Molecular analysis of Arachis interspecific hybrids.

Incorporation of genetic resistance against several biotic stresses that plague cultivated peanut, Arachis hypogaea (2n = 4x = 40), is an ideal option to develop disease resistant and ecologically safe peanut varieties. The primary gene pool of peanut contains many diploid wild species (2n = 2x = 20) of Arachis, which have high levels of disease and insect resistances. However, transfer of resistant genes from these species into A. hypogaea is difficult due to ploidy level differences and genomic incompatibilities. This study was conducted to monitor alien germplasm transmission, using Random Amplified Polymorphic DNA (RAPD) markers, from two diploid wild species, A. cardenasii and A. batizocoi, into A. hypogaea. Triploid interspecific hybrids were produced by crossing two A. hypogaea cultivars (NC 6 and Argentine) with the two species and by colchicine-treating vegetative meristems, fertility was restored at the hexaploid (C(o)) level in the four hybrids. Hexaploids were allowed to self-pollinate for four generations, each referred to as a cycle (C1, C2, C3, and C4). At each cycle, a backcross was made with the respective A. hypogaea cultivar as the maternal parent and only lineages tracing back to a single hexaploid hybrid were used for RAPD analysis. Analysis of mapped, species-specific RAPD markers in BC1F1 to BC1F3 hybrids indicated that alien germplasm retention decreased every generation of inbreeding, especially in Argentine and in A. batizocoi crosses. A similar trend was also observed for every cycle in BC1F2 and BC1F3 families, possibly, due to the loss of alien chromosomes following selfing of hexaploids. RAPD marker analysis of 40-chromosome interspecific hybrid derivatives from the four crosses supported previous reports that reciprocal recombination and/or translocations are the predominant mechanisms for exchange of chromosomal segments. No evidence was found for preferential transfer of alien chromosomal regions to specific linkage groups. The implications for developing disease resistant peanut breeding lines are discussed in light of these findings.

The phylogenetic relationship of possible progenitors of the cultivated peanut.

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid composed of A and B genomes. The phylogenetic relationship among the cultivated peanut, wild diploid, and tetraploid species in the section Arachis was studied based on sequence comparison of stearoyl-ACP desaturase and oleoyl-PC desaturase. The topology of the trees for both fatty acid desaturases displayed two clusters; one cluster with A genome diploid species and the other with B genome diploid species. The two homeologous genes obtained for each of the two fatty acid desaturases from the tetraploid species A. hypogaea and A. monticola were separated into the A and B genome clusters, respectively. The gene phylogenetic trees showed that A. hypogaea is more closely related to the diploid species A. duranensis and A. ipaensis than to the wild tetraploid species A. monticola, suggesting that A. monticola is not a progenitor of the cultivated peanut. In addition, for the stearoyl-ACP desaturase, the A. duranensis sequence was identical with one of the sequences of A. hypogaea and the A. ipaensis sequence was identical with the other. These results support the hypothesis that A. duranensis and A. ipaensis are the most likely diploid progenitors of the cultivated tetraploid A. hypogaea.

Characterization of a unique genomic clone located 5' upstream of the Oshsp16.9B gene on chromosome 1 in rice (Oryza sativa L. cv Tainung No. 67).

Small heat-shock proteins (sHSP) are the most abundant heat stress-induced proteins in plants. In rice, there are at least seven members of class-I sHSP. A 1.6-kb DNA fragment was isolated from the EcoRI-digested rice genomic library probed with the cDNA pTS1 encoding a 16.9-kDa class-I sHSP. This fragment was composed of 365-bp tandem direct repeats (DRs) and 441-bp near perfect long terminal inverted repeats (LTIRs). The DRs contain 123-bp regions with 99% nucleotide identity to the 5' coding region of the Oshsp16.9B gene. Two putative pseudogenes were deduced from the DRs. Using the LTIR as a specific probe, Southern-blotting analysis showed that there was a single copy of this 1.6-kb DNA fragment in the rice genome. By genomic walking, we located this fragment in proximity 5'-upstream of the Oshsp16.9B gene that was mapped on chromosome 1 with other two class-I sHSP genes, Oshsp16.9A and Oshsp16.9C. By comparative analysis of the nucleotide sequences of class-I sHSP genes clustered on chromosome 1 between Tainung No. 67 and Nipponbare cultivars, we confirmed our mapping results of these genes and only the promoter region of Oshsp16.9B was different. However, we found that the expression profile of Oshsp16.9B upon different heat stresses in Nipponbare was not significantly different relative to that in Tainung No. 67.

Microsatellite analysis of demographic genetic structure in fragmented populations of the tropical tree Symphonia globulifera.

