Microsatellite variation and population structure in the “Refractario” cacao of Ecuador

Utilization of germplasm for crop improvement is often hampered by absence of information regarding origin, genetic identity and genealogical relationships of germplasm groups or populations. Molecular marker technology offers an efficient tool to verify or reconstruct passport data. Using a high-throughput genotyping system with 15 microsatellite loci, we fingerprinted 482 accessions in 48 putative half-sib families of Refractario cacao (a group of germplasm collected from nine farms in Ecuador). Based on the multilocus profiles, a Bayesian method for individual assignment was applied to verify membership in each half-sib family. Multivariate statistical analysis showed that the Refractario genetic profile was different from other groups tested, except for the “Nacional” cacao from the coastal valley of Ecuador. Hierarchical partitioning of genetic variance in the Refractario cacao showed that 76% of the variation was contributed by intra-family difference, whereas the inter-family and inter-farm difference accounted for 15 and 9% of total variance, respectively. All three sources of variation were highly significant (P < 0.01). Cluster and Principal Coordinates Analyses revealed a population sub-structure in Refractario, which was also highly heterozygous, suggesting hybridization derived from Nacional cacao and multiple other parental varieties, which all shared a similar genetic background. The improved understanding of identities and structure in Refractario cacao will contribute to more efficient conservation and use of this germplasm group in cacao breeding.     

Combining-ability for cacao (Theobroma cacao L.) yield components under southern Bahia conditions

The objective of this study was to assess five cacao cultivars (selfs) and 20 hybrids with regard to their general-and specific-combining ability for yield components using method 1, model I, of the diallel analysis system. The selfings and the hybrids were obtained through controlled crossings, tested in the field in a random block design with four replications and plots containing 16 plants. The experiment was set up in the Centro de Pesquisas do Cacau, in Itabuna, Bahia, Brasil, in 1975. The characteristics studied were: the number of healthy and collected fruits per plant (NHFP and NCFP), the weight of humid seeds per plant and per fruit (WHSP and WHSF), and the percentage of diseased fruits per plant (PDFP), for 5 years (1986–1990). The F-test values, highly significant for general combining ability (GCA) and specific combining ability (SCA), demonstrated the existence of variability for both effects. However, the effects of SCA were greater than those of GCA, when compared in terms of the average squared effects. This condition held for the characteristics NHFP, NCFP and WHSP, which shows the relative importance of the non-additive genetic effects over the additive effects. The reciprocal effects did not show significance. Breeding methods which explore the additive portion of genetic variance should be employed for obtaining higher-yielding cacao and high seed weight. For this, the segregant populations should involve cultivars CEPEC 1, SIAL 169 and ICS 1. Combinations involving the cultivar ICS 1 presented the most favorable results for the characteristics WHSP and WHSF, where the hybrid SIAL 169 x ICS 1 and its reciprocal were outstanding.     

Genetic diversity in cocoa revealed by cDNA probes

The variability of the cocoa (Theobroma cacao) nuclear genome was investigated. A total of 203 cocoa clones was surveyed for restriction fragment length polymorphisms (RFLPs) using four restriction endonuclease and 31 seed cDNA probes. A high level of polymorphism has been found. This study points to a structuring of the species that fits with the distinction between the Criollo and Forastero populations. These results combined with previously obtained nuclear rDNA and mtDNA data allow us to propose new hypotheses on the origin and evolution of the different cocoa populations.     

Theobroma cacao L.: a genetic linkage map and quantitative trait loci analysis

A genetic linkage map of Theobroma cacao (cocoa) has been constructed from 131 backcross trees derived from a cross between a single tree of the variety Catongo and an F1 tree from the cross of Catongo by Pound 12. The map comprises 138 markers: 104 RAPD loci, 32 RFLP loci and two morphologic loci. Ten linkage groups were found which cover 1068 centimorgans (cM). Only six (4%) molecular-marker loci show a significant deviation from the expected 11 segregation ratio.The average distance between two adjacent markers is 8.3 cM. The final genome-size estimates based on two-point linkage data ranged from 1078 to 1112 cM for the cocoa genome. This backcross progeny segregates for two apparently single gene loci controlling (1) anthocyanidin synthesis (Anth) in seeds, leaves and flowers and (2) self-compatibility (Autoc). The Anth locus was found to be 25 cM from Autoc and two molecular markers co-segregate with Anth. The genetic linkage map was used to localize QTLs for early flowering, trunk diameter, jorquette height and ovule number in the BC₁ generation using both single-point ANOVA and interval mapping. A minimum number of 2–4 QTLs (P<0.01) involved in the genetic expression of the traits studied was detected. Coincident map locations of a QTL for jorquette height and trunk diameter suggests the possibility of pleiotropic effects in cocoa for these traits. The combined estimated effects of the different mapped QTLs explained between 11.2% and 25.8% of the phenotypic variance observed in the BC₁ population.     

