The genomes of Arachis hypogaea. 1. Cytogenetic studies of putative genome donors

Summary Cytological studies of wild diploid Arachis species in the same section of the genus (sect. Arachis) as the cultivated peanut A. hypogaea L. show, with one exception, a karyotype characterized by the presence of 9 pairs of larger chromosomes and one pair of small (A) chromosomes. The exceptional species A. batozocoi Krap. et Greg. has a more uniform karyotype. Interspecific hybrids between diploid species of similar karyotype have moderate to high pollen stainability, those involving A. batizocoi have zero pollen stainability and a very irregular PMC meiosis. Such infertile hybrids are the most likely to produce fertile, stable amphidiploids on doubling the chromosome complement. It is suggested that the cultivated peanut could have originated from such a sterile interspecific hybrid and on morphological and phytogeographic grounds the most likely genome donors are A. cardenasii (nomen nudum) and A. batizocoi of the species within section Arachis, which have been collected up to the present time.Paper number 5560 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27650     

Phylogenetic relationships in the genus Arachis based on seed protein profiles

Summary Seed protein profiles of 19 accessions representing seven sections of the genus Arachis were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The general profile showed appreciable homology between these taxa, supporting their classification based on morphology and cross-compatibility relationships. The accessions of section Arachis expressed a high variation confirming inferences from earlier studies. Variation between accessions of a species is limited. Accessions of the section Ambinervosae and Caulorhizae formed one cluster and accessions of sect. Erectoides and Procumbensae formed another. Whereas the representative accessions of sect. Triseminalae and Extranervosae formed two independent clusters. Using the percentage of dissimilarity in electrophoretic bands as a statistical genetic distance between accessions, sect. Arachis (containing the cultivated groundnut, A. hypogaea) is phylogenetically closest to sect. Erectoides followed by Procumbensae, Ambinervosae, Caulorhizae, Triseminalae and Extranervosae, respectively.Submitted as Journal Article No. 1485 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Patancheru, A.P. 502 324, India.     

RFLP analysis of genetic variation in species of section Arachis, genus Arachis (Leguminosae)

Four A-genome species of the genus Arachis (A. cardenasii, A. correntina, A. duranensis, A. kempff-mercadoi), three B genomes species (A. batizocoi, A. ipaënsis and A. magna),the AABB allotetraploid A. hypogaea (cultivated peanut) and introgression lines resulting from a cross between A. hypogaea and A. cardenasii were analyzed by RFLP. The A genome species (cytologically characterized by the presence of a small chromosome pair ‘A’) were closely similar to each other and shared a large number of restriction fragments. In contrast, the B genome species differed more from one another and shared few fragments. The results of this study indicate that the absence of the small chromosome pair is not a good criterion for grouping species of section Arachis as B genome species, since their genome might be quite distinct from the B genome of A. hypogaea.The lowest genetic variation was detected within accessions of A. duranensis (17 accessions), followed by A. batizocoi (4 accessions) and A. cardenasii (9 plants of accession GKP 10017).The high level of genetic variation found in A. cardenasii might indicate that not all accessions of wild species of Arachis are autogamous, as reported for A. hypogaea.     

Ribosomal DNA repeat unit polymorphism and heritability in peanut (Arachis hypogaea L.) accessions and related wild species

Repeat unit length and restriction site variation in ribosomal RNA geneclusters (rDNA) was surveyed in 77 Arachis accessions, includingsamples from 39 accessions of cultivated Arachis hypogaea(2n=4x=40), 36 accessions representing 15 related tetraploid and diploidwild species, and two synthetic amphidiploids. Total genomic DNA wasdigested with five restriction enzymes, and probed with three heterologousribosomal clones of wheat and broad bean. Four rDNA repeat unit lengthclasses were recognized in the Arachis species. Restriction site analysisshowed that some SacI, BamHI and TaqI cleavage sites in rDNA unit werehighly conserved. With few exceptions, the variable BamHI and EcoRV siteswere able to differentiate the taxonomic sections and species, respectively.Arachis hypogaea and A. duranensis accessions produced fourrDNA length classes. Among these, three were identical with those of otherArachis species. A SacI restriction site (s) from probe (Ver6-5) cangenerally distinguish the two subspecies A. hypogaea ssp. hypogaea and A. hypogaea ssp. fastigiata. Forty nine per centof bands were polymorphic across the A. hypogaea accessionsanalysed. This study does not support A. batizocoi to be a progenitorof A. hypogaea. For the gene array, the contribution from eachparental genome can be detected in the two synthetic amphidiploids.     

