Identification and analysis of single nucleotide polymorphisms (SNPs) in citrus

Most of the cultivated species of citrus have narrow genetic basis. Relationships among species and cultivars are obscured by sexual compatibility, polyembryony, apomixis and a high incidence of somatic mutations. DNA analysis is crucial in genetic studies not only for citrus breeding programs but also for characterization of hybrids and species. In this paper, single nucleotide polymorphisms ( SNPs) were investigated in 58 accessions of Citrus, hybrids and related genera. Genomic sequences of 'Pera IAC' sweet orange ( Citrus sinensis L. Osbeck) were used for primer design and selection of sequence tagged sites (STSs) for identification of SNPs. Analysis of 36 STSs showed identical sequences among 40 of the 41 sweet orange accessions studied. However, these accessions were heterozygous for many SNPs. Ten selected STSs were analyzed in 17 additional accessions from 13 species and hybrids. Comparing to the 'Pera IAC' sweet orange accession, a total of 150 polymorphic nucleotides were identified and most of the alterations were transitions ( 52.7%). The greatest number of SNPs was observed in Poncirus trifoliata ( L.) Raf. and the smallest in 'Ponkan' mandarin ( Citrus reticulata Blanco). At the intra-specific level, 'Bafa Gigante' ( Citrus sinensis L. Osbeck) was the only sweet orange accession with a divergent SNPs genotype, which corroborates the hypothesis of a hybrid origin for this accession. Although the STSs analyzed represent randomly sampled genomic sequences, they provided consistent information about the level of polymorphism and showed the potential of SNPs markers for characterization and phylogenetic studies.

A novel technique to produce polygenic resistance to anthracnose in Stylosanthes capitata

An improved cultivar, based on 17 genotypes of S. capitata and six of S.macrocephala, was developed at the wEmbrapa Beef Cattle Research Center, Campo Grande, Brazil. The aim was to create durable, race non-specific resistance to anthracnose controlled by polygenic factors. A mass hybridisation technique was employed to produce a high degree of genetic diversity and sizeable quantities of seed of hybrid-derived progenies of Brazilian and Venezuelan genotypes of S. capitata. Outcrossing resulted in a significant improvement in the forage production of progeny of Venezuelan accessions. The multicross was evaluated in multilocational trials, each representing a large tract of country in the Cerrados ecosystem along a north-south transect from lat.6degrees S to 20degrees S. The genetic shift that occurred in S. capitata was a key element in the formation of the new cultivar. It is a complex mixture of two species, and a recombination of much desired forage traits of Brazilian x Venezuelan genotypes, high forage and seed yields coupled with anthracnose resistance. The new cultivar with its diverse genetic make-up has a wide application in the acid-soil savannas of tropical America. It was released by Embrapa for the Cerrados in 2000.

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.

Genetic diversity of Stryphnodendron adstringens (Mart.) Coville determined by AFLP molecular markers

Bark extracts of Stryphnodendron adstringens (Mart) Coville a Leguminosae species, well known in Brazil as barbatimao, are popularly used as healing agent. The objective of this work was to determine the genetic diversity of S. adstringens populations and to correlate genetic distances to the production of tannins. S. adstringens accessions from populations found in Cerrado regions in the states of Goias, Minas Gerais and São Paulo were analyzed using the AFLP (Amplified Fragment Length Polymorphism) technique. A total of 236 polymorphic bands were scored and higher proportion of genetic diversity was found inter populations (70.9%), rather than intra populations (29.1%). F-ST value was found to be significantly greater than zero (0.2906), demonstrating the complex genetic structure of S. adstringens populations. Accessions collected in Cristalina, GO, showed higher percentage of polymorphic loci (87.3%) and the highest genetic diversity. The lowest genetic variability was detected among accessions from the population growing in Caldas Novas, GO. The genetic distance among populations was estimated using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), which grouped populations into 3 clusters. Moreover, chemotypes with tannin concentration above 40% showed higher genetic similarity. AFLP analysis proved to be an efficient gene mapping technique to determine the genetic diversity among remaining populations of S. adstringens. Obtained results may be employed to implement further strategies for the conservation of this medicinal plant. (C) 2011 Elsevier Ltd. All rights reserved.

Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty-five accessions of the species-rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species-level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0-3 bp). Comparisons to previously published mitochondrial cox2-3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2-3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.

Antioxidant content in guava (Psidium guajava) and araca (Psidium spp.) germplasm from different Brazilian regions

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação agronômica de sete acessos de Hemarthria altissima [poir.]stapf et C.E.Hubb.em Pindamonhangaba, SP

The experiment was carried out to evaluate seven accessions of limpograss Hermathria altissima (PI 364888, NO 158, PI 364863, IPEACO 336, UF 553, UF 347238, and IPEACO 337) as compared to coast-cross grass (Cynodon dactylon), to determine which grasses were best adapted ecologically to conditions at Pindamonhangaba. The experimental design was a randomized block with four replications. Five annual harvests were taken during the three-year experimental period, two in the dry season and three in the rainy season. The higher annual DM yields (kgDM/ ha) were obtained with accessions UF 553 (19996), NO 158 (18745), and PI 347238 (18267). Accessions UF 553 and IPEACO 336 had the most uniform production throughout the year with 37.4 and 33.2% during the dry season, respectively. Coast-cross had the highest percentage of crude protein (10.30%), while limpograss values varied around 7%. It was concluded that accession UF553 was the most promising grass for the Pindamonhangaba region.

