Genetic diversity and population structure of Musa accessions in ex situ conservation

Abstract Background Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa. Results From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis. Conclusions The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping.

Phylogeny of Lundia (Bignoniaceae) based on ndhF and PepC sequences

The circumscription of genera belonging to tribe Bignonieae (Bignoniaceae) has traditionally been complex, with only a few genera having stable circumscriptions in the various classification systems proposed for the tribe. The genus Lundia, for instance, is well characterized by a series of morphological synapomorphies and its circumscription has remained quite stable throughout its history. Despite the stable circumscription of Lundia, the circumscription of species within the genus has remained problematic. This study aims to reconstruct the phylogeny of Lundia in order to refine species circumscriptions, gain a better understanding of relationships between taxa, and identify potential morphological synapomorphies for species and major clades. We sampled 26 accessions representing 13 species of Lundia, and 5 outgroups, and reconstructed the phylogeny of the genus using a chloroplast (ndhF) and a nuclear marker (PepC). Data derived from sequences of the individual loci were analyzed using parsimony and Bayesian inference, and the combined molecular dataset was analyzed with Bayesian methods. The monophyly of Lundia nitidula, a species with a particularly complex circumscription, was tested using Shimodaira-Hasegawa (SH) test and the approximately unbiased test for phylogenetic tree selection (AU test). In addition, 40 morphological characters were mapped onto the tree that resulted from the analysis of the combined molecular dataset in order to identify morphological synapomorphies of individual species and major clades. Lundia and most species currently recognized within the genus were strongly supported as monophyletic in all analyses. One species, Lundia nitidula, was not resolved as monophyletic, but the monophyly of this species was not rejected by the AU and SH tests. Lundia sect. Eriolundia is resolved as paraphyletic in all analyses, while Lundia sect. Eulundia is monophyletic and supported by the same morphological characters traditionally used to circumscribe this section. The phylogeny of Lundia contributed important information for a better circumscription of species and served as basis the taxonomic revision of the genus.

Genetic variation of germination cold tolerance in Japanese rice germplasm

Low temperatures at the initial stages of rice development prevent fast germination and seedling establishment and may cause significant productivity losses. In order to develop rice cultivars exhibiting cold tolerance, it is necessary to investigate genetic resources, providing basic knowledge to allow the introduction of genes involved in low temperature germination ability from accessions into elite cultivars. Japanese rice accessions were evaluated at the germination under two conditions: 13 degrees C for 28 days (cold stress) and 28 degrees C for seven days (optimal temperature). The traits studied were coleoptile and radicle length under optimal temperature, coleoptile and radicle length under cold and percentage of the reduction in coleptile and radicle length due to low temperature. Among the accessions studied, genetic variation for traits related to germination under low temperatures was observed and accessions exhibiting adequate performance for all investigated traits were identified. The use of multivariate analysis allowed the identification of the genotypes displaying cold tolerance by smaller reductions in coleoptile and radicle lenght in the presence of cold and high vigour, by higher coleoptile and radicle growth under cold.

Genetic variation of the Ananas genus with ornamental potential

Brazil is one of the main centers of origin of pineapple species presenting the largest genetic variation of the Ananas genus. Embrapa Cassava and Fruits is a Brazilian Agricultural Research Corporation and has an ex-situ collection of 678 accessions of the Ananas genus and some other Bromeliaceae. The use of ornamental pineapple has increased in the last years demanding new varieties, mainly for the external market, due to the originality and colors of its tiny fruits. The main aim of the present study was describing accessions from the pineapple gene bank in order to quantify their genetic variation and identify possible progenitors to be used in breeding programs of ornamental pineapples. Eighty-nine accessions of Ananas comosus var. comosus, A. comosus var. bracteatus (Lindl.) Coppens et Leal, A. comosus var. ananassoides (Baker) Coppens et Leal, A. comosus var. erectifolius (L. B. Smith) Coppens et Leal, A. comosus var. parguasensis (Camargo et L. B. Smith) Coppens et Leal and A. macrodontes Morren were evaluated with 25 morphological descriptors. According to the results, the evaluated accessions were separated into the following categories: landscape plants, cut flower, potted plants, minifruits, foliage and hedge. The genetic distance among accessions was determined using the combined qualitative and quantitative data by the Gower algorithm. The pre-selected accessions presented genetic variation and ornamental potential for different uses. The multicategory analysis formed seven clusters through a classification method based on the average Euclidean distance between all accessions using the cut-point of genetic dissimilarity (D dg = 0.35). The genotypes A. comosus var. erectifolius were selected to be used as landscape plants, cut flower, minifruits and potted plants. Accessions of A. comosus var. bracteatus and A. macrodontes were selected as landscape plants and hedge. The highest variation was observed in A. comosus var. ananassoides genotypes, which presented high potential for use as cut flowers.

