Alessandra Pereira Fávero

Identification of candidate genome regions controlling disease resistance in Arachis

BackgroundWorldwide, diseases are important reducers of peanut (Arachis hypogaea) yield. Sources of resistance against many diseases are available in cultivated peanut genotypes, although often not in farmer preferred varieties. Wild species generally harbor greater levels of resistance and even apparent immunity, although the linkage of agronomically un-adapted wild alleles with wild disease resistance genes is inevitable. Marker-assisted selection has the potential to facilitate the combination of both cultivated and wild resistance loci with agronomically adapted alleles. However, in peanut there is an almost complete lack of knowledge of the regions of the Arachis genome that control disease resistance.ResultsIn this work we identified candidate genome regions that control disease resistance. For this we placed candidate disease resistance genes and QTLs against late leaf spot disease on the genetic map of the A-genome of Arachis, which is based on microsatellite markers and legume anchor markers. These marker types are transferable within the genus Arachis and to other legumes respectively, enabling this map to be aligned to other Arachis maps and to maps of other legume crops including those with sequenced genomes. In total, 34 sequence-confirmed candidate disease resistance genes and five QTLs were mapped.ConclusionCandidate genes and QTLs were distributed on all linkage groups except for the smallest, but the distribution was not even. Groupings of candidate genes and QTLs for late leaf spot resistance were apparent on the upper region of linkage group 4 and the lower region of linkage group 2, indicating that these regions are likely to control disease resistance.

Genetic mapping of wild introgressions into cultivated peanut: a way toward enlarging the genetic basis of a recent allotetraploid

BackgroundPeanut (Arachis hypogaea L.) is widely used as a food and cash crop around the world. It is considered to be an allotetraploid (2n = 4x = 40) originated from a single hybridization event between two wild diploids. The most probable hypothesis gave A. duranensis as the wild donor of the A genome and A. ipaënsis as the wild donor of the B genome. A low level of molecular polymorphism is found in cultivated germplasm and up to date few genetic linkage maps have been published. The utilization of wild germplasm in breeding programs has received little attention due to the reproductive barriers between wild and cultivated species and to the technical difficulties encountered in making large number of crosses. We report here the development of a SSR based genetic map and the analysis of genome-wide segment introgressions into the background of a cultivated variety through the utilization of a synthetic amphidiploid between A. duranensis and A. ipaënsis.ResultsTwo hundred ninety eight (298) loci were mapped in 21 linkage groups (LGs), spanning a total map distance of 1843.7 cM with an average distance of 6.1 cM between adjacent markers. The level of polymorphism observed between the parent of the amphidiploid and the cultivated variety is consistent with A. duranensis and A. ipaënsis being the most probable donor of the A and B genomes respectively. The synteny analysis between the A and B genomes revealed an overall good collinearity of the homeologous LGs. The comparison with the diploid and tetraploid maps shed new light on the evolutionary forces that contributed to the divergence of the A and B genome species and raised the question of the classification of the B genome species. Structural modifications such as chromosomal segment inversions and a major translocation event prior to the tetraploidisation of the cultivated species were revealed. Marker assisted selection of BC1F1 and then BC2F1 lines carrying the desirable donor segment with the best possible return to the background of the cultivated variety provided a set of lines offering an optimal distribution of the wild introgressions.ConclusionThe genetic map developed, allowed the synteny analysis of the A and B genomes, the comparison with diploid and tetraploid maps and the analysis of the introgression segments from the wild synthetic into the background of a cultivated variety. The material we have produced in this study should facilitate the development of advanced backcross and CSSL breeding populations for the improvement of cultivated peanut.

Identification of Fungus Resistant Wild Accessions and Interspecific Hybrids of the Genus Arachis.

Peanut, Arachis hypogaea L., is a protein-rich species consumed worldwide. A key improvement to peanut culture involves the development of cultivars that resist fungal diseases such as rust, leaf spot and scab. Over three years, we evaluated fungal resistance under field conditions of 43 wild accessions and three interspecific hybrids of the genus Arachis, as well as six A. hypogaea genotypes. In the first year, we evaluated resistance to early and late leaf spot, rust and scab. In the second and third years, we evaluated the 18 wild species with the best resistance scores and control cultivar IAC Caiapó for resistance to leaf spot and rust. All wild accessions displayed greater resistance than A. hypogaea but differed in their degree of resistance, even within the same species. We found accessions with as good as or better resistance than A. cardenasii, including: A. stenosperma (V15076 and Sv 3712), A. kuhlmannii (V 6413), A. kempff-mercadoi (V 13250), A. hoehnei (KG 30006), and A. helodes (V 6325). Amphidiploids and hybrids of A. hypogaea behaved similarly to wild species. An additional four accessions deserve further evaluation: A. magna (V 13751 and KG 30097) and A. gregoryi (V 14767 and V 14957). Although they did not display as strong resistance as the accessions cited above, they belong to the B genome type that is crucial to resistance gene introgression and pyramidization in A. hypogaea.

