Diversity of seed storage protein patterns in wild peanut (Arachis,Fabaceae) species

55 accessions of wild peanuts (Arachis spp.) introduced from South America were analyzed for seed storage protein composition using SDS-PAGE electrophoresis. The objectives of the study were to evaluate variability within sect.Arachis and to classify taxa based on protein composition. 25 different band positions were resolved. Individual accessions had 11 to 18 bands which included the conarachin region (MW > 50 kD), two to five bands in the acidic arachin region (MW 38–49.9 kD), three to seven in the intermediate MW region (23 to 37.9 kD), two to five bands in the basic arachin region (18–22.9 kD), and one to three bands in the low MW protein region (14–17.9 kD). These data were utilized in a principal coordinate analysis based on the matrix of genetic distances between all pairs of the 55 accessions. Several groups of accessions conformed to expected species classification includingA. batizocoi, A. stenosperma, andA. monticola; whileA. duranensis, A. cardenasii, A. helodes, andA. correntina did not form good groups. The study showed that great diversity exists for protein profiles and seed storage proteins have potential for aiding species classification and for serving as markers for interspecific hybridization studies.     

Genetic relationships between peanut and wild species ofArachis sect.Arachis (Fabaceae): Evidence from RAPDs

Twenty-six accessions of wildArachis species and domesticated peanuts,A. hypogaea, introduced from South America were analyzed for random amplified polymorphic DNA (RAPD). The objective of the study was to investigate inter- and intraspecific variation and affinities among species of sect.Arachis which have been proposed as possible progenitors for the domesticated peanut. Ten primers resolved 132 DNA bands which were useful for separating species and accessions. The most variation was observed among accessions ofA. cardenasii andA. glandulifera whereas the least amount of variation was observed inA. hypogaea andA. monticola. The two tetraploid species could not be separated by using RAPDs.Arachis duranensis was most closely related to the domesticated peanut and is believed to be the donor of the A genome. The data indicated thatA. batizocoi, a species previously hypothesized to contribute the B genome toA. hypogaea, was not involved in its evolution. The investigation showed that RAPDs can be used to analyze both inter- and intraspecific variation in peanut species. Southern hybridization of RAPD probes to blots containing RAPD of theArachis species provided information on genomic relationships and revealed the repetitive nature of the amplified DNA.     

Flow cytometric analysis of genome size variation in cultivated and wildPisum sativum (Fabaceae)

Fifteen cultivars, landraces, and wild accessions ofPisum sativum subspecies, and one accession ofP. abyssinicum were analysed with flow cytometry (DAPI staining) usingP. sativum Kleine Rheinländerin as internal standard. Applying the method of jointly isolating, staining, and measuring nuclei of individual seedlings of test and standard material, it was found that in allP. sativum comparisons G 1 and G 2 peaks were invariably unimodal and symmetric at coefficients of variation mostly less than 2%. This is strong evidence for absence of significant genome size variation in theP. sativum strains analysed. These data are markedly at variance to results of other authors reporting considerable genome size variation withinP. sativum. However, inP. abyssinicum flow cytograms and Feulgen densitometric measurements indicate 4–8% more DNA, at same chromosome number (2n = 14), than inP. sativum. This result demonstrates that genome size variation is indeed existent in the genus and requires further examination.     

An analysis of the B genome speciesArachis batizocoi (Fabaceae)

Arachis batizocoi Krap. & Greg. is a suggested B genome donor to the cultivated peanut,A. hypogaea L. Until recently, only one accession of this species was available in U.S.A. germplasm collections for analyses and species variability had not been documented. The objective of this study was to determine the intraspecific variability ofA. batizocoi to better understand phylogenetic relationships in sect.Arachis. Five accessions of the species were used for morphological and cytological studies and then F₁ intraspecific hybrids analyzed. Some variation was observed among accessions—for example, differences in seed size, plant height and branch length. The somatic chromosomes of accessions 9484, 30079, and 30082 were nearly identical, whereas, the karyotypes of accessions 30081 and 30097 have several distinct differences. For example, 30081 had significantly more asymmetrical chromosomes 2 and 6 and more median chromosomes 7 and 10, and 30097 had significantly more asymmetrical chromosomes 3 and 10 and more median chromosomes 1 and 5 than accessions 9484, 30079, and 30082. All F₁ hybrids among accessions were highly fertile. Meiotic observations indicated that hybrids among accessions 9484, 30079, or 30082 had mostly bivalents. However, quadrivalents were observed when either 30081 or 30097 was crossed with the above three accessions and 30081 × 30097 had quadrivalents, hexavalents and octavalents. The presence of translocations is the most likely cause of multivalent formation inA. batizocoi hybrids. Cytological evolution via translocations has apparently been an important mechanism for differentiation in the species.Paper No. 12382 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643.     

