Population Structure and Genetic Diversity of Sweet Cassava Accessions from the Midwestern, Southeastern and Southern Regions of Brazil

Abstract The objective of this work was to screen sweet cassava accessions collected in smallholding areas in the Midwestern, Southeastern and Southern regions of Brazil, using 15 SSR molecular markers, to determine population structure and genetic diversity. Polymorphism was detected in every loci analyzed, with mean of 6.33 alleles per locus, and mean polymorphism information content (PIC) of 0.6057, pointing out that the primers were highly informative. The observed heterozygosity ranged from 0.0709 (SSRY 101) to 0.9398 (GA 12), with a mean of 0.6511, and mean genetic diversity of 0.6578, ranging from 0.3592 (GA 136) to 0.8116 (SSRY 21). The most dissimilar combinations observed were BGM526PR-BGM596MS, BGM526PR-BGM622MS and BGM526PR-BGM629MS. The traditional cassava cultivars assessed were divided into four distinct groups: two with cultivars from the South, one from the Southeast and one from the Midwestern region of Brazil. The variances among and within groups determined by the analysis of molecular variance were 44 and 56%, respectively. The PhiPT parameter (analogue to Fst) of 0.44 indicates high differentiation among groups. Broad genetic diversity was found among the traditional sweet cassava cultivars assessed, and the most divergent groups were formed by cultivars from the South and the Midwestern regions of Brazil.

Molecular identification of Lactobacillus spp. associated with puba, a Brazilian fermented cassava food

Puba or carimã is a Brazilian staple food obtained by spontaneous submerged fermentation of cassava roots. A total of 116 lactobacilli and three cocci isolates from 20 commercial puba samples were recovered on de Man, Rogosa and Sharpe agar (MRS); they were characterized for their antagonistic activity against foodborne pathogens and identified taxonomically by classical and molecular methods. In all samples, lactic acid bacteria were recovered as the dominant microbiota (7.86 ± 0.41 log10 CFU/g). 16S-23S rRNA ARDRA pattern assigned 116 isolates to the Lactobacillus genus, represented by the species Lactobacillus fermentum (59 isolates), Lactobacillus delbrueckii (18 isolates), Lactobacillus casei (9 isolates), Lactobacillus reuteri (6 isolates), Lactobacillus brevis (3 isolates), Lactobacillus gasseri (2 isolates), Lactobacillus nagelii (1 isolate), and Lactobacillus plantarum group (18 isolates). recA gene-multiplex PCR analysis revealed that L. plantarum group isolates belonged to Lactobacillus plantarum (15 isolates) and Lactobacillus paraplantarum (3 isolates). Genomic diversity was investigated by molecular typing with rep (repetitive sequence)-based PCR using the primer ERIC2 (enterobacterial repetitive intergenic consensus). The Lactobacillus isolates exhibited genetic heterogeneity and species-specific fingerprint patterns. All the isolates showed antagonistic activity against the foodborne pathogenic bacteria tested. This antibacterial effect was attributed to acid production, except in the cases of three isolates that apparently produced bacteriocin-like inhibitory substances. This study provides the first insight into the genetic diversity of Lactobacillus spp. of puba.

Evaluation of cassava plants generated by somatic embryogenesis at different stages of development using molecular markers

Background: Cassava (Manihot esculenta Crantz) is a crop that is high in carbohydrates in the roots and in protein in the leaves, important for both human consumption and animal feed, and also has a significant industrial use for its starches. In this study we evaluated the genetic variability with molecular markers in different stages in micropropagated plants from somatic embryos of Venezuelan native clone 56. Results: Three markers were used: ISTR, AFLP and SSR, finding that ISTR showed the highest polymorphism among individuals tested. With AFLP a high similarity between the evaluated individuals was observed and with SSR total monomorphism was seen. Using cluster analysis it was found that individuals from an embryo labeled as fasciated at the beginning of the somatic embryogenesis process were grouped as independent of the other plants when analyzed at the acclimatization stage. The differences found with the different markers used are discussed. In field trials, micropropagated plants had a yield between 4 and 5 times the average yield of cassava in Venezuela. Conclusion: Despite variability in terms of DNA markers, somatic embryogenesis is suitable for mass propagation of highly performing cassava clones.