CARACTERIZAÇÃO MORFOLÓGICA DE ACESSOS DE GERMOPLASMA DE QUATRO ESPÉCIES BRASILEIRAS DE AMENDOIM-SILVESTRE

Caracterizaram-se morfologicamente os acessos de germoplasma de especies silvestres brasileiras de amendoim do genero Arachis L., Sect. Arachis e analisaram-se a similaridade genetica entre acessos da mesma especie e entre as especies. Realizou-se o experimento nos anos agricolas de 1993 a 1996, no Nucleo Experimental de Campinas, do Instituto Agronomico (IAC). Avaliaram-se os acessos disponiveis no Banco Ativo de Germoplasma de Especies Silvestres de Arachis, da Embrapa Recursos Geneticos e Biotecnologia (CENARGEN - Brasilia, DF), das especies A. palustris Krapov., W.C. Gregory & Valls, A. decora Krapov., W.C. Gregory & Valls, A. praecox Krapov., W.C. Gregory & Valls e A. stenosperma Krapov. & W.C. Gregory, efetuando-se anotacoes fenotipicas quantitativas e qualitativas, conforme lista de descritores morfologicos. Observou-se que os acessos de A. stenosperma sao semelhantes, apesar da sua grande distância geografica, e diferem das demais especies, formando um grupo mais coeso. Caracteres como o diâmetro do eixo central e o comprimento dos frutos e das sementes serviram para distingui-la das demais especies. Arachis decora apresentou alta variacao entre acessos nos varios caracteres morfologicos estudados. A. palustris apresentou alta variacao morfologica entre acessos, ainda que tenham sido analisados apenas dois, para altura da planta, largura da semente, dimensoes do esporao, istmo, foliolo, raque e eixo central e quanto a presenca e ausencia de tricomas no foliolo. Arachis praecox, representada por um unico acesso, aproximou-se mais de A. decora que das demais especies.

Avaliação de espécies silvestres e cultivares de amendoim para resistência a Enneothrips flavens Moulton

In Brazil, various pests are reported to infest peanut crop, being the thrips Enneothrips flavens the most important. Accesses of wild peanut species, anfidiploide and peanut cultivars were evaluated in field conditions at Pindorama, Sao Paulo State, at the agricultural year of 2007/2008. The experiment consisted of a complete ramdomized block design with 48 treatments and five replications. The material was sown in small bags under greenhouse conditions. Plantings were carried out in October/November 2007. Starting at 30 days after planting in the field, evaluations were done at 15-day intervals, in five closed leaves of each plant. The following data were obtained: presence or absence of thrips in each leaf; symptoms of thrips damage in recently opened leaves, based on a 1-5 scale where 1 = without damage, 2 = 1 to 25% of the leaf surface with damage (grooves and deformations), 3 = 26 to 50%, 4 = 51 to 75% and 5 = 76 to 100% of damage symptoms. The accesses with the least damages and insect presence were: VS 14957 (A. gregoryi), V13832 (A. stenosperma), V8979 (A. kuhlmannii), V9912 (A. kuhlmannii), V7639 (A. kuhlmannii) e VMiIrLbGv14309 (A. villosa). The most sensitive accesses were V12549 (A. hypogaea), Ac2562 (A. hypogaea) and the commercial A. hypogaea genotypes IAC Caiapo and IAC Runner 886.

Successful crosses between fungal-resistant wild species of Arachis (section Arachis) and Arachis hypogaea

Peanut (Arachis hypogaea) is the fifth most produced oil crop worldwide. Besides lack of water, fungal diseases are the most limiting factors for the crop. Several species of Arachis are resistant to certain pests and diseases. This study aimed to successfully cross the A-genome with B-K-A genome wild species previously selected for fungal disease resistance, but that are still untested. We also aimed to polyplodize the amphihaploid chromosomes; cross the synthetic amphidiploids and A. hypogaea to introgress disease resistance genes into the cultivated peanut; and analyze pollen viability and morphological descriptors for all progenies and their parents. We selected 12 A-genome accessions as male parents and three B-genome species, one K-genome species, and one A-genome species as female parents. Of the 26 distinct cross combinations, 13 different interspecific AB-genome and three AA-genome hybrids were obtained. These sterile hybrids were polyploidized and five combinations produced tetraploid flowers. Next, 16 combinations were crossed between A. hypogaea and the synthetic amphidiploids, resulting in 11 different hybrid combinations. Our results confirm that it is possible to introgress resistance genes from wild species into the peanut using artificial hybridization, and that more species than previously reported can be used, thus enhancing the genetic variability in peanut genetic improvement programs.

Rooting performance from leaf petioles of accessions and hybrids of wild Arachis species

The genus Arachis has 69 species, the majority native to Brazil. Arachis spp. seedling root tips have been used to obtain dividing cells for cytological preparations. Several wild species produce small amounts of seeds and consequently few root meristems, making the cytogenetic characterization very difficult. The purpose of this study was to evaluate the rooting capacity of several wild species of Arachis using the detached leaves technique for obtaining roots from petioles. One hundred and thirty accessions and 27 interspecific hybrids were studied. Three evaluations were performed at 15, 31, and 49 days after planting. Leaf rooting observed in the wild species confirmed the great genetic variability typical of the Arachis genus, indicating that the number of days interferes with the rooting percentage. The Arachis Section presented the highest rate of rooting leaves. The Caulorrhizae Section showed the same pattern observed in the Erectoides Section. Accessions from Heteranthae, Trierectoides, Extranervosae, and Rhizomatosae Sections did not differ among each other. Differences were not observed in Erectoides Section when compared to Rhizomatosae and Extranervosae Sections. The number and morphology of roots obtained through this technique were promising to be used on cytological preparations.