Detection of Differentiation Among BB,CC and EE Genomes in the Genus Oryza by Two-probe Genomic in situ Hybridization (GISH)

双探针原位杂交揭示稻属BB、cc 和EE基因组之间的分化 李常宝1 张大明1* 葛颂1 卢宝荣2 洪德元1     

Genomic in situ hybridization analysis between Rubus coreanus and its relatives in Rubus (Sect. Idaeobatus)

To further investigate the phylogenetic relationship between Rubus coreanus and its relatives in the section Idaeobatus, we used genomic in situ hybridization (GISH) to ascertain the degree of their genomic homology. Genomic DNA from R. parvifolius and R. inopertus hybridized throughout the centromeric and sub-terminal regions on 14 and 12 chromosomes of R. coreanus, respectively. The probes from R. niveus and R. ellipticus var. obcordatus gave robust signals at the same region of eight chromosomes. R. ellipticus and R. pinfaensis generated strong signals at the centromeric and sub-terminal parts of six chromosomes. The hybridization signals from the R. tsangii and R. corchorifolius probes existed only at the telomeric parts of four chromosomes. The two signals at the sub-terminal region on chromosome 6 of R. coreanus might be 45S rDNA repeats. These results indicated that R. coreanus and R. parvifolius shared many repeat sequences. It could be deduced that the genome of R. parvifolius was most closely related to that of R. coreanus among the species tested, R. inopertus came next, while R. tsangii and R. corchorifolius showed the farthest relationship. The phylogenetic relationships between R. parvifolius and R. coreanus, as well as among the five subsections were mainly discussed.     

An improved method of genomic in situ hybridization (GISH) for distinguishing closely related genomes of tetraploid and hexaploid wheat species

An improved modification of genomic in situ hybridization (GISH) was proposed. It allows clear and reproducible discrimination between closely related genomes of both tetraploid and hexaploid wheat species due to preannealing of labeled DNA probes and prehybridization of chromosomal samples with blocking DNA. The method was applied to analyze intergenomic translocations 6A:6B and 1A:6B identified in the IG46147 and IG116188 samples of tetraploid wheat Triticum dicoccoides by C-banding. The structure of the rearranged chromosomes was defined for two translocation variants, and the breakpoints were identified on the chromosome arms. Possible application of the developed GISH variant to study genome reorganizations during speciation of allopolyploid plants in evolution is discussed.     

A new species of the genusLotus (Fabaceae) in North Africa

A new species,Lotus digii, has been found in Morocco growing on the coastal sandy soils. Further localities are from Algeria and Egypt. It should be expected also in Libya.     

Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens

Summary Crossability between the diploid species S. circaeifolium subsp. circaeifolium (crc) and other diploid species, primarily diploid S. tuberosum subsp. tuberosum (tbr-2x), was studied. Forty-seven hybrids were obtained from crosses between crc as female parent and tbr-2x and some other species from series Tuberosa as male parents. Of these hybrids 17% were diploids; the other 83% were triploids, probably carrying two genomes of crc. Female fertility was sufficient to obtain offspring from backcrosses with the cultivated parent. Pollen stainability of the f₁ varied, and micro-pollen as well as unreduced pollen occurred. During meiosis of the diploids and triploids a rather high proportion of univalents was found, and in the triploids on average two or three trivalents per cell were found. All hybrids were resistant to Globodera pallida pathotypes 2 and 3, and 75% of the tested genotypes were highly resistant to Phytophthora infestans. Solanidine, tomatidine, tomatidenol, and demissidine glycosides were found in tubers of the hybrids. Comparisons with somatic hybrids between crc and tbr-2x are made. It is concluded that crc is a valuable Solanum species that can and should be included in potato breeding programs.     

