Genetic variation of cassava (Manihot esculenta Crantz) cultivated by Chibchan Amerindians of Costa Rica

This study focuses on the genetic diversity of the cassava (Manihot esculenta Crantz) grown by the Chibchan Amerindians of Costa Rica. The authors collected cassava in various locations within two Amerindian Reserves: Talamanca, inhabited by Cabecares and Bribris, and Coto Brus, inhabited by Guaymi. Through the use of isozyme electrophoretic techniques we found variation for six out of nine systems analyzed, namely DIA, EST, IDH, MDH, PGI, and SKD. No variation was found in ADH, PGD, and PGM systems. We analyzed the distribution of variation within and between the reserves, and found most of the variation occurred within reserves (Gst = 0.1084). Only low levels of genetic differentiation were found between reserves (Nei’s genetic distance = 0.0088). The high levels of genetic variation within reserves that we found concur with results of previous studies on cassava grown by South American Amerindians and farmers. The role of the breeding system of cassava and the agricultural practices of Amerindians in the maintenance of high levels of genetic diversity are discussed.     

Biodiesel production from algal oil using cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) as feedstock

As a potential source of biomass supplies, cassava (Manihot esculenta Crantz) has been studied for bioethanol production, but not for the production of biodiesel. In this study, we used cassava hydrolysate as an alternative carbon source for the growth of microalgae (Chlorella protothecoides) which accumulated oil in vivo, with high oil content up to 53% by dry mass under a 5-L scale fermentation condition. The oils were extracted and converted into biodiesel by transesterification. The biodiesel obtained consisted of mainly unsaturated fatty acids methyl ester (over 82%), cetane acid methyl ester, linoleic acid methyl ester, and oleic acid methyl ester. This work suggests the feasibility of an alternative choice for producing biodiesel from cassava by microalgae fermentation. We report herewith the optimized condition for the fermentation and for the hydrolysis of cassava as the carbon source.     

Diversity of LTR-retrotransposons and <Emphasis Type="Italic">Enhancer/Suppressor</Emphasis><Emphasis Type="Italic">Mutator</Emphasis>-like transposons in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava (Manihot esculenta Crantz), though a major world crop with enormous potential, is very under studied. Little is known about its genome structure and organisation. Transposable elements have a key role in the evolution of genome structure, and can be used as important tools in applied genetics. This paper sets out to survey the diversity of members of three major classes of transposable element within the cassava genome and in relation to similar elements in other plants. Members of two classes of LTR-retrotransposons, Ty1/copia-like and Ty3/gypsy-like, and of Enhancer/Suppressor Mutator (En/Spm)-like transposons were isolated and characterised. Analyses revealed 59 families of Ty1/copia, 26 families of Ty3/gypsy retrotransposons, and 40 families of En/Spm in the cassava genome. In the comparative analyses, the predicted amino acid sequences for these transposon classes were compared with those of related elements from other plant species. These revealed that there were multiple lineages of Ty1/copia-like retrotransposons in the genome of cassava and suggested that vertical and horizontal transmission as the source of cassava Mecops may not be mutually exclusive. For the Ty3/gypsy elements network, two groups of cassava Megyps were evident including the Arabidopsis Athila lineage. However, cassava En/Spm-like elements (Meens) constituted a single group within a network of plant En/Spm-like elements. Hybridisation analysis supported the presence of transposons in the genome of cassava in medium (Ty3/gypsy and En/Spm) to high (Ty1/copia) copy numbers. Thus the cassava genome was shown to contain diverse members of three major classes of transposable element; however, the different classes exhibited contrasting evolutionary histories.     

Batch ethanol production from cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz.) flour using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells immobilized in calcium alginate

The objective of this research was to saccharify cassava flour by acid-acid and acid-enzyme hydrolysis and further conversion of the resulting sugar into ethanol by fermenting with the immobilized (in Ca-alginate) cells of Saccharomyces cerevisiae. The saccharification resulted in higher total sugar recovery by acid-enzyme hydrolysis (72.88 %) than by enzyme-enzyme hydrolysis (58.1 %). Further study on ethanol production was carried out using the hydrolysate obtained from acid-enzyme hydrolysis. The growth of the yeast started in the log phage and maximum ethanol (189?±?3.1 g ethanol/kg flour) production was achieved with 94.74?±?2.187 % sugar conversion during the stationary phase.     