Infestation of cassava genotypes by Neosilba perezi (Romero & Ruppell) (Diptera: Lonchaeidae)

The objectives of this work were to assess the infestation of ten genotypes of cassava by the shoot fly Neosilba perezi (Romero & Ruppell) and to investigate effects of plant age, temperature or precipitation on cassava plants infestation by the shoot fly. Thirty-two individuals of each cassava genotype were planted and analyzed every two weeks in order to calculate the percentage of plants infested by shoot fly larvae at each sampling event and per genotype. Infestation by the fly was different across the genotypes. Genotype IAC Caapora 105-66 and genotype IAC Cascuda were resistant to shoot fly infestation, whereas the genotype IAC 15 was the most susceptible to this insect. Plant age may have an influence on infestation by shoot flies. Advanced plant age apparently favors lower or even inexistent infestation rates. However, infestation rate does not seem to be affected by temperature or precipitation.

Genetic characterization of cassava (Manihot esculenta) landraces in Brazil assessed with simple sequence repeats

Based on nine microsatellite loci, the aim of this study was to appraise the genetic diversity of 42 cassava (Manihot esculenta) landraces from selected regions in Brazil, and examine how this variety is distributed according to origin in several municipalities in the states of Minas Gerais, São Paulo, Mato Grosso do Sul, Amazonas and Mato Grosso. High diversity values were found among the five above-mentioned regions, with 3.3 alleles per locus on an average, a high percentage of polymorphic loci varying from 88.8% to 100%, an average of 0.265 for observed heterozygosity and 0.570 for gene diversity. Most genetic diversity was concentrated within the regions themselves (HS = 0.52). Cluster analysis and principal component based scatter plotting showed greater similarity among landraces from São Paulo, Mato Grosso do Sul and Amazonas, whereas those from Minas Gerais were clustered into a sub-group within this group. The plants from Mato Grosso, mostly collected in the municipality of General Carneiro, provided the highest differentiation. The migration of human populations is one among the possible reasons for this closer resemblance or greater disparity among plants from the various regions.

Wild Manihot species: botanical aspects, geographic distribution and economic value

A total of 98 Manihot species have been recognized in the genus. All of them are native to the tropics of the New World, particularly Brazil and Mexico. The cultigen, Manihot esculenta Crantz (cassava), grows throughout the lowland tropics. Wild species vary in growth habit from acaulescent or short shrubs to tree-like. Because of their adaptations to different conditions, they are gene reservoirs for tackling many abiotic and biotic stresses such as improving root quality and resistance to diseases. They have been used successfully by the first author for improving protein content, seed-fertility, apomixis, resistance to mealy bug, and tolerance to drought. A table of the most important species from an economic viewpoint is presented.

Anatomical alterations due to polyploidy in cassava, Manihot esculenta Crantz

Information on anatomical structure is needed by breeders working on improvement for drought tolerance. For studying the effect of polyploidy on cassava anatomy and its significance to tolerance to drought, we induced a polyploidy type of a selected clone (UnB 530) by applying an aqueous solution of 0.2% colchicine on lateral buds for a period of 12 h. The stem identified as tetraploid was propagated to produce the whole plant. Free-hand cross-sections of the median portion between stem internodes were made. They were clarified using 50% sodium hypochlorite solution, stained with 1% safranin-alcian blue, passed through an ethanol series and butyl acetate and mounted in synthetic resin. The tetraploid type showed more prismatic and druse crystals in the cortical parenchyma, and its pericycle fibers had thicker walls. The secondary xylem of tetraploid types was wider than diploid ones, having thinner walls and less starch.