We developed genetic markers for three microsatellite loci in the tropical tree Symphonia globulifera and used them to examine the demographic genetic consequences of forest fragmentation. High levels of genetic variation were revealed in samples of adults, saplings, and seedlings. The more-variable loci exhibited less stability in allelic composition across sites and stages. The number of alleles per hectare (ha) of forest was similar when continuous forest plots were compared to plots from fragmented forest for all three stages. This pattern also held for the number of unique multilocus adult and sapling genotypes, but the number of unique seedling genotypes per ha of fragmented forest greatly exceeded expectations based on continuous forest data, probably due to the concentration of seeds into remnant forest patches by foraging bats. Significant inbreeding and genetic differentiation were most often associated with the fragmented forest and the seedlings. Finally, principal component analysis reaffirmed that a bottleneck, acting in concert with pre-existing genetic structure in the adults, had led to enhanced and rapid divergence in the seedlings following deforestation, a result that is of central interest for landscape management.

Independent amplification of two classes of Tourists in some Oryza species.

Transposable elements similar to Tourist elements from maize were isolated from the rice genome. The elements were about 300 bp, exhibited short terminal inverted repeats (TIR), and appeared to show preferential insertion at TAA sites. Some rice Tourist elements seem to have recently transposed. Based on the sequences of cloned elements, two classes of rice Tourist elements have been identified. Members of these two classes apparently amplified independently at different times in the past.

Identification of RAPD, SCAR, and RFLP markers tightly linked to nematode resistance genes introgressed from Arachis cardenasii into Arachis hypogaea.

Two dominant genes conditioning resistance to the root-knot nematode Meloidogyne arenaria were identified in a segregating F2 population derived from the cross of 4x (Arachis hypogaea x Arachis cardenasii)-GA 6 and PI 261942. Mae is proposed as the designation for the dominant gene restricting egg number and Mag is proposed as the designation for the dominant gene restricting galling. The high levels of resistance in GA 6 were introgressed from A. cardenasii and, therefore, a search to identify A. cardenasii specific RAPD markers that are tightly linked to these resistance genes was conducted utilizing bulked segregant analysis. One RAPD marker (Z3/265) was linked at 10 +/- 2.5 (SE) and 14 +/- 2.9 cM from Mag and Mae, respectively. The marker was mapped to linkage group 1 at 5 cM from Xuga.cr239 in the backcross map in an area where introgression from A. cardenasii had previously been reported. This fragment was cloned and used to generate a pair of primers that specifically amplified this locus (sequence characterized amplified region, SCAR) and as a RFLP probe. Their close linkage with the resistance genes will be useful in marker-based selection while transferring nematode resistance from introgression lines into elite breeding lines and cultivars. The Z3/265 marker associated with the genes Mae or Mag was not found in other highly resistant Arachis species (Arachis batizocoi or Arachis stenosperma), in progenies of interspecific crosses with A. cardenasii that were moderately resistant, or in the resistant A. hypogaea lines PI 259634 and PI 259572. These represent the first molecular markers linked with a resistant gene in peanut and the first report of two physiological responses to nematode attack associated with two genetic factors.

Genome duplication in soybean (Glycine subgenus soja).

Restriction fragment length polymorphism mapping data from nine populations (Glycine max x G. soja and G. max x G. max) of the Glycine subgenus soja genome led to the identification of many duplicated segments of the genome. Linkage groups contained up to 33 markers that were duplicated on other linkage groups. The size of homoeologous regions ranged from 1.5 to 106.4 cM, with an average size of 45.3 cM. We observed segments in the soybean genome that were present in as many as six copies with an average of 2.55 duplications per segment. The presence of nested duplications suggests that at least one of the original genomes may have undergone an additional round of tetraploidization. Tetraploidization, along with large internal duplications, accounts for the highly duplicated nature of the genome of the subgenus. Quantitative trait loci for seed protein and oil showed correspondence across homoeologous regions, suggesting that the genes or gene families contributing to seed composition have retained similar functions throughout the evolution of the chromosomes.

Genetic diversity within the species Arachis duranensis Krapov. &W.C. Gregory, a possible progenitor of cultivated peanut.

Eighteen accessions of a diploid wild peanut species (Arachis duranensis) were analyzed using morphological, intercrossing, cytological, and RFLP data. Abundant variation was found for morphological characters and for RFLP patterns both between and within accessions, and each accession could be uniquely identified by RFLP pattern. Several plants were found to be F1 hybrids between different accessions, indicating that intercrossing had occurred when these were planted for seed increase. Patterns of RFLP diversity were found to correspond with geographic distribution. Analysis of the number of RFLP fragments observed per accession indicates that additional field collections of this complex of taxa will yield additional genetic variability.

Analysis of annual Medicago species using RAPD markers.