Onion chromosome nomenclature and homoeology relationships — workshop report

Summary Participants in a workshop on onion chromosome nomenclature held during the Eucarpia 4th Allium Symposium, agreed to consider the nomenclature for the chromosomes of Allium cepa L. proposed by E.R. Kalkman (1984) as the standard. Recommendations were formulated for the chromosome nomenclature of species which are cross compatible with onion.     

Assessment of the degree of genetic variation in beet based on RFLP analysis and the taxonomy of Beta

Summary Clones derived from Beta vulgaris and Beta maritima were assayed for their ability to detect restriction fragment length polymorphisms (RFLPs) in different beet accessions. The clones able to detect polymorphism were used as genetic markers to assess the degree of genetic variation existing between and within different species of the genus Beta. The data support the current taxonomy of the Beta vulgaris section, while the great genetic similarity found between Beta webbiana and Beta procumbens indicates that they could belong to the same species.Enough variation was found between Beta vulgaris cultivars, allowing the isolation of a sufficient number of genetic markers for the construction of detailed genetic maps.     

Aseptic culture of gynophores to obtain peanut intersectional hybrids

Summary Cross-incompatibility between cultivated peanuts and their wild relatives outside the section Arachis has impeded the utilization of many species possessing high resistances or good qualities. Despite the great efforts made to culture immature ovules or embryos, few hybrid offspring have been obtained. In this study, gynophores from Arachis hypogaea L. pollinated with A. glabrata Benth. were cultured and F₁ hybrids seeds were harvested, and F₂ and F₃ generations produced. The characters of F₂ generation exhibited a wide range of segregation. Leaf peroxidase isozyme PAGE analysis revealed that the hybrids were quite different from their parents in relation to band number, width and isozyme activity. The zymograms of the hybrids and their parents were partially alike. This verified the authenticity of the hybrids.     

Identification and Molecular Phylogenetic Relationships of Δ12-Fatty Acid Desaturase in Arachis

Summary On the basis of the conservative sequence of Δ12-fatty acid desaturase, a cDNA encoding the enzyme was isolated from different genotypes of Arachis. The cDNA consisted of 1140 bp and encoded a polypeptide of 379 amino acid resides with a calculated molecular weight of 42 KD. The predicted amino acid sequence had three histidine conservative regions sharing integral-protease specificity. Hydrophobicity analysis showed that the sequence of the encoded amino acids had two hydrophobic structures sharing the characteristics of membrane-anchored protein, which totally crossed the membrane four times. These analyses revealed that the obtained sequence was Δ12-fatty acid desaturase. Phylogenetic-tree analysis demonstrated that the found Δ12-fatty acid desaturase sequences in Arachis was conservative and was highly homogenous to the Δ12-fatty acid desaturase sequences in other species. The present research provided molecular evidence of phylogenetic relationships among the accessions in Arachis.     

Morphological, cytological and disease resistance studies of the intersectional hybrid between Arachis hypogaea L. and A. glabrata Benth.

Arachis glabrata Benth, variety glabrata collector GK 10596 (PI276233; ICG 8176) belonging to section Rhizomatosae has resistance to rust, late leaf spot and viral diseases. A. hypogaea L. cv MK 374 (section Arachis) is susceptible to rust, late leaf spot and to the viral diseases peanut stripe, peanut mottle and peanut bud necrosis. Hybrids between A. hypogaea cv MK 374 and A. glabrata were produced after inter specific pollinations and embryo culture. The hybrids produced had morphological characters of both parents plus floral abnormalities not seen in either parent. It was possible to identify the hybrids by esterase isozyme analysis when still in culture. Cytological research showed variable chromosome association and also homeology between the genomes of A. hypogaea and A. glabrata. The hybrids inherited resistance to rust, late leaf spot, peanut bud necrosis and peanut stripe diseases from the pollen parent A. glabrata. This revised version was published online in August 2006 with corrections to the Cover Date.     