Cassava genetic resources and their utilization for breeding of the crop

Wild cassava relatives are perennials and vary in growth pattern from nearly acaulescent subshrubs to small trees. They have been used as a source of useful characters such as high protein content, apomixis, resistance to mealybug and mosaic disease, and tolerance to drought. Indigenous clones are a potential source of beta-carotene and lycopene. Apomixis genes have been transferred to the crop successfully through interspecific hybridization, and apomictic clones arising from these hybrids are now being grown at the Universidade de Brasília. Interspecific hybrids produced earlier were polyploidized and had their fertility restored. Different useful types of chimera were also produced.

Cassava diversity in Brazil: the case of carotenoid-rich landraces

In Brazil, the center of cassava origin, cassava landraces have acquired through their domestication a large diversity in relation to many economic traits such as high content of carotenoids and excellent palatability among other characters. One of these clones, which has been grown by indigenous Brazilian farmers and is now being maintained in the University of Brasília gene bank, showed a high level of lycopene content (5 mg/kg viz. a viz. zero in common cultivars, and 12-20 mg/kg in tomato, a lycopene-rich vegetable). A second landrace called UnB 400 had a high content of b-carotene, which reached 4 mg/kg.

Amino acid profile in cassava and its interspecific hybrid

Cassava roots have a low-protein content (0.7-2%). Amino acids such as lysine and methionine are also low, and some research reports have indicated the absence of methionine. The amino acid profiles of a common cassava cultivar and an interspecific hybrid, namely ICB 300, were determined using the computerized amino acid analyzer Hitachi L-8500. The interspecific hybrid has 10 times more lysine and 3 times more methionine than the common cassava cultivar: lysine content was 0.010 g per 100 g in the common cassava cultivar while it reached 0.098 in the interspecific hybrid. Methionine in the common cassava cultivar was 0.014 g per 100 g whereas it reached 0.041 g per 100 g in the interspecific hybrid. Total amino acid content in the common cassava cultivar was 0.254 g per 100 g viz. a viz. 1.664 g per 100 g in the interspecific hybrid. The genetic variability of the profile and quantity of amino acids indicate the feasibility of selecting interspecific hybrids that are rich in both crude protein and amino acids. This is the first report of high true protein in cassava root.

Molecular analysis of apomixis in cassava

Cassava is the main staple for more than 800 million people in the tropics. It is propagated vegetatively by stem cuttings, which maintains superior genotypes but favors disease accumulation and spread. In this report, we present the results of the screening of the progeny and the second generation of the clone UnB 307 for apomixes using microsatellites. A total of 29 plants were screened, representing the maternal plant, its first and second generations that were left to open pollination. About 20% of the offspring were rated as genetically identical plants. This result confirms the facultative apomictic nature of cassava, with high environmental effect.

The synthesis of a new cassava-derived species, Manihot vieiri Nassar

A new species was synthesized artificially by chromosome doubling in a hybrid. The ensuing polyploid type exhibits an apomictic nature and maintains its morphological characteristics in the progeny. It showed a frequency of multiembryonic sacs of 29% in the ovules examined, whereas sacs were absent in the diploid type.

Cassava in South America, Brazil's contribution and the lesson to be learned from India

South America is responsible for about half of the cassava world production. In the 1970's productivity of the crop on the continent was about 15 ton/ha, and dropped continuously until reaching 12 ton/ha in 2004. India's productivity of cassava increased from 10 ton/ha in the 1970's to 28 ton/ha in 2004. Brazil contributed significantly to improving cassava crops through the Instituto Agronômico de Campinas in the 1960's and 1970's. The Universidade de Brasília released high-protein content hybrids, apomictic clones and explored the potential of indigenous landraces.

