Genetic variation within and among species of genus Arachis, section Rhizomatosae

The genus Arachis is endemic to South America and comprises 80 species, 69 of which have already been described and eleven not yet published. The genus includes the cultivated peanut (A. hypogaea) and several forage species, the most important ones being A. glabrata and A. pintoi. Accessions of section Rhizomatosae, including three tetraploid species 2n = 4x = 40 (A. glabrata, A. pseudovillosa and A. nitida nom. nud.) and one diploid species 2n = 2x = 20 (A. burkartii), were evaluated using RAPD markers to assay genetic variability within and among species. The ten random primers used yielded a total of 113 polymorphic bands. The data were scored as the presence or absence of each band in each sample. A distance matrix and dendrogram were obtained using Link's coefficient and the neighbor-joining method. Most accessions analyzed grouped into two major clusters: the first comprised most accessions of A. glabrata and accessions of A. nitida, and the second cluster comprised accessions of A. burkartii. Arachis pseudovillosa and a few accessions of A. glabrata and A. nitida were placed between these major clusters. The diploid and tetraploid species were grouped quite separately, suggesting that the tetraploids did not originate from the diploid species analyzed.     

Characterization of resveratrol content in ten wild species of section Arachis, genus Arachis

Peanut is one of the few plants that synthesizes resveratrol, a phenolic compound of the stilbene class, which has been associated with reduced risk of developing chronic diseases, such as cancer, cardiovascular diseases, skin diseases, pulmonary diseases, diabetes and neurological diseases. Resveratrol was detected in different parts of the peanut plant, including roots, leaves, seeds and their derivatives. The wild species of the Arachis section are also strong candidates to synthesize resveratrol because they are phylogenetically closely related to cultivated peanut. Our objective was to characterize the resveratrol content in ten wild species of Arachis with three different genomes (A, B and K). The plant material was composed of leaves of the ten species treated (test) and not treated (control) with ultraviolet (UV) radiation. The test and control samples were extracted and the identification and quantification of resveratrol was performed using high performance liquid chromatography (HPLC). All species studied synthesized resveratrol and the concentrations ranged from 299.5 μg/g in A. kempff-mercadoi to 819.9 μg/g in A. cardenasii. DPPH antioxidant activity varied between 18.7 % for A. duranensis and 48.2 % in A. simpsonii. The results showed that wild Arachis species are a potential source of alleles for improvement of cultivated peanut, with the aim of achieving higher resveratrol content in leaves.     

Genetic variation within and among species of five sections of the genus Arachis L. (Leguminosae) using RAPDs

Some Arachis species are widely used as commercial plants, e.g. the groundnut A. hypogaea, an important source of good quality protein and oil, and A. pintoi and A. glabrata, that are utilized as forage species. Germplasm of most Arachis species is available in germplasm banks. However, little it is known about the genetic attributes of this germplasm, and mainly about its genetic variability, which is very important for its maintenance. In the present study RAPDs were used to assay the genetic variation within and among 48 accessions of five sections of the genus Arachis and to establish the genetic relationships among these accessions. Ten of 34 primers tested were selected for DNA amplification reactions since they yielded the largest numbers of polymorphic loci. A dendrogram was constructed based on data from the 10 primers selected. Eighty RAPD polymorphic bands were analyzed among the accessions studied. The relationships among species based on RAPDs were similar to those previously reported based on morphological, cytological and crossability data; demonstrating that RAPDs can be used to determine the genetic relationships among species of the different sections of the genus Arachis. In general, wide variation was found among accessions and low variation was found within the accessions that had two or more plants analyzed. However, higher polymorphism was found in the section Trierectoides and in one accession of A. major, indicating that generalizations should be avoided and each species should be analyzed in order to establish collection and maintenance strategies.     

