromosome pairing are underway, but the production of the several F2 progenies at least raises A. ipaënsis to the same status attributed by Singh and Smartt (1998) to A. batizocoi, additionally emphasizing the closest association of A. ipaënsis to A. hypogaea, based on a broad array of investigative approaches.
The fact that fertile hybrids were obtained between the synthetic A. ipaënsis x A. duranensis amphidiploid and representatives of both subspecies and even more described varieties of A. hypogaea (Krapovickas and Gregory, 1994) than those used by Singh (1988) documents their high potential for the crop improvement, especially when recent studies based on more accurate techniques (Ferguson et al., 2004; Moretzsohn et al., 2004) start to unveil the obvious genetic diversity, previously not well understood, of Arachis germplasm resources.
Received for publication October 17, 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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  G. Seijo, G. I. Lavia, A. Fernandez, A. Krapovickas, D. A. Ducasse, D. J. Bertioli, and E. A. Moscone Genomic relationships between the cultivated peanut (Arachis hypogaea, Leguminosae) and its close relatives revealed by double GISH Am. J. Botany, December 1, 2007; 94(12): 1963 - 1971. [Abstract] [Full Text] [PDF]
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