Diversity in selected wild and cultivated species of pigeonpea using RFLP of mtDNA

Diversity in 28 accessions representing 12 species of the genus, Cajanus arranged in 6 sections including 5 accessions of the cultivated species, C. cajan, and 4 species of the genus Rhyncosia available in the germplasm collection at ICRISAT was assessed using RFLP with maize mtDNA probes. Cluster analysis of the Southern blot hybridization data with 3 restriction enzymes – 3 probe combinations placed the genus Rhyncosia in a major group well separated from all the species belonging to the genus Cajanus. Within the genus Cajanus, the 4 accessions of C. platycarpus belonging to section Rhynchosoides formed a separate group in contrast to those in other sections of pigeonpea. In the section, Cajanus all the 5 accessions of C. cajan were grouped together and C. cajanifolius belonging to the same section was in a subgroup by itself closer to the main group. The four accessions of C. scarabaeoides, were together and the other species belonging to section Cantharospermum were in different subgroups. The intra-specific variation was seen even within accessions of certain pigeonpea wild species such as C. scarabaeoides, C. platycarpus, C. acutifolius, and even the cultivated species of C. cajan. This study suggests that RFLP of mtDNA can be used for the diversity analysis of pigeonpea and it gives some indications on the maternal lineage among the species. The variations in the mitochondrial DNA hybridization patterns also suggest the extensive rearrangement of the organelle genome among the Cajanus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

A pest risk assessment for Copitarsia spp., Insects associated with importation of commodities into the United States

Summary Eggs and larvae of Copitarsia spp. (Lepidoptera: Noctuidae) are frequently intercepted on fresh commodities arriving in the United States from Mexico, Central America, and South America. Copitarsia spp. are not known to occur in the US and, thus, are considered actionable pests by the US Department of Agriculture, Animal and Plant Health Inspection Service (USDA, APHIS). Whenever the genus is detected in imported goods, shipments must be disinfested, destroyed, or returned to the country of origin. Inspections and interdictions might be unnecessary if Copitarsia spp. were unlikely to establish in the US or if consequences of Copitarsia establishment were trivial. Consequently, we prepared a qualitative pest risk assessment to characterize the degree of risk posed by the genus to US agricultural and natural ecosystems. Published literature was consulted to describe the biology and ecology of the genus. Trade statistics and interception records were summarized to identify pathways of introduction. With this information, experts assigned risk ratings to each of eleven elements identified by USDA, APHIS that pertained to the likelihood or consequence of exotic-pest establishment. The likelihood of Copitarsia spp. becoming established in the US was considered high, but confidence in this assessment was low. Similarly, consequences of Copitarsia establishment were rated high, but confidence in this assessment was moderate. Overall, the assessment revealed that Copitarsia pose a high degree of risk to the US and phytosanitary measures to exclude the pest seem warranted. However, additional research is needed to address critical data gaps and refine assessments of risk for individual species within the genus.     

Inter simple sequence repeat analysis of genetic relationships in the genus Vigna

The utility of inter simple sequence repeat (ISSR) DNA polymorphisms to distinguish taxa within the genus Vignawas investigated. Nineteen primers, most containing either aGA or CA repeat, generated amplification products that differed among the taxa examined. The ISSR polymorphisms produced by 15 of these primers were very effective for distinguishing taxa at the species level or below. The Vigna unguiculataaccessions analyzed formed a cohesive group and appeared to be most closely related to V. triphylla and V. reticulata.In contrast, ISSR analysis was not able to clearly differentiate subgeneric divisions within Vigna. We attribute this loss of resolution at the subgeneric level to the high rate of evolution of the sequences we examined. Several probable instances of misclassification or hybrid origin of an accession were identified.     

PCR-based markers to differentiate the mitochondrial genomes of petaloid and male fertile carrot (Daucus carota L.)

Cytoplasmic male sterility (CMS) is essential for the development of highly adapted and uniform hybrid varieties of carrot and other crops. The most widely used type of CMS in carrot is petaloidy, in which the stamens are replaced by petals or bract-like structures. We have developed a series of mitochondria-specific PCR markers to distinguish cytoplasms inducing petaloidy (Sp) and male-fertility (N). The markers target the atp1,atp6, atp9, orfB (atp8), nad6 and cob loci from the mitochondrial genomes of a diverse collection of male fertile and petaloid carrots. We report 14 primer pairs that amplify marker fragments from either Sp or N cytoplasms and three primer pairs that amplify fragments with length polymorphism. The amplification products span sites of insertions, deletions or recombinations adjacent to or within the coding regions of the targeted genes. The markers reported here are useful tools to identify the type of cytoplasm in cultivated carrot and to evaluate variation in the mitochondrial genomes within the genus Daucus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of Paspalum germplasm for variation in leaf wax and heat tolerance

