Cultivated peanut (Arachis hypogaea) is an allotetraploid with closely related subgenomes of a total size of ∼2.7 Gb. This makes the assembly of chromosomal pseudomolecules very challenging. As a foundation to understanding the genome of cultivated peanut, we report the genome sequences of its diploid ancestors (Arachis duranensis and Arachis ipaensis). We show that these genomes are similar to cultivated peanut's A and B subgenomes and use them to identify candidate disease resistance genes, to guide tetraploid transcript assemblies and to detect genetic exchange between cultivated peanut's subgenomes. On the basis of remarkably high DNA identity of the A. ipaensis genome and the B subgenome of cultivated peanut and biogeographic evidence, we conclude that A. ipaensis may be a direct descendant of the same population that contributed the B subgenome to cultivated peanut.

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The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut.

Nuclear restriction fragment length polymorphism (RFLP) analysis was used to determine the wild diploid Arachis species that hybridized to form tetraploid domesticated peanut. Results using 20 previously mapped cDNA clones strongly indicated A. duranensis as the progenitor of the A genome of domesticated peanut and A ipaensis as the B genome parent A large amount of RFLP variability was found among the various accessions of A duranensis, and accessions most similar to the A genome of cultivated peanut were identified. Chloroplast DNA RFLP analysis determined that A duranensis was the female parent of the original hybridization event Domesticated peanut is known to have one genome with a distinctly smaller pair of chromosomes ("A"), and one genome that lacks this pair. Cytogenetic analysis demonstrated that A duranensis has a pair of "A" chromosomes, and A. ipaensis does not The cytogenetic evidence is thus consistent with the RFLP evidence concerning the identity of the progenitors RFLP and cytogenetic evidence indicate a single origin for domesticated peanut in Northern Argentina or Southern Bolivia, followed by diversification under the influence of cultivation

RFLP and Cytogenetic Evidence on the Origin and Evolution of Allotetraploid Domesticated Peanut, Arachis hypogaea (Leguminosae)

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5' and 3' ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.

Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica).

G e n e t i c molecular markers are DNA segments that behave as landmarks for genome analysis. These segments usually represent variant or polymorphic sites that can be identified using general strategies such as molecular hybridization or enzymatic amplification of DNA. For years, DNA-based diagnostic markers have been used in general organismal identification and in the construction of genetic linkage maps. (1) The widely used restriction fragment length polymorphisms (RFLPs), for example, are molecular markers identified by endonuclease restriction and blot hybridization of DNA. (z) DNA amplification using PCR (3'4) has also been used extensively in many applications to study polymorphic loci, like hypervariable minisatellites (s) or microsatellites harboring simple sequence repeats, (6-8) and to generate sequence-tagged sites (STSs) (9) for genetic and physical mapping. Several strategies involving DNA replication, DNA ligation, or RNA transcription have been used for in vitro amplification of nucleic acids. (1~ However, PCR remains the most widely accepted amplification tool. Primer-directed amplification of DNA, at first used in PCR to amplify cognate regions present at very low levels in the genome, has extended DNA analysis to regions adjacent to sequenced DNA segments, to unknown DNA, and even to the study of RNA-expressed sequences. (~s-~7) Amplification strategies can be grouped according to the mechanism of the amplification process (Table 1). Amplification with specific primers in the PCR, for example, is a determinate process that requires prior knowledge of the template sequence and targets usually one defined amplification site. Similarly, amplification of interspersed repetitive sequences (IRS), like Alu-PCR (18) or REP-PCR, (19) are determinate processes that target multiple sites of defined sequence in both DNA strands. In contrast, amplification with degenerate primers in random indeterminate amplification processes, like random primed amplification (RPA), (2~ primer-extension preamplification (PEP), (21) and random PCR (rPCR), (22) take advantage of stochastic annealing events that randomly amplify nucleic acid stretches or even whole genomes by targeting sites of a noncognate nature. These random DNA amplification procedures are generally used for radioactive or fluorescent labeling of nucleic acids, to increase the amount of DNA in the construction of representative cDNA libraries, or for PCR typing of single haploid cells. Other kinds of amplification processes, although still determinate, do not require prior knowledge of template sequence and can target single or multiple sites in a genome or template molecule. These strategies can use either one arbitrary primer in conjunction with a specific primer (23) or one or more arbitrary primers (24-26) to study single or multiple amplicons, respectively, and can even be extended to the analysis of RNA populations. (27-28) In particular, strategies like gene-walking PCR (23) and differential cDNA PCR (27) study specific template regions arbitrary in length but juxtaposed to a known DNA segment defining a specific primer. Like anchored PCR, (29) these hemispecific reactions allow analysis of unknown genomic regions corresponding to mRNA sequences, or adjacent to STSs or sequenced stretches of DNA.

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Amplifying DNA with arbitrary oligonucleotide primers.

Twenty-one random and 29 SSR primers were used to assess genetic variation and interrelationships among subspecies and botanical varieties of cultivated peanut, Arachis hypogaea (2n = 4x = 40), and...

RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/-) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F2 progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F2 progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.

I Tom Stalker

Papers

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.).