Evaluation of genetic relationships among botanical varieties of cultivated peanut (Arachis hypogaea L.) using AFLP markers

Forty-four accessions of cultivated peanut (Arachis hypogaea L.) representing sixbotanical varieties of two subspecies along with three accessions ofthe wild relative A. monticola Krapov et Rigoni were evaluated for their genetic relationships using theAFLP marker technology. Fifteen AFLP primer pairs (EcoRI/MseI) generated 28distinct polymorphic markers that were employed to develop uniqueprofiles of all accessions and to construct a phenogram. The resultsshowed that the botanical varieties aequatoriana and peruviana werecloser to subspecies hypogaea than subspeciesfastigiata Waldr. to which they belong, and the wildA. monticola was notdistinct from the cultivated A.hypogaea. Although the extent of geneticdiversity in peanut is low compared to many other crops, our studiesshow that by employing the AFLP approach, sufficient DNA variationcan be detected in the cultivated peanut germplasm to conductevolutionary studies.     

Genomic affinities of <Emphasis Type="Italic">Arachis</Emphasis> genus and interspecific hybrids were revealed by SRAP markers

The Arachis genus is native to South America, and contains 70–80 described species assembled into nine sections. A better understanding of the level of speciation and taxonomic relationships is a prerequisite to the effective use of Arachis species in peanut breeding programs. Forty-eight genotypes representing 19 species in 6 sections were evaluated to assay the genetic variability within and among species, and 10 recombinant lines and those parents were identified with introgression of Arachis species chromosome segments into A. hypogaea genome using SRAP markers. Sixty of sixty-four SRAP primers tested were selected for DNA amplification reactions. A dendrogram and principal component analysis were constructed based on 353 SRAP polymorphic bands of the accessions. The number of scored polymorphic bands per each primer combination varied from 1 to 25 with an average of 5.9 per reaction. Estimates of genetic distance among the 48 accessions Arachis species ranged from 0.11 to 0.76. A-genome accessions 475845 (A. duranensis), and 331197 (A. villosa) were most closely associated to A. hypogaea. The first two PCAs accounted for 77.74% (62.02 and 15.72%) of the total variation observed and separated the different genomic groups. SRAPs also identified introgression of Arachis species chromosome segments into A. hypogaea. genome with 10 recombinant lines and those parents. The present results indicated that SRAPs can be used to determine the genetic relationships among species of the different sections of the genus Arachis and to identify introgression of Arachis genus chromosome segments into A. hypogaea genome.     

Development of new sources of tetraploid <Emphasis Type="Italic">Arachis</Emphasis> to broaden the genetic base of cultivated groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Groundnut, an important crop of many countries of the world, is susceptible to a range of diseases and pests. High levels of resistances are not available in the cultivated gene pool as the crop is said to have a narrow genetic base. Narrow genetic base is attributed to the evolution of the crop which took place by the combination of A and B genome species, and later doubling their chromosome number, giving rise to tetraploid cultivated groundnut. Direct utilization of cross-compatible wild relatives, which are diploids, to broaden the genetic base and introduction of useful traits, is not a straight-forward process due to ploidy differences between the cultivated species and wild relatives. Hence amphiploids and autotetraploids were created by not only combining the putative genomes, but many other A and B genome species, thus producing a highly variable population of tetraploid groundnuts also called new sources of Arachis hypogaea. This study describes the development and characterization of newly generated tetraploid groundnuts and the level of molecular diversity as assessed by DArT markers.     

Strontium accumulation in whole grain of Aegilops and Triticum species

Plant species differ in their ability to accumulate mineral elements. Differences also exist among genotypes, lines, and varieties within species. Strontium (Sr) enters the food chain primarily via plants, and wheat products are amongst the most important pathways by which Sr enters the human body. The aim of this study was to analyze the accumulation of Sr in whole grain of various Aegilops and Triticum species with different genomes (AA, BB, BBAA, BBAADD, and DD). During three experimental years plants were grown in temperate continental climate on a calcareous, gleyic chernozem soil. The results reveal that the studied species and genomes differed significantly in Sr accumulation in the grains. The average Sr concentration of the grains ranged, depending on the year of study and genotype, from 0.70 to 3.89 mg kg ⁻¹ DM. The grain of wild diploid Aegilops speltoides with BB genome contained significantly more Sr than all of the other analyzed genotypes. Grains of wild and cultivated tetraploid wheat genotypes had the lowest Sr concentration. The modern cultivated hexaploid wheat varieties displayed substantial variation in Sr concentration in the grains. The examined genotypes also differed significantly with respect to their mass of 1000 grains, which was the smallest in Aegilops speltoides . There was a significantly negative correlation between the average mass of a 1000 grains and concentration of Sr. The results show that even when the background concentration of Sr was relatively low, wheat genotypes accumulated it in the grains, although to a different extent. Therefore, selection of wheat genotypes which accumulate less Sr in the grain could contribute to improve food quality.     