Breeding cassava for apomixis

Apomixis genes have been successfully transferred to cassava (Manihot esculenta) by hybridizing it with the wild species, M. glaziovii. The interspecific hybrid of cassava and M. glaziovii was exposed to open pollination during three subsequent generations. Seven sibs and the maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs from M. glaziovii proved to be identical when examined by the same microsatellite loci. This evidence leads to the conclusion that apomixis does occur in wild-cassava relatives and apparently has played an important role in Manihot speciation. This is the first report of nearly 100% apomixis.

Gene flow between cassava, Manihot esculenta Crantz, and wild relatives

Controlled and natural hybridization between cassava and wild relatives does occur. Barriers within the genus appear to be weak due to recent evolution of the group. All Manihot species examined cytogenetically have a chromosome number of 2n = 36. However, they behave meiotically as diploids. The weak interspecific barriers have led to an extremely heterozygous gene pool that may begin a sequence of hybridization followed by speciation. Introgression from cassava into a number of wild species (M. neusana, M. alutacea, M. reptans and M. anomala) has been detected by both morphological marker genes and molecular techniques. Winged fruit, setaceous bracteoles, and wide leaf sinus were dominant genes that came from cassava and appeared in the hybrids. The characteristic protein bands of cassava were recognized in the hybrid seed protein electrophoresis

Cassava, Manihot esculenta Crantz genetic resources: VI. Anatomy of a diversity center

Central Brasil is one of the four centers of diversity of Manihot species. Of 26 wild Manihot species reported to occur in this region, 20 were collected from two limited areas, each less than 100 km in diameter. These two areas are Goiás Velho and Corumbá de Goiás. Considering the Harlan concept of geographic patterns of variation of cultivated crops, it is assumed that the two areas are microcenters of cassava diversity. Topographic, soil and ethnological data of these species were collected and compared

Cassava, Manihot esculenta Crantz, genetic resources: origin of the crop, its evolution and relationships with wild relatives

About 98 species of Manihot are known. All of them are native to the New World and are concentrated in four regions in Brazil and Central America. All the Manihot species so far examined have 2n = 36 chromosomes. Interspecific hybrids between cassava and its wild relatives show relatively normal meiosis, and further generations can be obtained. Electrophoresis shows affinity among wild species of different sections, and between some of them and cassava. Both polyploidy and apomixis may have contributed to speciation in this genus. Polyploidy produced genetic variability, while apomixis is responsible for perpetuating new hybrid types adapted to different environments. Cassava may have originated by hybridization between two wild Manihot species, followed by vegetative reproduction of the hybrid

Apomixis and cassava

Apomixis means seed formation without fertilization. In cassava (Manihot esculenta) it is an alternative to reproduction by cuttings, which normally transmits pathogens and leads to an accumulation of viral and bacterial diseases. Apomixis also assures preservation of heterosis and avoids genetic segregation. It occurs in wild relatives of cassava and has been transferred successfully from Manihot glaziovii and M. neusana. It is facultative, and occurs at a low frequency, ranging from 1-2, and apparently is genetically different from apomixis in other crops. With selection, the frequency can reach 13. Apomixis in cassava is frequently associated with aneuploidy but it does occur in some diploid types. It is due to the formation of aposporic sacs, which can easily be detected by clearing tissue preparations. Apomixis appears to have played an important role in speciation during the evolution of Manihot, since it leads to the maintenance and perpetuation of sterile interspecific hybridization. The use of apomixis in cassava breeding could lead to a boom in line improvement and commercial production. In addition to preserving superior genotypes, avoiding contamination of new plants, it would enable international programs to export their germplasm to destination countries. This would allow the use of superior genotypes even if apomixis occurs at a low frequency. A scheme to maximize benefits is to use diploid apomictic clones as maternal parents, which can be crossed with pollinators of polyploid interspecific hybrids, followed by selection among the progeny of new apomictic types that combine the heteroses of both interspecific hybridization and polyploidy. In addition, they acquire favored genes that have been transferred from the wild to the commercial crop