Annual species of the genus Medicago have attracted interest as green manure and temporary forage crops. This study was conducted to determine if randomly amplified polymorphic DNA (RAPD) markers could be used to assess the variability within and among species. Several accessions of each six species (M. scutellata Mill., M. disciformis DC., M. murex Willd., M. truncatula Gaertn., M. polymorpha L., and M. rugosa Desr.) were studied. A phylogeny reconstructed with the computer program Phylogenetic Analysis Using Parsimony (PAUP) showed the same relationships as traditional taxonomy. Variation was present among accessions of all species. Several accessions were considerably different from others within the species (one of each M. scutellata and M. polymorpha) and four accessions of M. murex were differentiated by both morphology and RAPD banding patterns from the other accessions. These accessions may be useful to include in a core collection. Variation within accessions was present. Although the species are autogamous, the original seed collections may have been made from a number of plants in the same area. Also, some outcrossing or seed mixing may have occurred. Finally, at least 10 RAPD primers appear to be necessary in order to develop reliable estimates of relatedness among annual Medicago accessions.

Introgression analysis of an interspecific hybrid population in peanuts (Arachis hypogaea L.) using RFLP and RAPD markers.

Forty-six introgression lines (F10C9) from a cross between Arachis hypogaea L. (2n = 4x = 40) and A. cardenasii Krapov. & W. C. Gregory (2n = 2x = 20) were analyzed for the introgression of A. cardenasii chromosome segments. Seventy-three RFLP probes and 70 RAPD primers, expressing from one to four A. cardenasii-specific bands, were used to evaluate the set of introgression lines. Thirty-four RFLP probes and 45 RAPD primers identified putative A. cardenasii introgressed chromosome segments in one or more lines. Introgressed segments were detected by RFLP analysis in 10 of the 11 linkage groups; the smallest introgressed fragments were detected by single RFLP markers and the largest were detected by three or four adjacent markers and represented introgressed segments of 30-40 cM. Similar results were obtained with RAPD markers, although markers detecting introgressed fragments could not be placed on the peanut linkage map. Introgression into both A. hypogaea genomes was detected and its implication in breeding for disease resistance is discussed.

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa).

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F2 progeny, which were derived from a single F1 plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.

Comparative RFLP mapping of a wild rice, Oryza officinalis, and cultivated rice, O. sativa.

A comparative RFLP map was constructed in a wild rice, Oryza officinalis, by using 139 genomic and cDNA probes that had been used previously to map RFLPs in O. sativa. Nine of the 12 chromosomes of O. officinalis were highly homosequential to those of O. sativa. A major rearrangement of gene order was detected in chromosome 1 and small inversions were found in chromosomes 3 and 11. Fourteen translocated RFLP markers were found, and chromosome 11 contained a high frequency of such translocated segments. Results were consistent with meiotic and trisomic analysis, which suggested that the genomes of O. officinalis and O. sativa were similar. Applications of comparative maps in plant breeding and gene cloning are discussed.

Molecular characterization of a strain-specific repeated DNA sequence in the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm moth, Spodoptera frugiperda, is a migratory species composed of sympatric corn and rice strains. The strains are indistinguishable in morphology but can be recognized by molecular markers. We have cloned and characterized seven monomer units of a repeated DNA sequence, called FR, which is found exclusively in the genome of the rice strain individuals. The 189 bp FR units are tandemly organized in arrays longer than 30 kb. Female individuals possess over 100-fold more of the FR sequence than male individuals. The repeated sequence is not methylated at GGCC sites, and shows high sequence similarity among repeat units.

A study of genetic variation and evolution of Phyllostachys (Bambusoideae: Poaceae) using nuclear restriction fragment length polymorphisms.

Phylogenetic and taxonomic difficulties are common within the woody bamboos, due to their unique life cycle, which severely limits the availability of floral characters. To addresss some of these problems, 20 species of woody bamboos in the genus Phyllostachys were analyzed using nuclear restriction fragment length polymorphisms (RFLPs). The RFLP data were used to generate genetic distances between all pairs of taxa and to examine the degree of genetic variation within and among bamboo species. The genetic distances were also used to create dendrograms of accessions and species. These trees supported the current division of the genus into two sections and provided some information on the thorny taxonomic problems in this group. We show that RFLPs can be used for species identification and the delineation of species limits.

Clusters of interspersed repeated DNA sequences in the rice genome (Oryza).