Molecular phylogeny of banana cultivars from Thailand based on HAT-RAPD markers

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the wild progenitors of the cultivated banana, they are highly variable in Thailand. The genetic system is relatively unknown and complicated due to interspecific hybridization, heterozygosity and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, especially when sterile. The high annealing temperature-random amplified polymorphic DNA (RAPD) technique was used to estimate the genetic relationship between 22 selected banana cultivars, utilizing 14 random primers. Phylogenetic relationship was determined by unweighted pair group method with arithmetical averages cluster analysis. The dendrogram constructed from the similarity data showed that all the 22 cultivars analysed were closely related with a narrow genetic base. There were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram grouped all the AA, BB, AAA, AAB and ABB genomes into a major cluster. Several subgroups are recognized within the major clade. As expected, Ensete glauca Roxb. (Musaceae) and Strelitzia reginae Banks (Strelitziaceae) were clearly differentiated from the analysed edible bananas. Our study showed that RAPD markers are sufficiently abundant to classify and readily dissect genetic differences between the closely related Musa germplasm and provide a basis for the selection of parents for improvement of this germplasm.     

Dual origin of the cultivated rice based on molecular markers of newly collected annual and perennial strains of wild rice species, Oryza nivara and O. rufipogon

The direct ancestor of rice (Oryza sativa L.) is believed to be AA genome wild relatives of rice in Asia. However, the AA genome wild relatives involve both annual and perennial forms. The distribution of the retrotransposon p-SINE1-r2, a short interspersed nuclear element (SINE) at the waxy locus was analyzed in diverse accessions of the AA genome wild relatives of rice (O. rufipogon sensu lato). Most annual wild rice accessions had this retrotransposon, while most perennial types lacked this element, contradicting results to the previous studies. Results presented here suggest that O. sativa has dual origin that lead to indica-japonica differentiation. Results suggest the indica line of rice varieties evolved from the annual genepool of AA genome and the japonica varieties from the perennial genepool of AA genome wild rice.     

Genetic Diversity in AA and CC Genome Oryza Species in Southern South Asia

The CC genome of Oryza is found in nine species of Oryza that are distributed on all continents having a tropical climate. Three diploid Oryza species with CC genome are found in Africa and Asia to Papua New Guinea. In southern South Asia these three CC genome diploid species can be found, O. eichingeri and O. rhizomatis in Sri Lanka and O. officinalis in India. AA genome wild relatives of rice are also found in the same geographic region. Germplasm of both diploid CC and AA genome Oryza germplasm has recently been collected from Sri Lanka. AFLP analysis was used to compare the genetic diversity of the two Oryza genomes from a similar geographic region in southern South Asia. In addition, the diploid CC Oryza germplasm was also analyzed by RAPD and SSR methodologies and the combined results were analyzed. The results show that in southern South Asia the diploid CC genome species have a high level of genetic diversity compared to the diploid AA genome species. Molecular marker analysis revealed that populations of O. rhizomatis from northern and southeastern Sri Lanka are genetically differentiated. One accession of O. rhizomatis was aligned with O. eichingeri. This accession was collected from the site of O. rhizomatis that is the closest to a population of O. eichingeri. O. eichingeri showed lower genetic diversity than the other two diploid Oryza CC genome species. O. officinalis accessions from Assam, India, and China were genetically less diverged from O. eichingeri and O. rhizomatis than two accessions of O. officinalis from Kerala state, India. The first two authors contributed equally to this research     

Sodium azide induced high-oleic peanut (Arachis hypogaea L.) mutant of Virginia type