ISOLATION AND CHARACTERISTICS OF EIGHT NOVEL POLYMORPHIC MICROSATELLITE LOCI IN LIPPIA ALBA (VERBENACEAE)

Premise of the study: A set of eight microsatellite (simple sequence repeat [SSR]) markers for Lippia alba, an important medicinal and cosmetic plant, was developed to aid studies of genetic diversity and to define efficient strategies for breeding programs. Methods and Results: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 11 SSR loci were developed and optimized in L. alba. Of the 11 loci, eight were found to be polymorphic after screening 61 accessions from two populations. The parameters used to characterize loci were expected heterozygosity (H-e) and number of alleles. A total of 44 alleles were identified, with an average of 5.5 alleles per loci, which were moderately to highly informative according to H-e. Conclusions: These new SSR markers have potential for informing genetic diversity, allele mining, and mapping studies and will be used to generate information for breeding programs of L. alba

Viability, production and morphology of pollen grains for different species in the genus Manihot (Euphorbiaceae)

(Viability, production and morphology of pollen grains for different species in the genus Manihot (Euphorbiaceae)). The objective of this work was to characterize the viability, production and morphology of pollen for different species in the genus Manihot. Floral buds from Manihot accessions were collected from two germplasm banks at Embrapa Cassava & Fruits. The viability of the pollen was assessed via colorimetric, in vitro and in vivo assays. The diameter of the pollen grains was determined by measuring the transversal length of the grain. The experimental design was entirely randomized. Studies on pollen ultrastructure were performed via scanning electron microscopy. Pollen viability was high in the colorimetric tests and intermediate in vivo tests; there was no germination in the in vitro tests. The average production for all accessions was 1,253 pollen grains per floral bud. The size of the pollen grains varied from 132 to 163 pm in the wild accessions, and 129 to 146 pm in the cultivated accessions. The pollen grains for all accessions were very large, apolar, spherical as well as inaperturate, with an exine ornamented with pila organized in a Croton pattern. The wild accessions, in general, produced more and larger pollen grains compared with the cultivated accessions.

DEVELOPMENT OF MICROSATELLITE PRIMERS FOR JATROPHA CURCAS (EUPHORBIACEAE) AND TRANSFERABILITY TO CONGENERS

Premise of the study: Microsatellite primers were developed for Jatropha curcas (Euphorbiaceae), a tree species with large potential for biofuel production, to investigate its natural genetic diversity and mating system to facilitate the establishment of tree improvement and conservation programs. Methods and Results: Using a protocol for genomic library enrichment, 104 clones containing 195 repeat motifs were identified. Primer pairs were developed for 40 microsatellite loci and validated in 41 accessions of J. curcas from six provenances. Nine loci were polymorphic revealing from two to eight alleles per locus, and six primers were able to amplify alleles in the congeners J. podagrica, J. pohliana, and J. gossypifolia, but not in other Euphorbiaceae species, such as Hevea brasiliensis, Manihot esculenta, or Ricinus communis. Conclusions: The primers developed here revealed polymorphic loci that are suitable for genetic diversity and structure, mating system, and gene flow studies in J. curcas, and some congeners.