Identification of Haynaldia villosa chromosomes added to wheat using a sequential C-banding and genomic in situ hybridization technique

Genomic in situ hybridization (GISH) offers a convenient and effective method for cytological detection, but can not determine the identity of the chromosomes involved. We integrated C-banding with GISH to identify Haynaldia villosa chromosomes in a wheat background. All chromosomes of H. villosa showed C-bands, either in telomeric regions or in both telomeric and centromeric regions, which allowed unequivocal identification of each H. villosa chromosome. The seven pairs of H. villosa chromosomes were differentiated as 1–7 according to their characteristic C-bands. Using a sequential C-banding and GISH technique, we have analyzed somatic cells of F₃ plants from the amphiploid Triticum aestivum-H. villosa x Yangmai 158 hybrids. Three plants (94009/5-4,94009/5-8 and 94009/5-9) were shown to contain H. villosa chromosome(s). 94009/5-4 (2n = 45) had three H. villosa chromosomes (2, 3 and 4); 94009/5-8 (2n = 45) possessed one chromosome 4 and a pair of chromosome 5, and 94009/5-9 (2n = 43) was found to have one chromosome 6 of H. villosa. The combination of GISH with C-banding described here provides a direct comparison of the cytological and molecular landmarks. Such a technique is particularly useful for identifying and localizing alien chromatin and DNA sequences in plants.     

Chromosome investigations in annual Medicago species (Fabaceae) with emphasis on the origin of the polyploid Medicago rugosa and M. scutellata

Chromosome number variations play an important role in the genus Medicago. In addition to polyploidy there are cases of dysploidy as evidenced by two basic numbers, x = 8 and x = 7, the latter limited to five annual species having 2n = 14. Annuals are diploid with the exception of Medicago scutellata and Medicago rugosa which have 2n = 30 and are considered the result of crosses between the 2n = 16 and 2n = 14 species. However, this hypothesis has never been tested. This study was carried out to investigate the 2n = 14 and 2n = 30 karyotypes and verify the allopolyploid origin of M. scutellata and M. rugosa. Fluorescence in situ hybridization (FISH) of rDNA probes and genomic in situ hybridization (GISH) were performed. FISH showed that all five diploids with 2n = 14 have one pair of 45S and one pair of 5S rDNA sites. M. scutellata displayed four sites of 45S and four sites of 5S rDNA, while in M. rugosa only one pair of each of these sites was found. GISH did not produce signals useful to identify the presumed progenitors with 14 chromosomes. This result suggests alternative evolutionary pathways, such as the formation of tetraploids (2n = 32) and subsequent dysploidy events leading to the chromosome number reduction.     

Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens

Summary Somatic fusions between the cultivated potato Solanum tuberosum and the wild species S. circaeifolium subsp. circaeifolium Bitter were produced in order to incorporate desirable traits into the potato gene pool. Selection of the putative hybrids was based on a difference in callus morphology between the hybrids and their parents, with the hybrids showing typical purple-colored cells in otherwise green calli. In all, 17 individual calli regenerated to plants. Of the nine plants that could be transferred to the greenhouse, eight showed a hybrid and one a parental morphology. Restriction fragment length polymorphism (RFLP) analysis confirmed the hybrid character in the former group. Chloroplast counts in stomatal guard cells and flow cytometric determination of nuclear DNA content showed that four hybrid plants were tetraploid (4x), one was mixoploid (5x–8x), and the others were polyploid (6x; 8x). Three out of four tetraploid hybrids were found to be fully resistant to Phytophthora infestans, and all four hybrids were resistant to Globodera pallida pathotypes Pa2 and Pa3. It was further observed that the type and amount of steroidal glycoalkaloids varied among the tubers of the parents and the hybrids. Using the hybrids as female parents in crosses with S. tuberosum, viable seeds could be obtained. This demonstrates the potential of these hybrids in practical plant breeding.     