Phosphomannose-isomerase (<Emphasis Type="Italic">pmi</Emphasis>) gene as a selectable marker for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of rapeseed

Nodal segments (4 ± 1 mm long) of Hibiscus moscheutos (hardy hibiscus) were excised from in vitro proliferating microshoots and utilized to evaluate initial factors involved in bulk alginate encapsulation. The factors evaluated were; storage vessel type, volume and multiple rinse effects of CaCl₂ solutions, and sodium alginate concentrations (2.5, 2.75, 3.0 or 3.25%) for bulk alginate encapsulation. Results indicate that vessels utilized for bulk alginate encapsulation should have a lower base area (L × W) to height ratio to reduce the amount of alginate matrix shrinkage resulting in exposure of nodal segments. Increased volumes and multiple 50 mM CaCl₂ solution rinses did not have an effect on alginate solidification. Shoot length, root number, and root length significantly decreased in a linear manner from nodal explants stored for 4 weeks with increasing concentrations of sodium alginate. This research suggests an innovative technique for alginate encapsulation of H. moscheutos utilizing bulk methods as an alternative to single bead alginate encapsulation.     

Proteome characterization of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) somatic embryos,plantlets and tuberous roots

Background  

Proteomics is increasingly becoming an important tool for the study of many different aspects of plant functions, such as investigating the molecular processes underlying in plant physiology, development, differentiation and their interaction with the environments. To investigate the cassava (Manihot esculenta Crantz) proteome, we extracted proteins from somatic embryos, plantlets and tuberous roots of cultivar SC8 and separated them by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

A simple hydroponic hardening system and the effect of nitrogen source on the acclimation of <Emphasis Type="Italic">in vitro</Emphasis> cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Plant tissue culture technology is being widely used for large-scale, rapid, clonal multiplication and genetic transformation in cassava. The main limitation of this technology is the period of acclimation of the fragile in vitro plants after they have been multiplied or regenerated. Most losses of in vitro plants occur when the plantlets are moved directly from the test tubes to the ex vitro conditions. Our aim was to design a simple, rapid, low-maintenance hydroponic system to improve survival rate of transplanting to the ex vitro conditions through the rapid acclimation process of in vitro plants. In this paper, we have developed a simple hydroponic system to accelerate the cassava acclimation and multiplication process. This system considerably increased the survival percentage of in vitro and/or transgenic lines and reduces the time requirement for multiplication by hydroponic acclimation. In order to assess the effectiveness of the acclimation of seedlings on their establishment, we analyzed plant growth and field survival rate with response to different nitrogen (N) sources using different cassava accessions. Nitrogen sources of NO₃ ^? and NH₄NO₃ increased plant growth and root length compared to NH₄ ⁺ alone, or water treatments. The greenhouse and field survivability of N-hardened plants, including transgenic lines, were significantly different in growth and development. We present a simple NO₃ ^? hydroponic acclimation system that can be quickly and cheaply constructed and used by the cassava community around the world. The efficiency of our proposed N hydroponic acclimation system is validated in the transgenic development pipeline which will enhance the cassava molecular breeding.     

A Multiplex Microsatellite Marker Kit for Diversity Assessment of Large Cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) Germplasm Collections

Current methods for molecular fingerprinting of cassava (Manihot esculenta Crantz) have limited throughput or are costly, thus preventing the characterization of large germplasm collections such as those held by the International Agricultural Research Centers or National Research Institutions, which comprise hundreds to thousands of accessions. Here, we report the development of a fluorescence-based multiplex simple sequence repeat (SSR) marker kit that enables accurate and cost-effective cassava fingerprinting. The kit comprises 16 SSR markers assembled into five multiplex panels and was tested on 21 cassava cultivars alongside one accession of Manihot epruinosa, a wild relative. A total of 68 alleles were detected with, on average, 4.25 alleles per locus and a polymorphism information content of 0.53. The marker kit reported here is comparable to previously published amplified fragment length polymorphism and SSR marker systems in terms of discriminating power and informativeness while offering significant advantages in speed and cost of marker analysis. Previous molecular genetic diversity studies have suggested that cassava germplasm collections contain duplicate entries based on the occurrence of identical genetic profiles. Using the newly developed microsatellite kit, three out of six putative duplicate accessions could be readily differentiated, showing that these are distinct genotypes. The relevance of these findings with respect to the characterization and management of large cassava germplasm collections is discussed.     