Less prone to oxidation than its conventional counterpart, high-oleic peanut (Arachis hypogaea L.) containing?≥?72% oleate and?<?8% linoleate is preferred by processors, seed sellers and consumers. Development of high-oleic peanut cultivars with suitable pod/seed size and shape may satisfy the need from food processors manufacturing whole kernel or halve products. In this study, a high-oleic Virginia type peanut mutant was identified through screening of a 15 mmol/L sodium azide mutagenized M3 population with near infrared spectroscopy. Sequencing of the mutated and wild type FAD2A/FAD2B genes detected 2 point mutations. The G448A mutation in FAD2A was the same as in previous reports, causing an amino acid change of D150N. G558A in FAD2B was a novel mutation, resulting in a stop codon and premature termination of protein synthesis. 16 promising lines with acceptable productivity and pod/seed characters have been tentatively bred, which will be evaluated further in yield tests with replications after seed increase.

     

A retrotransposon-based probe for fingerprinting and evolutionary studies in rice (Oryza sativa)

A genomic DNA fragment (pAA7-2) amplified by a random amplified polymorphic DNA primer from the rice cultivar IR68 was used to assess the genetic variation and genetic relationships among the species of genus Oryza. A Southern hybridization experiment of diverse upland rice accessions using a single restriction enzyme HindIII generated unique DNA fingerprint for each accession. The differential hybridization pattern reflecting the copy number variation of pAA7-2 in a collection of wild species and cultivated species of rice provided insight about the genetic relationships among them. All AA genome species exhibited clear banding pattern suggesting presence of fewer copies of this sequence. Strongest hybridization signal was obtained in species belonging to BB, CC, GG, BBCC, CCDD genomes, whereas weakest hybridization signal was visible in EE, FF, and HHJJ genome species. Oryza brachyantha was the most divergent species. Clear difference in banding pattern was evident between Oryza schlechteri and Oryza coarctata belonging to HHKK genome. Although pAA7-2 had no repetitive sequences often associated with hypervariable loci, homology to a putative unclassified expressed retrotransposon distributed over several rice chromosomes was responsible for the complex banding patterns. There were more sites homologous to pAA7-2 sequence in corn and sorghum genome compared with the rice genome. The study demonstrates the potential of pAA7-2 as a powerful molecular tool for DNA fingerprinting, genetic diversity, phylogenetic, and evolutionary studies in Oryza sativa and its wild relatives and other grasses.     

The Weedy Rice Complex in Costa Rica. I. Morphological Study of Relationships Between Commercial Rice Varieties,Wild <Emphasis Type="Italic">Oryza</Emphasis> Relatives and Weedy Types

Weedy rice is a complex of Oryza morphotypes widely distributed in commercial rice fields, which interfere with rice cultivation, seed production, industrial processing and commercialization of this crop in several countries. The objective of this study was to characterize the weedy rice complex of Costa Rica by comparing it with the cultivated and wild rice species found in the country. A collection of weedy rice accessions, representative of the morphotypes found in the country, was established and characterized. Their morphometric relationships were established by comparing 27 morphological traits with commercial rice cultivars, landraces and wild Oryza species and by performing a multivariate analysis. Twenty-one weedy rice morphotypes were identified among 735 weedy accessions by using a three-digit code based on seed characters. Three principal components (PCs) explained 66.25% of the variation observed. The first PC accounted for 36.21% of the variation and separated CCDD genome type Oryza latifolia and O. grandiglumis from AA genome species O. sativa, O. glumaepatula, O. rufipogon and O. glaberrima. The second (18.9%) and third (11.14%) PCs separated the weedy morphotype groups from the AA genome species O.sativa, O. glaberrima and O. rufipogon. The weedy morphotypes were scattered between the indica commercial rice varieties, the cluster landraces–glaberrima and O. rufipogon. Additionally, a group of morphotypes showed intermediate characteristics between O. sativa and O. rufipogon, suggesting that hybridization could have taken place in the past between these species. None of the morphotypes collected in Costa Rica clustered with the allotetraploids CCDD species or O. glumaepatula.     