We have characterized a repeated DNA sequence (RTL122) from rice (Oryza sativa L.) with respect to its organization in the rice genome and its distribution among rice and other plants. The results indicate that the RTL122 sequence is interspersed in the rice genome and limited to the genus Oryza. It is highly polymorphic and can be used to fingerprint rice varieties. A structure was observed in which several repeated sequences were clustered in DNA regions of 15-20 kb. We characterized three bacteriophage lambda clones that contained the RTL122 sequence. Southern analysis using probes derived from restriction fragments of the three lambda clones indicated that all fragments except one are interspersed repeated sequences and belong to different repeated sequence families. Subsequent slot blot hybridization showed that most of them are only present within the genus Oryza. Some of the Oryza-specific, physically linked sequences show the same phylogenetic distribution, which suggests that these sequences might have evolved in a coordinate fashion. On the other hand, some of the repeated sequences have a different distribution even though they are physically adjacent in the genome. We speculate that such blocks of interspersed repeated sequences may serve as hotspots for rapid changes in the rice genome.

Genetic relatedness among rabbiteye blueberry (Vaccinium ashei) cultivars determined by DNA amplification using single primers of arbitrary sequence.

Most improved cultivars of commercially important hexaploid rabbiteye blueberry were developed from only four native selections collected from the wild; thus, many cultivars are closely related by lineage. The consanguinity among major cultivars is a potential problem, as the rabbiteye blueberries are highly self-incompatible natural outcrossers with potential inbreeding depression. We investigated the extent of genetic relatedness among 15 improved cultivars and four wild selections by the technique of random amplified polymorphic DNA, also referred to as arbitrarily primed polymerase chain reaction. Single decanucleotides of arbitrary sequence revealed polymorphism among cultivars and wild selections. Genetic distances were estimated based on the amount of band sharing. Cluster analysis of genetic distance estimates tended to group siblings with each other and with one or both parents. The average genetic distance between improved cultivars decreased relative to the four wild parental selections, which might indicate progression towards inbreeding. The significance of increased genetic relatedness among the improved cultivars of rabbiteye blueberry and the application of molecular methods in breeding and commercial cultivation is discussed.

Phylogenetic relationships of the pigeonpea (Cajanus cajan) based on nuclear restriction fragment length polymorphisms.

Nuclear restriction fragment length polymorphisms (RFLPs) were used to determine phylogenetic relationships in the genus Cajanus using 15 random genomic probes and six restriction enzymes. Twenty-four accessions representing 12 species of four genera (Cajanus, Dunbaria, Eriosema, and Rhynchosia) were examined to determine phylogenetic relationships in the genus Cajanus. Eriosema parviflorum was selected as the out-group. Sufficient RFLP polymorphisms were detected among species to resolve in-group taxa into distinct clusters. Topologies of trees from parsimony and similarity matrix analyses were similar but not identical, and clustering patterns agreed broadly with published phylogenies based on seed protein data and, to a lesser extent, data from cytology and breeding experiments. Accessions of cultivated C. cajan shared more DNA fragments with C. scarabaeoides than with C. cajanifolia. Inconsistencies in taxonomic relationships based on data from morphology, cytology, crossability, and RFLPs are discussed.

Phylogenetic distribution and genetic mapping of a (GGC)n microsatellite from rice (Oryza sativa L.).

DNA microsatellites are ubiquitously present in eukaryotic genomes and represent a vast source of highly informative markers. We describe in this article a (GGC)n microsatellite which is widely distributed in eukaryotic genomes. Using polymerase chain reaction (PCR) techniques and DNA sequencing, we demonstrated for the first time in plant species that a (GGC)n microsatellite locus is moderately polymorphic. Six alleles are present at this locus in rice and length polymorphisms are caused by variation in the number of tandem GGC repeats. By scoring a backcross mapping population, we were able to demonstrate that this locus is stably inherited and does not link to any known RFLP markers on the rice RFLP map. Our results suggest that DNA microsatellites should be useful in plants for construction of genetic linkage maps, extension of the existing genetic linkage maps, linkage analysis of disease and pest resistance genes, and the study of population genetics.

Development of an RFLP linkage map in diploid peanut species.

An RFLP linkage map of peanut has been developed for use in genetic studies and breeding programs aimed at improving the cultivated species (Arachis hypogaea L.). An F2 population derived from the interspecific hybridization of two related diploid species in the sectionArachis (A. stenosperma ×A. cardenasii) was used to construct the map. Both random genomic and cDNA clones were used to develop the framework of the map. In addition, three cDNA clones representing genes coding for enzymes involved in the lipid biosynthesis pathway have been mapped in peanut. Of the 100 genomic and 300 cDNA clones evaluated, 15 and 190, respectively, revealed polymorphisms among the parents of our mapping population. Unfortunately, a large number of these produced complex banding patterns that could not be mapped. Of the 132 markers analyzed for segregation, 117 are distributed among 11 linkage groups, while 15 have not yet been associated with any other marker. A total map distance of approximately 1063 cM has been covered to-date.