Candidate gene sequencing and validation of SNP markers linked to carotenoid content in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava is a widely grown staple in Sub-Saharan Africa and consumed as a cheap source of calories, but the crop is deficient in micronutrients including pro-vitamin A carotenoids. This challenge is currently being addressed through biofortification breeding that relies on phenotypic selection. Gene-based markers linked to pro-vitamin A content variation are expected to increase the rate of genetic gain for this critical trait. We sequenced four candidate carotenoid genes from 167 cassava accessions representing the diversity of elite breeder lines from IITA. Total carotenoid content was determined using spectrophotometer and total β-carotene was quantified by high-performance liquid chromatography. Storage root yellowness due to carotenoid pigmentation was assessed. We carried out candidate gene association analysis that accounts for population structure and kinship using genome-wide single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing. Significant SNPs were used to design competitive allele-specific PCR assays and validated on the larger population for potential use in marker-assisted selection breeding. Candidate gene sequencing of the genes β-carotene hydroxylase (crtRB), phytoene synthase (PSY2), lycopene epsilon cyclase (lcyE), and lycopene beta cyclase (lcyB) yielded a total of 37 SNPs. Total carotenoid content, total β-carotene, and color parameters were significantly associated with markers in the PSY2 gene. The SNPs from lcyE were significantly associated with color while those of lcyB and crtRB were not significantly associated with carotenoids or color parameters. These validated and breeder-friendly markers have potential to enhance the efficiency of selection for high β-carotene cassava, thus accelerating genetic gain.     

The analysis of candidate genes and loci involved with carotenoid metabolism in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) using SLAF-seq

Carotenoids in cassava storage roots play important roles in benefiting people’s health in the tropics because they provide essential nutrients and antioxidants. Although the related genes and loci associated with carotenoid metabolism in many species are well reported, in cassava they are poorly understood. In the present study, GWAS base on SLAF-seq was used in detecting the related genes and loci correlated to carotenoid contents in 98 accessions from a cassava F₁ mapping population. The 98 accessions were divided into four subgroups. On the basis of general linear and compressed linear models, 144 genes were detected by selective sweep analysis, and 84 SNPs and 694 genes were detected by association mapping, in which Manes.04G164700 (XanDH) and Manes.11G105300 (AAO) were probably involved in the downstream pathway of carotenoid metabolism, and their expressions in six cassava genotypes were confirmed. Our results will be useful in yellow-root cassava variety improvement and provide the most effective and sustainable approach to maximize the nutritional and health benefits of carotenoid to a large number of populations.     

Construction of a genetic linkage map using simple sequence repeat markers from expressed sequence tags for cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava (Manihot esculenta) is an economically important crop that is grown in tropical and sub-tropical regions. Use of molecular technology for genetic improvement of cassava has been limited by the lack of a large set of DNA markers and a genetic map. Therefore, the aims here were to develop additional simple sequence repeat (SSR) markers from the public expressed sequence tags (ESTs), and to construct a genetic linkage map. In this study, we designed 425 EST-SSR markers from sequences obtained from the cassava EST database in GenBank, and integrated them with 667 SSR markers from a microsatellite-enriched genomic sequence received from the International Center for Tropical Agriculture (CIAT). Of these, 107 EST-SSR and 500 genomic SSR primer pairs showed polymorphic patterns when screened in two cassava varieties, Hauy Bong 60 and Hanatee, which were used as female and male parental lines, respectively. Within the 107 and 500 primer pairs, 81 and 226 EST-SSR and SSR primer pairs were successfully genotyped with 100 samples of F₁ progeny, respectively. The results showed 20 linkage groups consisting of 211 markers—56 EST-SSR and 155 SSR markers—spanning 1,178 cM, with an average distance between markers of 5.6 cM and about 11 markers per linkage group. These novel EST-SSR markers provided genic PCR-based co-dominant markers that were useful, reliable and economical. The EST-SSRs were used together with SSR markers to construct the cassava genetic linkage map which will be useful for the identification of quantitative trait loci controlling the traits of interest in cassava breeding programs.     

New species in <Emphasis Type="Italic">Barleria</Emphasis> sect. <Emphasis Type="Italic">Stellatohirta</Emphasis> (<Emphasis Type="Italic">Acanthaceae</Emphasis>) from Africa

Summary  Three new species are described in Barleria L. sect. Stellatohirta M. Balkwill from tropical Africa: B. aristata from south-central Tanzania, B. aenea from south-western Tanzania and northeast Zambia, and B. purpureotincta from south-western Zambia. Their affinities and conservation status are discussed.     

Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop’s biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55 296 clones with an insert size range of 40–150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25–250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2–3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.     