Variation in resistance to the cabbage aphid ( Brevicoryne brassicae) between and within wild and cultivated Brassica species

Seven Brassica species were evaluated for their resistance to the cabbage aphid, Brevicoryne brassicae, in a series of field experiments. Four wild Brassica species, two 8 chromosome species with similarities to the B genome of Brassica nigra (Brassica fruticulosa and Brassica spinescens) and two 9 chromosome species containing the C genome (Brassica incana and Brassica villosa) were identified as possessing consistently high levels of antibiosis mediated resistance to B. brassicae. None of the species were shown to possess consistently high levels of antixenosis resistance. In more detailed glasshouse experiments one B-like genome species, B. fruticulosa, showed considerable variation between accessions collected from different sites for resistance to B. brassicae. In addition, individual accessions of one A genome species (Brassica rapa) and one C genome species (Brassica alboglabra) were shown to be highly variable in their resistance to B. brassicae, some plants of each accession being highly resistant and others very susceptible. The implications of the variability in resistance to B. brassicae within wild Brassica species for exploitation in Brassica breeding programmes are discussed.     

Population size and N2-fixing activity of native peanut rhizobia in soils of Thailand

Summary The objective of this study was to assess the number and effectiveness of peanut rhizobia in soils of the major peanut-growing areas of Thailand. Three cropping areas, (1) continuously cropped with peanuts, (2) continuously cropped with non-legumes, and (3) non-cultivated fields, were chosen in each region. Peanut rhizobia were found in the soil at 38 to 55 sites sampled. Cultivated fields with a peanut cultivation history contained (as estimated by most probable numbers) an average of 1.6×10³ cells g^-1 of soil. The numbers of peanut rhizobia in most of the fallow fields and some of the noncultivated shrub or forest locations were much the same as at the sites where Arachis hypogaea was cultivated. In contrast, there were no or few (28–46 cells g^-1 soil) peanut rhizobia in the majority of fields continuously cultivated with sugarcane, cassava, corn, and pineapple. It appears that in these areas the indigenous peanut rhizobial populations are not adequate in number for a maximal nodulation of peanuts. A total of 343 Bradyrhizobium isolates were tested for effectiveness and were found to vary widely in their ability to fix N₂. In some areas the majority of rhizobia were quite effective while in others they were less effective than the inoculum strain THA 205 recommended in Thailand.     

Genetic diversity of Curcuma alismatifolia Gagnep.(Zingiberaceae) in Thailand as revealed by allozymepolymorphism

Allozyme polymorphism at seven loci (TPI, G6PD-2,IDH-1, SKD-2, MDH-1, GOT-1, andGOT-2) was employed to detect the level of geneticdiversity in C.alismatifolia populations from both cultivatedand wild habitats in Thailand. High diversity was observed in allpopulations with relatively lower values in cultivated populations.Percentage of polymorphic loci (P)varied from 85.7–100% in cultivated populations comparedwith 100% in all natural populations. Allele number per locus(A _L) was 3.14 in cultivatedpopulations, and from 2.86–4 in natural populations. Allelenumber per polymorphic locus(A _P) of cultivated andnatural populations ranged from 3.14–3.5 and 2.86–4,respectively. Genetic diversity within populations(H _S) varied from0.586–0.611 in cultivated and from 0.621–0.653 in naturalpopulations. The genetic identity(I _SP) for the species was0.833. The cultivated populations yielded higher value of geneticidentity with highland populations(I _{C /H} =0.776) than with the lowland ones(I _{C /L} =0.754). The analysis of genetic similarities with theNeighbor-Joining algorithm results in the separation ofcultivated populations from all wild populations. One highlandpopulation from the tourist spot, H2, was placed in a separatecluster between the cultivated and other wild populations. It isconsidered as the possible origin of the cultivatedpopulations.     