Fluorescence in situ hybridization with specific oligonucleotide rRNA probes distinguishes the sibling species Stylonychia lemnae and Stylonychia mytilus (Ciliophora, Spirotrichea)

Based on morphological and morphogenetic characters alone, the sibling species Stylonychia lemnae and Stylonychia mytilus, members of the Stylonychia mytilus complex, can hardly be distinguished. However, biochemical investigations of the isoenzyme pattern of different enzymes showed a distinct differentiation between these two species. In the last few years, fluorescence in situ hybridization (FISH) techniques have become a suitable and reliable tool for identification and differentiation of closely related species of protozoa, such as ciliates. To distinguish the sibling species, a set of specific oligonucleotide probes were developed. In the present study, the SSU rDNA of 7 clones of Stylonychia lemnae and 13 clones of Stylonychia mytilus, isolated from different geographic regions, were sequenced. Comparing all SSU rDNA sequences of both species, only one single difference within the whole gene was detected. Based on this difference, a set of two oligonucleotide probes, targeting the SSU rRNA of each species (Stylonychia mytilus and Stylonychia lemnae) was designed. These probes were successfully tested by applying the FISH techniques on preserved cells of different clones of both species.     

The genetic status and conservation management of two cultivated bulb species extinct in the wild: Tecophilaea cyanocrocus (Chile) and Tulipa sprengeri (Turkey)

Tecophilaea cyanocrocus and Tulipasprengeri are both extinct in the wild as aresult of overharvesting by commercialcollectors. Stocks of both species survive incultivation in amateur collections, commercialnurseries and botanic garden collections.Whilst both species share a similarconservation history, the distribution ofgenetic diversity within cultivated materialwas unknown. To support the long-termconservation of both species the geneticdiversity of the surviving stocks was assessedusing amplified fragment length polymorphisms(AFLPs). This study revealed different geneticstructures for the two bulb species. The Kewcollections of T. cyanocrocus, originallyobtained from a commercial nursery, VanTubergen, are genetically highly uniform andthe addition of samples from other collectionshas dramatically increased the level ofvariation. In contrast, the collections ofT. sprengeri held at Kew since the early20th century include representativegenotypes covering the whole range of geneticvariation found in this species and areprobably the source of all other cultivatedmaterial. The results are discussed in relationto the management of these species to ensuretheir continued survival in cultivation.     

Characterization of Thinopyrum bessarabicum chromosome segments in wheat using random amplified polymorphic DNAs (RAPDs) and genomic in situ hybridization

Ten random amplified polymorphic DNA (RAPD) markers specific to chromosome 5E^b of Thinopyrum bessarabicum were detected. Genomic in situ hybridization and standard cytological observations revealed that six of the markers are located on the 5E^b short arm and four are located on the 5E^b long arm. These RAPD markers have been used to confirm the identity of putative 5E^b (5A) and 5E^b (5D) substitution individuals. The potential of RAPDs for the detection of wheat/alien recombinants is discussed.     

Taxonomy of North and Central American diploid wild potato (Solanum sect. Petota) species: AFLP data

Solanum section Petota, the potato and its wild relatives, includes about 200 wild species distributed from the southwestern United States to central Argentina and adjacent Chile, with about 30 species in North and Central America. The North/Central American region and the South American region all include diploids, tetraploids, and hexaploids. Chloroplast DNA restriction enzyme data from a prior study showed that 13 of the North/Central American species formed a clade containing only diploids, but there was low resolution within the clade. This Amplified Fragment Length Polymorphism (AFLP) study is conducted to provide additional resolution within the North/Central American diploids and complements the chloroplast results, and prior morphological results. Wagner parsimony and phenetic analyses mostly agreed with the morphological data in supporting currently recognized species except that they suggest that S. brachistotrichium and S. stenophyllidium are conspecific. Our new AFLP data, in combination with the cpDNA and morphological data, also support sister taxon relationships for the following diploid species from North and Central America: 1) S. cardiophyllum subsp. ehrenbergii and S. stenophyllidium, 2) S. tarnii and S. trifidum, 3) S. jamesii and S. pinnatisectum, 4) S. lesteri and S. polyadenium, and 5) S. clarum and S. morelliforme.This work represents partial fulfillment for the requirements of a Ph.D. degree in Plant Breeding and Genetics at the University of Wisconsin-Madison. We thank committee members Paul Berry, Michael Havey, Thomas Osborn, and Kenneth Sytsma. We also thank John Bamberg and Staff of the Unites States Potato Genebank for germplasm and locality data; Charles Nicolet and staff of the University of Wisconsin Biotechnology Center for technical help; Lynn Hummel and staff at Walnut Street Greenhouse for help in growing plants; and lab partners Brian Karas, Iris Peralta, Celeste Raker, and Sarah Stephenson for technical advice. This study was supported by CONACYT (Mexico) scholarship number 116742 granted to Sabina I. Lara-Cabrera, and the United States Department of Agriculture. Names are necessary to report data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable.     