Summary The genus Paspalum contains a number of important forage grasses, including P. dilatatum Poir. (dallisgrass) and P. notatum Flugge (bahiagrass). Even though many Paspalum species are adapted to moist, humid areas, they frequently are subjected to extended periods of drought stress which reduces forage productivity. This study was initiated to determine the range of variation for different indicators of drought tolerance in several Paspalum species. Fifteen species representing a broad cross section of germplasm were grown in a replicated field nursery and sampled for leaf epicuticular wax content on four dates over three years. Wax was extracted with chloroform and quantified by the dichromate oxidation technique. Heat tolerance (solute leakage technique) was also determined by calculating the ratio of the conductivity of the external bathing solution of leaves following a heat treatment to the conductivity of the bathing solution after leaves were boiled. Wax values of the entries ranged from 5.7 to 0.88 mg/dm². Both relative rankings and absolute wax values were very consistent across all sampling dates. The accessions of Paspalum nicorae Parodi and P. quandrifarum Lam. were consistently high in wax content, while the accessions of P. commune Lillo and P. unispicatum (Scribn. et Merr.) Nash were consistently low. The heat tolerance testing demonstrated that the accession of P. notatum var. saurae Parodi was the most heat tolerant of the entries. Results from this investigation demonstrate the wide range of diversity that exists for both epicuticular wax and heat tolerance within the genus Paspalum.     

The genetic diversities of 4 species of subg. Lithocerasus (Prunus, Rosaceae) revealed by RAPD analysis

RAPD analysis was applied to reveal the genetic diversities of 4 speciesof subg. Lithocerasus within the genus Prunus using 40accessions representing the subgenera Prunophora, Amygdalus,Lithocerasus and Cerasus. The accession of subg. Lithocerasus are phenotypically similar to members of subgenus Cerasus but different with them in interspecific crossing tests and isozymeprofiles. Two major clusters, `Prunophora and Amygdalus group' and `Cerasus group' were constructed in the phenogram. We revealthat the examined 4 species of subg. Lithocerasus species; P.tomentosa Thunb., P. japonica Thunb., P. glandulosa Thunb.and P. besseyi Bailey, were genetically closer to the members ofsubgenera Prunophora and Amygdalus than to subg. Cerasus.     

Insect resistance in potatoes: sources,evolutionary relationships,morphological and chemical defenses,and ecogeographical associations

Summary The past 25 years, 1686 potato accessions, representing 100 species in the genus Solanum L., subgenus Potatoe, section Petota, were evaluated for field resistance to one or more of the following insect pests: green peach aphid, Myzus persicae (Sulzer); potato aphid, Macrosiphum euphorbiae (Thomas); Colorado potato beetle, Leptinotarsa decemlineata (Say); potato flea beetle, Epitrix cucumeris (Harris); and potato leafhopper, Empoasca fabae (Harris). Accessions highly resistant to green peach aphid were identified within 36 species, to potato aphid within 24 species, to Colorado potato beetle within 10 species, to potato flea beetle within 25 species, and to potato leafhopper within 39 species. Resistance levels were characteristic within Solanum species. Insect resistance appears to be a primitive trait in wild potatoes. Susceptibility was most common in the primitive and cultivated Tuberosa. Insect resistance was also characteristic of the most advanced species. The glycoalkaloid tomatine was associated with field resistance to Colorado potato beetle and potato leafhopper. Other glycoalkaloids were not associated with field resistance at the species level. Dense hairs were associated with resistance to green peach aphid, potato flea beetle, and potato leafhopper. Glandular trichomes were associated with field resistance to Colorado potato beetle, potato flea beetle, and potato leafhopper. Significant correlations between insect score and altitude of original collection were observed in six of thirteen species. Species from hot and arid areas were associated with resistance to Colorado potato beetle, potato flea beetle, and potato leafhopper. Species from cool or moist areas tended to be resistant to potato aphid.Abbreviations EBN Endosperm Balance Number     

Evaluation of wild Arachis species for abiotic stress tolerance: I. Thermal stress and leaf water relations