Recombinants from the crosses between amphidiploid and cultivated peanut (Arachis hypogaea) for pest-resistance breeding programs.

Peanut is a major oilseed crop worldwide. In the Brazilian peanut production, silvering thrips and red necked peanut worm are the most threatening pests. Resistant varieties are considered an alternative to pest control. Many wild diploid Arachis species have shown resistance to these pests, and these can be used in peanut breeding by obtaining hybrid of A and B genomes and subsequent polyploidization with colchicine, resulting in an AABB amphidiploid. This amphidiploid can be crossed with cultivated peanut (AABB) to provide genes of interest to the cultivar. In this study, the sterile diploid hybrids from A. magna V 13751 and A. kempff-mercadoi V 13250 were treated with colchicine for polyploidization, and the amphidiploids were crossed with A. hypogaea cv. IAC OL 4 to initiate the introgression of the wild genes into the cultivated peanut. The confirmation of the hybridity of the progenies was obtained by: (1) reproductive characterization through viability of pollen, (2) molecular characterization using microsatellite markers and (3) morphological characterization using 61 morphological traits with principal component analysis. The diploid hybrid individual was polyploidized, generating the amphidiploid An 13 (A. magna V 13751 x A. kempff-mercadoi V 13250)4x. Four F1 hybrid plants were obtained from IAC OL 4 × An 13, and 51 F2 seeds were obtained from these F1 plants. Using reproductive, molecular and morphological characterizations, it was possible to distinguish hybrid plants from selfed plants. In the cross between A. hypogaea and the amphidiploid, as the two parents are polyploid, the hybrid progeny and selves had the viability of the pollen grains as high as the parents. This fact turns the use of reproductive characteristics impossible for discriminating, in this case, the hybrid individuals from selfing. The hybrids between A. hypogaea and An 13 will be used in breeding programs seeking pest resistance, being subjected to successive backcrosses until recovering all traits of interest of A. hypogaea, keeping the pest resistance.

Apomixis-related genes identified from a coexpression network in Paspalum notatum, a Neotropical grass

Apomixis is a highly desirable trait in modern agriculture; however, incorporating it into breeding programs requires a deeper comprehension of apomictic regulatory mechanisms. Paspalum notatum is considered a good model for such studies because it exhibits both sexual and apomictic cytotypes. A transcriptomic approach to identifying differentially expressed genes between these cytotypes would constitute an important tool for the identification of genes in the apomictic pathway. We generated leaf and inflorescence transcriptomes of apomictic tetraploids and sexual diploids/tetraploids of P. notatum accessions. We de novo assembled and annotated 114,306 unigenes. Coexpression network and ontological annotation were applied to recover the interactions and biological processes of exclusive and differentially expressed genes from the apomictic libraries. Additionally, genes potentially linked to the apomixis-controlling region and several genes reported in the literature were identified. The reference transcriptome obtained in this study represents a robust set of expression data for P. notatum. Our approach to sequencing florets and leaves from different cytotypes enabled us to isolate a set of candidate genes. Using a gene expression network, we recovered transcriptionally coordinated genes related to mechanisms regulating apomixis. These findings provide an important contribution to our knowledge of apomixis and are valuable for Paspalum breeding programs.

Resistance to thrips (Enneothrips flavens) in wild and amphidiploid Arachis species

Thrips (Enneothrips flavens) is a pest that causes severe damage and yield losses to peanut crop if not properly controlled. The main control method currently used by farmers is bi-weekly application of insecticides during crop development, which, in addition to its toxicity, is very costly. Thus, new sources of resistance must be identified in order to reduce the use of insecticides and effectively manage the pest. This study aimed to evaluate the occurrence and symptoms of E. flavens infestations in 12 accessions of 10 wild species of Arachis and nine amphidiploids, as well as to compare their morphoagronomic characteristics to those of commercial cultivars. To this end, we conducted experiments during two summer seasons, using a randomized block design with four replications. We conducted evaluations of the severity of infestation, noting visual symptoms of E. flavens and morphological and reproductive characteristics of the Arachis plants. Results indicated that wild accessions V 7635 (A. vallsii), V 13250 (A. kempff-mercadoi), K 9484 (A. batizocoi), Wi 1118 (A. williamsii), V 14167 (A. duranensis) and V 13751 (A. magna) are the most promising for obtaining useful new amphidiploids. Among the amphidiploids, An 12 (A. batizocoi x A. kempff-mercadoi)4x, An 9 (A. gregoryi x A. stenosperma) 4x, and An 8 (A. magna x A. cardenasii)4x showed high level of resistance to E. flavens. The identified thrips resistant wild and amphidiploid Arachis species may be used in future breeding program to produce thrips resistant peanut cultivars.