RFLP and cytogenetic evidence on the origin and evolution of allotetraploid domesticated peanut, Arachis hypogaea (Leguminosae)

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

Genetic variation between several species of sections Extranervosae, Caulorrhizae, Heteranthae, and Triseminatae (genus Arachis) estimated by DNA polymorphism

Genetic variation within and among accessions of the genus Arachis representing sections Extranervosae, Caulorrhizae, Heteranthae, and Triseminatae was evaluated using RFLP and RAPD markers. RAPD markers revealed a higher level of genetic diversity than did RFLP markers, both within and among the species evaluated. Phenograms based on various band-matching algorithms revealed three major clusters of similarity among the sections evaluated. The first group included the species from section Extranervosae, the second group consisted of sections Triseminatae, Caulorrhizae, and Heteranthae, and the third group consisted of one accession of Arachis hypogaea, which had been included as a representative of section Arachis. The phenograms obtained from the RAPD and RFLP data were similar but not identical. Arachis pietrarellii, assayed only by RAPD, showed a high degree of genetic similarity with Arachis villosulicarpa. This observation supported the hypothesis that these two species are closely related. It was also shown that accession V 7786, previously considered to be Arachis sp. aff. pietrarellii, and assayed using both RFLPs and RAPDs, was possibly a new species from section Extranervosae, but very distinct from A. pietrarellii.

Genetic variation and phylogenetic relationships based on RAPD analysis in section Caulorrhizae, genus Arachis (Leguminosae)

Wild Arachis germplasm includes potential forage species, such as the rhizomatous Arachis glabrata and the stoloniferous A. pinto and A. repens. Commercial cultivars of A. pintoi have already been released in Australia and in several Latin American countries, and most of these cultivars were derived from a single accession of A. pintoi (GK 12787). Arachis repens is less productive as a forage plant than is A. pintoi. However, it can be crossed with A. pintoi, and thus has good potential as germplasm for the improvement of A. pintoi. Arachis repens is also used as an ornamental plant and ground cover. Many new accessions of these two stoloniferous species are now available, and they harbor significant genetic variability beyond that available in the few older accessions, previously available. Therefore, these new accessions need to be conserved, documented and considered in terms of their potential for crop improvement and direct commercial use. Sixty-four accessions of this new germplasm were analyzed using RAPD analysis. Most of the accessions of A. repens grouped together into a clearly distinct group. In general, the accessions from the distinct valleys of the Jequitinhonha, Sao Francisco and Parana rivers did not group together, suggesting there is not a tight relation between dispersion by rivers and the geographic distribution of genetic variation in these species.

Isolation and characterization of microsatellite loci from the forage species Arachis pintoi (Genus Arachis)

Arachis pintoi is an alternative to forage production in the tropics. Its germplasm comprises more than 150 accessions, that could be used to improve it. Our objective was the isolation and characterization of microsatellite loci in A. pintoi to be used to molecular evaluation of this germplasm and of A. repens (section Caulorrhizae). Seven loci were analyzed using five accessions of A. repens and 20 accessions of A. pintoi. The high variation found makes clear the high potential of this marker in genetic studies in these species. The developed markers showed total transferability to A. repens.

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 the development of Fafia - Pfaffia glomerata (Spreng.) Pedersen

The aim of this experiment is to evaluate the development in height of the plants of four accessions (2 from Parana - accession 1 and 2, one from São Paulo - accession 3, and one from Mato Grosso do Sul State- accession 4). The experimental design was made of randomized blocks, with four treatments (accessions) and four replications. In each plot were planted 24 seedlings. Five evaluations of the height were accomplished, with an interval of two months, starting from the second month after the planting of the seedlings. The results show a significant difference between accession 4 and other three accessions in first evaluation (141.42 % higher than accession 2, the lowest of three and 44.16 % higher than accession 3, the highest of three), demonstrating a higher vigour of this accession on initial stage of its development. In the second and third evaluation, all accessions have no statistical difference, demonstrating a similar level of development at intermedial stages of development. At 5 th evaluation, all accessions have almost the same result, with no statistical difference among them. This is a clear result of similar level of development of this species in these accessions, representing a stabilization of their height development at the end of first year. We concluded that fáfia possesses a strong initial growth, with visible difference among the accessions in the first months. From the eight month on, we observed a slight decrease in the height. This suggests the end of annual cycle.

Chemical and biochemical characterization of guava and araçá fruits from different regions of Brazil

Guava and araçá, species of the Psidium genus, are important options for Brazilian agribusiness, especially the former species, due to their fruit characteristics, such as appearance, taste and richness in minerals and phenolic compounds. These fruits can be consumed in natura or in several processed forms. The active germplasm bank is an important tool for genetic resource characterization and plant breeding studies. Sixty guava and ten araçá accessions of the Psidium active germplasm, sampled in 44 different Brazilian regions and grown at Embrapa Semiarid, were chemically and biochemically characterized in order to support breeding programs. The accessions were grown in a randomized block design, with two replications and three plants/plot. The sugar, proteins, soluble solids, titratable acidity, calcium, magnesium, iron and phosphorus contents were determined. Large variations were observed in the analyzed compounds, which could be attributed to the diversity of genotypes and also to the environmental conditions, which affect the plant metabolism. The high variability observed in most parameters of the accessions is an important factor for the improvement of these species. Most guava accessions showed higher titratable acidity and soluble solids than those found in commercial cultivars and, in araçá, these levels were even higher, which makes them promising for commercial exploitation. Moreover, fruits of the guava and araçá accessions present good sources of sugars and minerals. Special attention should be given to some guava and araçá accessions from Maranhão and Pernambuco States, respectively, which showed high levels for titratable acidity, soluble solids, SS/TA ratio, total soluble sugars, calcium, magnesium and iron, should be targets of breeding programs for new Psidium cultivars. © ISHS.

Caracteres estruturais e dimensão fractal da folha de espécies de Arachis e sua relação com a cercosporiose

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)