Leaf proteomic analysis in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>, Crantz) during plant development,from planting of stem cutting to storage root formation

Tuberization in cassava (Manihot esculenta Crantz) occurs simultaneously with plant development, suggesting competition of photoassimilate partitioning between the shoot and the root organs. In potato, which is the most widely studied tuber crop, there is ample evidence suggesting that metabolism and regulatory processes in leaf may have an impact on tuber formation. To search for leaf proteins putatively involved in regulating tuber generation and/or development in cassava, comparative proteomic approaches have been applied to monitor differentially expressed leaf proteins during root transition from fibrous to tuberous. Stringent cross comparison and statistical analysis between two groups with different plant ages using Student’s t test with 95% significance level revealed a number of protein spots whose abundance were significantly altered (P < 0.05) during week 4 to week 8 of growth. Of these, 39 spots were successfully identified by ion trap LC–MS/MS. The proteins span various functional categories from antioxidant and defense, carbohydrate metabolism, cyanogenesis, energy metabolism, miscellaneous and unknown proteins. Results suggested possible metabolic switches in the leaf that may trigger/regulate storage root initiation and growth. This study provides a basis for further functional characterization of differentially expressed leaf proteins, which can help understand how biochemical processes in cassava leaves may be involved in storage root development.     

Initiation of embryogenic cell suspensions of taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> var. <Emphasis Type="Italic">esculenta</Emphasis>) and plant regeneration

Embryogenic callus was initiated by culturing in vitro taro corm slices on agar-solidified half-strength MS medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by transfer to 1.0 mg/L thidiazuron (TDZ). Callus was subsequently proliferated on solid medium containing 1.0 mg/L TDZ, 0.5 mg/L 2,4-D and 800 mg/L glutamine before transfer to liquid medium containing the same components but with reduced glutamine (100 mg/L). After 3 months in liquid culture on an orbital shaker, cytoplasmically dense cell aggregates began to form. Somatic embryogenesis was induced by plating suspension cells onto solid media containing reduced levels of hormones (0.1 mg/L TDZ, 0.05 mg/L 2,4-D), high concentrations of sucrose (40–50 g/L) and biotin (1.0 mg/L). Embryo maturation and germination was then induced on media containing 0.05 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-acetic acid (IAA). Histological studies of the developing embryos revealed the presence of typical shoot and root poles suggesting that these structures were true somatic embryos. The rate of somatic embryos formation was 500–3,000 per mL settled cell volume while approximately 60% of the embryos regenerated into plants.     

Somatic embryogenesis,organogenesis and plant regeneration in taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> var. <Emphasis Type="Italic">esculenta</Emphasis>)

Callus was initiated in three different “esculenta” taro cultivars by culturing corm slices in the dark on half-strength MS medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by subculture of all corm slices to half-strength MS medium containing 1.0 mg/l thidiazuron (TDZ). Depending on the cultivar, 20–30% of corm slices produced compact, yellow, nodular callus on media containing TDZ. Histological studies revealed the presence of typical embryogenic cells which were small, isodiametric with dense cytoplasms. Somatic embryos formed when callus was transferred to hormone-free medium and ~72% of the embryos germinated into plantlets on this medium. Simultaneous formation of roots and shoots during germination, and the presence of shoot and root poles revealed by histology, confirmed that these structures were true somatic embryos. Plants derived from somatic embryos appeared phenotypically normal following 2 months growth in a glasshouse. This method is a significant advance on those previously reported for the esculenta cultivars of taro due to its efficiency and reproducibility.     

Two new species and a new combination in <Emphasis Type="Italic">Elaphoglossum</Emphasis> sect. <Emphasis Type="Italic">Polytrichia</Emphasis> subsect. <Emphasis Type="Italic">Apoda</Emphasis> (Dryopteridaceae) from Costa Rica and Panama

Two new species, Elaphoglossum alvaradoanum and E. pendulum, are described. The first species is endemic to Cocos Island, and the second to the mountains of Costa Rica. Elaphoglossum auripilum var. longipilosum is elevated to species rank as E. longipilosum.     

Arbutoid mycorrhizas of the genus <Emphasis Type="Italic">Cortinarius</Emphasis> from Costa Rica

Arbutoid mycorrhizas of Comarostaphylis arbutoides (Arbutoidea, Ericaceae) from neotropical montane forests are rarely described. To date, only mycorrhizal associations with the fungal species Leccinum monticola, Leotia lubrica and Sebacina sp. are known from literature. The genus Cortinarius is one of the most species-rich ectomycorrhizal taxa with over 2000 assumed species. In this study, two sites in the Cordillera de Talamanca of Costa Rica were sampled, where Com. arbutoides is endemic and grows together with Quercus costaricensis. Using a combined method of rDNA sequence analysis and morphotyping, 33 sampled mycorrhizal systems of Cortinarius were assigned to the subgenera Dermocybe, Phlegmacium and Telamonia. Specific plant primers were used to identify the host plant. Here, we present the phylogenetic data of all found Cortinarii and describe four of the arbutoid mycorrhizal systems morphologically and anatomically.