Genetic variability of banana with ornamental potential

The Musa germplasm collection at Embrapa Cassava and Fruits detains accessions from different sections of the Musa genus. The objective of the present study was to identify and morphologically characterize banana accessions from the banana germplasm with ornamental potential, as well as to quantify their genetic variability; and identify possible progenitors to be used in breeding aiming to achieve ornamental crossbreeds. The accessions were evaluated with the use of 32 morphological descriptors. Then, they were the following grouped into categories: landscape plants, cut flower, potted plants, and male inflorescence minifruits. The pre-selected accessions presented great genetic variability and ornamental potential for different uses. The accessions of the Rhodochlamys and Callimusa sections were selected to be used as landscape plants, cut flowers, potted plants, male inflorescence and minifruits. Most of the diploids from the Eumusa section evaluated in this study are indicated for the production of ornamental minifruits, except for 'Lidi' and Cici, which can also be indicated as landscape plants. The BB diploids have great potential for the use of the male inflorescence in floral arrangements, and did not offer any other indication.

Selection and Use Recommendation in Hybrids of Ornamental Banana

The ornamental market is dynamic and demands constant novelties. The use of fruit crops as ornamental plants can be an interesting alternative with very differentiated and original products. The banana germplasm bank at Embrapa Cassava and Fruits has been primarily used in the breeding program for generating new cultivars as food. To diversify and expand the use of this collection, accessions with ornamental potential have been selected to obtain new hybrids. This work was aimed at characterizing the progeny of ornamental Musa L. spp. by grouping the hybrids according to the following uses: landscape plants, potted plants, cut flower, or minifruits. Forty-two hybrids were evaluated with 14 quantitative and 12 qualitative descriptors in three production cycles. In addition, assays for resistance to black and yellow Sigatoka and to Fusarium wilt were performed. Variability was observed for all the characteristics evaluated within progenies, especially with regard to leaf color, fruit, peduncle, rachis, and heart. All evaluated hybrids were resistant to yellow Sigatoka and to Fusarium wilt and were resistant or showed reduced symptoms of susceptibility to black Sigatoka. Most hybrids (82%) presented reduced plant height. After clustering by use category, the hybrids RM 09, RM 38, RM 37, and RM 33 were selected and recommended to be used as cut flowers, minifruits, or landscaping plants.

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 Sao 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.

New microsatellite markers for garlic, Allium sativum (Alliaceae)

Premise of the study: A new set of microsatellite or simple sequence repeat (SSR) markers for garlic, an important medicinal spice, was developed to aid studies of genetic diversity and to define efficient strategies for germplasm conservation. Methods and Results: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 16 SSR loci were developed and optimized in garlic. Ten loci were found to be polymorphic after screening 75 accessions. The parameters used to characterize the loci were observed and expected heterozygosity, number of alleles, Shannon Index, and polymorphism information content (PIC). A total of 44 alleles were identified, with an average of 4.4 alleles per loci. The vast majority of loci were moderate to highly informative according to PIC and the Shannon Index. Conclusion: The new SSR markers have the potential to be informative tools for genetic diversity, allele mining, mapping and associative studies, and in the management and conservation of garlic collections.

Genotipagem de polimorfismos associados com sistemas de macho-esterilidade em acessos de cebola adaptados ao Brasil

A produção em escala comercial de sementes híbridas de cebola (Allium cepa) tem sido conduzida com o emprego de dois sistemas de macho-esterilidade do tipo genética-citoplasmática (CMS-S e CMS-T) em associação ao citoplasma normal (macho-fértil). No entanto, a análise molecular desses diferentes tipos citoplasmáticos ainda não está disponível para um grande número de acessos de cebola adaptados para cultivo em regiões tropicais. Além de adaptação às condições edafoclimáticas do Brasil, muitos desses acessos apresentam tolerância a doenças, sendo de potencial valor como genitores de híbridos. O presente trabalho visou identificar os tipos citoplasmáticos de acessos de cebola de diferentes grupos morfoagronômicos de interesse para o melhoramento genético no Brasil, usando a reação da polimerase em cadeia (PCR) com 'primers' específicos para regiões polimórficas do genoma mitocondrial de cebola. Foi observada, nos 66 acessos amostrados, a presença dos três principais tipos de citoplasma descritos para cebola (S, N e T). Foi constatada maior frequência do citoplasma S (56%) seguido do citoplasma T (25,8%). Em 18,2% das amostras, foi encontrado exclusivamente o citoplasma N. Essa caracterização pode ser útil para guiar a escolha de materiais genéticos dentro dos programas de melhoramento com objetivo de desenvolver cultivares híbridas adaptadas às condições tropicais.