The Indian groundnut cultivars have a narrow genetic base. Hence, it was of interest to investigate the genetic variability among wild Arachis species and their accessions for tolerance to thermal stress. A wide variation was observed in leaf morphological characters such as colour, shape, hairiness, length and width and thickness (SLA). The temperature and time required for 50% leaf injury was worked out with limited number of genotypes and was found to be 54°C for 50 min. Among 36 genotypes (having SLA in the range of 66 and 161 cm² g⁻¹) screened, the inherent potential for cold as well as heat tolerance in terms of relative leaf injury (RI) was observed. Thus, based on RI-values, A. glabrata 11824 and A. paraguariensis 12042 were identified as heat-tolerant and cold-tolerant genotypes, respectively while A. appresipila 11786 was found to be susceptible to both heat and cold. Correlation between SLA and RI values for heat (r = 0.38, P < 0.05) and cold (r = 0.52, P < 0.05) tolerance was positive, indicating that thicker the leaf the lower the injury or higher the tolerance. Among six species and 13 accessions, comprising both heat-tolerant and heat- susceptible genotypes, the concentrations of various leaf chemical constituents such as total protein, phenols, sugars, reducing sugar, amino acids, proline, epicuticular wax load and chlorophyll varied significantly. The epicuticular wax load ranged between 1.1 and 2.5 mg dm² among 13 A.glabrata accessions. These accessions were categorized into two groups, i.e. high-wax (range: 2.0–2.5 mg dm²) and low-wax types (range: 1.1–1.6 mg dm²). The high-wax type showed a higher diffusion resistance (dr) as compared to low-wax type; though the transpiration rate (tr) in high-wax type was moderate (between 9.5 and 11.6 μg cm⁻² s⁻¹). Genetic variability in parameters such as canopy temperature, dr and tr was also distinct. The fully turgid leaves with relative water content ≥91%, showed leaf water potential (ψ_leaf) between −0.7 and −1.2 MPa. Results indicated that the plants with thicker leaves are better protected from heat injuries. Further, epicuticular wax load seems to help in maintaining stomatal regulation and leaf water relations, thus affording adaptation to wild Arachis species to thrive under water-limited environments. The sources of tolerance, as identified in this study, could be utilized to improve thermal tolerance of the groundnut cultivars by intra-specific hybridization, following either conventional breeding using embryo rescue techniques, if required or utilizing biotechnological tools.     

2n Gamete formation in the genus Brachiaria (Poaceae: Paniceae)

Microsporogenesis of several Brachiaria species of the Brazilian collection at Embrapa Beef Cattle has been analyzed in detail. This paper reports abnormal cytokinesis in three accessions of three different species (Brachiaria humidicola, 2n = 4x = 36, Brachiaria decumbens, 2n = 4x = 36, and Brachiaria dura, 2n = 6x = 54). Chromosomes paired in bi-, tri-, and quadrivalents in these accessions, whereas chromosome segregation at meiosis I was characterized by exclusion of laggards as micronuclei. In a high number of meiocytes, the first sign of cytokinesis appeared only in metaphase II and did not divide the meiocyte into a dyad. Total absence of cytokinesis was also detected among meiocytes in the second division. Since in both cases the two metaphase plates were very close, they favored the rejoining of chromosome sets after anaphase II and formed a restitutional nucleus in telophase II. Second cytokinesis occurred after telophase II in most meiocytes. Monads, dyads, and triads with n or 2n nuclei were observed among meiotic products. The 2n gametes observed correspond to the first division restitution (FDR). The number of affected cells in each accession was variable, but the number of microspores with restitutional nucleus, including those scored in tetrads and the released ones, did not exceed 9%. Although polyploidy is common in the genus Brachiaria, its origin is still unclear. Current results suggest that 2n gametes may have contributed to the evolutionary history of the genus.     

Photomorphogenic mutants of tomato

Summary Two cloned cassava genes (pCAS6, pHNL) and a barley gene (pBLT63) which is highly conserved in higher plants, were evaluated as potential multilocus probes for RFLP analysis of phylogenetic relationships of species within the genus Manihot. For M. rubricaulis, M. chlorosticta and M. esculenta subsp. flabellifolia dendrograms based on 13 probe-restriction enzyme combinations show very little variation between accessions of the same species. A close relationship is demonstrated between the Mexican species M. chlorosticta and the M. esculenta subsp. flabellifolia accessions, which does not support the classification of flabellifolia as a true South American wild species.     

Isozyme differentiation of Azolla Lam

Summary Enzyme electrophoresis was used to differentiate sections within the genus Azolla Lam. and demonstrate the value of this method in fingerprinting taxa. Polyacrylamide gel methodology has been developed to identify suspected species of this genus. Preliminary examination of selected isolates indicated that allozyme diversity was particularly evident in section Azolla, where specific classification by morphological means is difficult. Leaf tissue was preferable to root tissue for this purpose because of the ease of tissue preparation and the superior resolution and staining intensity of enzymes. Contribution of enzyme bands by the cyanobiont to zymograms was not significant.Scientific paper no. 7962. College of Agriculture and Home Economics Research Center, Washington State University, Pullman WA, Project Number 0708.