A genetic model for the endosperm balance number (EBN) in the wild potato Solanum acaule Bitt. and two related diploid species

Solanum acaule Bitt. is a disomic tetraploid potato which has been assigned two endosperm balance numbers (EBN). It readily crosses with diploids but does not cross with other tetraploid species, although exceptions have been reported. The genetic basis of this behavior was studied in intra- and interspecific crosses involving plants of four introductions of this species and plants of one introduction of 2x S. commersonii Dun., one of 2x S. gourlayi Haw., and two of 4x S. gourlayi Haw., which have been assigned one EBN, two EBN, and four EBN respectively. Some of the pollinated pistils were used to analyze pollen-pistil compatibility reactions; the rest were left in the plants for seed production. At harvest, seeds were sorted according to size and plumpness, and the ploidy of the resulting plantlets determined from root tips. A model is proposed to explain the results of these crosses as well as the exceptions previously reported. It is based on the presence of two independent loci controlling the EBN, with two alleles in homozygosity: 1/2 and 0. This model, which is extended to cmm and grl, also explains the behavior of 3x (cmm x grl) hybrids in crosses with one-EBN, two-EBN, and four-EBN species reported in a previous work.     

Interspecific hybridization and the analysis of meiotic chromosome pairing inDahlia (Asteraceae — Heliantheae) species with x = 16

The successful production of a large number of artificial hybrids betweenDahlia species based on x = 16 has allowed a detailed study of their genomic relationships. Chromosome behaviour in these artificial hybrids was extremely similar to that observed in parental species suggesting that there is a considerable degree of homology between the genomes of theseDahlia species. Using GISH it can be demonstrated that in these hybrids bivalent formation involved pairing only between parental genomes. The ability of GISH to differentiate between parental genomes in artificial hybrids was variable, indicating that molecular divergence of highly repeated sequences has accompanied the evolution of these species. However, the extent of chromosome pairing and chiasma formation in the hybrids does not reflect the differences that can be detected by GISH. Seyeral of the new hybrid combinations have resulted in horticulturally interesting plants.     

The repetitive component of the A genome of peanut (Arachis hypogaea) and its role in remodelling intergenic sequence space since its evolutionary divergence from the B genome

Background and Aims

Peanut (Arachis hypogaea) is an allotetraploid (AABB-type genome) of recent origin, with a genome of about 2·8 Gb and a high repetitive content. This study reports an analysis of the repetitive component of the peanut A genome using bacterial artificial chromosome (BAC) clones from A. duranensis, the most probable A genome donor, and the probable consequences of the activity of these elements since the divergence of the peanut A and B genomes.

Methods

The repetitive content of the A genome was analysed by using A. duranensis BAC clones as probes for fluorescence in situ hybridization (BAC-FISH), and by sequencing and characterization of 12 genomic regions. For the analysis of the evolutionary dynamics, two A genome regions are compared with their B genome homeologues.

Key Results

BAC-FISH using 27 A. duranensis BAC clones as probes gave dispersed and repetitive DNA characteristic signals, predominantly in interstitial regions of the peanut A chromosomes. The sequences of 14 BAC clones showed complete and truncated copies of ten abundant long terminal repeat (LTR) retrotransposons, characterized here. Almost all dateable transposition events occurred <3·5 million years ago, the estimated date of the divergence of A and B genomes. The most abundant retrotransposon is Feral, apparently parasitic on the retrotransposon FIDEL, followed by Pipa, also non-autonomous and probably parasitic on a retrotransposon we named Pipoka. The comparison of the A and B genome homeologous regions showed conserved segments of high sequence identity, punctuated by predominantly indel regions without significant similarity.

Conclusions

A substantial proportion of the highly repetitive component of the peanut A genome appears to be accounted for by relatively few LTR retrotransposons and their truncated copies or solo LTRs. The most abundant of the retrotransposons are non-autonomous. The activity of these retrotransposons has been a very significant driver of genome evolution since the evolutionary divergence of the A and B genomes.