Cross-species Transferability of Rice Microsatellites in its Wild Relatives and the Potential for Conservation Genetic Studies

Here, we investigated the transferability of 60 microsatellite markers characterized for cultivated rice Oryza sativa L. in three wild Oryza species representing different genome types: O. rufipogon Griff. (AA), O. officinalis Wall. et Watt. (CC), and O. granulate Nees et Arn. ex Watt. (G). The results indicate the 60 rice SSR loci tested produced homologous amplification products to different extents in O. rufipogon (100%), O. officinalis (90%) and O. granulata (73.3%). Proportions of polymorphism for successfully amplified loci ranged from 0.983 via 0.667 to 0.364 in O. rufipogon, O. officinalis and O. granulata, respectively. The utility of these microsatellite markers was tested for the characterization of genetic diversity in 117 genotypes of these four Oryza species. The values of genetic diversity in cultivated rice are higher than the other two wild species O. officinalis and O. granulata, suggesting microsatellites tend to have more variability in the focal species than in non-focal species to which they are applied. However, much lower levels of genetic variation were observed in rice than in its wild progenitor O. rufipogon, which indicates severe loss of genetic variation may reflect the ‘domestication bottleneck’ through which rice passed. The observation that most of the rice microsatellites are able to detect allelic polymorphisms at different extent in Oryza species suggest that rice microsatellite loci should be useful for the analysis of genetic diversity and inter- and intra-specific relationships in the genus. Therefore, high rates of successful cross-amplification of rice microsatellites among Oryza species with different genome types will offer excellent opportunities to investigate the population genetic structure of wild rice species and explore their conservation genetics.     

Genetic relationships among cultivated and wild <Emphasis Type="Italic">Vigna angularis</Emphasis> (Willd.) Ohwi et Ohashi and relatives from Korea based on AFLP markers

Genetic variation and relationships among members of the azuki bean complex (Vigna angularis) including wild (V. angularis var. nipponensis), weedy, and cultivated types (V. angularis var. angularis), V. nakashimae, and rice bean (V. umbellata) from Korea were examined using the Amplified fragment length polymorphism (AFLP) method. AFLP analysis of 50 accessions revealed 333 (72.1%) polymorphic fragments out of 462 fragments amplified using seven primer combinations. The number of polymorphic fragments within each species was 70 in the azuki bean complex and 41 in V. nakashimae, but there was no polymorphism in rice bean. The number of shared fragments among species ranges from 142 between the azuki bean complex and V. nakashimae to 166 between the azuki bean complex and rice bean. Within the azuki bean complex, the range of shared bands was from 231 between cultivated and weedy types to 238 between cultivated and wild types. A dendrogram generated from Jaccard’s similarity matrix was divided into three groups, which correspond to V. nakashimae, azuki bean complex, and rice bean. The relationship between azuki bean and rice bean is closer than between azuki bean and V. nakashimae. Phenetic distances averaged 0.502 between the azuki bean complex and V. nakashimae and 0.467 between the azuki bean complex and rice bean. Within the azuki bean complex, the weedy type was more closely related to wild than cultivated types. But UPGMA dendrogram of the azuki bean complex reveals that each type is not clearly isolated. These results will help to understand genetic diversity and evolutionary dynamics of Vigna in Korea.     

Divergent evolution of wild and cultivated subspecies of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> as revealed by the study of <Emphasis Type="Italic">PolA1</Emphasis> gene

Triticum timopheevii (genome symbol AAGG) comprises two subspecies, cultivated ssp. timopheevii, and wild ssp. armeniacum. These two subspecies are considered as allotetraploids of AA genome from Triticum diploid species and SS genome from Aegilops species. The difference in genome symbol (G vs. S) is due to wide genetic variations among four SS genome species, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. speltoides. In order to study the origin of T. timopheevii, we compared 19th intron (PI19) sequence of the PolA1 gene, encoding the largest subunit of RNA polymerase I. Two different sized DNA fragments containing PI19 sequences (PI19A and PI19G) were amplified both in ssp. timopheevii and ssp. armeniacum. Shorter PI19A (112 bp) sequences of both subspecies were identical to PI19 sequences of two AA species, T. monococcum and T. urartu. Interestingly, the longer PI19G (241–243 bp) sequences of ssp. armeniacum showed more similarity to PI19 sequences of Ae. speltoides whereas ssp. timopheevii showed more similarity to PI19 sequences of other three SS genome species. The results indicated that two subspecies of T. timopheevii, ssp. armeniacum or ssp. timopheevii, might have arisen independently by allotetraploidization of AA genome with Ae. speltoides or one of the remaining three Aegilops species, respectively.