Assessing threats and mapping sandal resources to identify genetic ‘hot-spot’ for in-situ conservation in peninsular India

Santalum album L. is an economically important tropical tree species. Owing to extensive logging, changes in land-use patterns and poor natural regeneration, the natural sandal populations are rapidly dwindling. It is feared that such threats could easily undermine the genetic diversity of sandal populations. Effective measures to prevent such loss are prerequisite. We have developed baseline data on the levels of genetic diversity and its distribution on a geographic scale for sandal in India. Based on Geographic Information System, distribution map of sandal was developed and found that the populations were geographically more concentrated in the Deccan plateau. It was found that, for past 53 years, there was a monotonic decrease in the extraction of sandal in the state of Karnataka, India. Using allozyme markers, the genetic diversity of 19 sandal populations in peninsular India were determined and over all observed heterzygosity (H_o) was found to be 31%. The percent H_o was positively correlated with the density of the sandal populations (r = 0.44) and with increasing longitude it was found to be negatively correlated (r = −0.51). The dendrogram analysis indicated a clear clustering of sandal populations based on their geographic occurrence. The Deccan plateau populations were found to be genetically the most diverse and seemed to represent the ‘hot-spot’ of sandal genetic resources in peninsular India. Our results have important implications for the conservation strategies for sandal populations in peninsular India and can be applied for the conservation of other taxa as well.     

Abscisic acid metabolism and episodic growth in cocoa

Abscisic acid (ABA) levels and metabolism were investigated in relation to shoot growth in cocoa (Theobroma cacao L.). ABA levels were high (14 nmoles/g fwt) in young flush leaves during shoot growth but gradually declined during the subsequent dormant period. ABA levels were low (1–2 nmoles/g fwt) in mature leaves when the terminal bud re-initiated growth. ABA-glucose ester (ABA-glu) levels were low (3–4 nmoles/g fwt) in the flush leaves during shoot growth and dormancy, however, ABA-glu levels increased more than 7-fold in these same leaves during the next flush cycle. ABA-glu levels then dropped significantly during the dormant period. Radiolabeled-ABA was metabolized to three products in cocoa leaves: ABA-glu, phaseic acid and dihydrophaseic acid. Catabolism of radiolabeled ABA was significantly greater in mature leaves during the dormant period when endogenous levels of ABA were high as compared to the period of active shoot growth when endogenous levels of ABA in mature leaves were low.     

Genetic transformation of cocoa leaf cells using Agrobacterium tumefaciens

Leaf strips from cocoa tree (Theobroma cacao L.) clones ICS-16 and SIC-5 were cocultivated with the supervirulent Agrobacterium tumefaciens strain A281-Kan. A281-Kan contains a wild-type Ti plasmid and an additional plasmid, pGPTV-Kan, which confers kanamycin resistance to transformed plant cells after integration and expression of the neomycin phosphotransferase II (nptII) gene. Transformed cells were selected on callusing medium containing 100 g ml^-1 kanamycin. NptII assays confirmed that kanamycin-resistant cultures of ICS-16 and SIC-5 expressed the nptII gene, whereas control cultures did not. Genomic Southern blot analyses demonstrated single T-DNA insertions into ICS-16 and SIC-5. T-DNA/cocoa DNA border regions from transformed cultures were cloned and sequenced, revealing that in both transformed cell lines, the right T-DNA border was at the 5 end of the 25 bp right border repeat. Cocoa DNA probes from the T-DNA/cocoa DNA insertion sites were used in Southern blot analyses and showed that T-DNA from pGPTV-Kan had inserted into a unique region in ICS-16 and into a repetitive region in SIC-5. This study establishes that foreign genes can be inserted and expressed in cocoa using A. tumefaciens-mediated gene transfer.     

The vesicular-arbuscular (VA) mycorrhiza and its effects on growth of vegetatively propagated Theobroma cacao L.

A greenhouse experiment was conducted to study the effects of mycorrhizal inoculation on growth and nutrient uptake of vegetatively propagated cocoa plants from the Sabah hybrid (NA 33 × PA 7) raised on unsterilized Munchong soils. Inoculation with mixed species of Scutellospora and Glomus, resulted in higher dry matter yield and stem diameter of mycorrhizal plants obtained through budding, air layering or marcotting (a form of asexual plant propagation using vegetative parts and the selection of a healthy branch and scraping off the bark at the lower end of the branch, forcing it to root by covering it with a ball of soil) and stem cutting. Budded and marcotted mycorrhizal plants gave a significant increase in P content of shoots. In contrast, only budded mycorrhizal plants (on both young and old rootstocks) gave a significantly higher Ca concentration in tops, compared to uninoculated plants.     

Headspace volatile markers for sensitivity of cocoa (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) somatic embryos to cryopreservation

The mechanisms that reduce the viability of plant somatic embryos following cryopreservation are not known. The objective of the present study was to evaluate the sensitivity of cocoa (Theobroma cacao L.) somatic embryos at different stages of an encapsulation–dehydration protocol using stress-related volatile hydrocarbons as markers of injury and recovery. The plant stress hormone ethylene and volatile hydrocarbons derived from hydroxyl radicals (methane) and lipid peroxidation (ethane) were determined using gas chromatography headspace analysis. Ethylene and methane were the only volatiles detected, with both being produced after each step of the cryogenic protocol. Ethylene production was significantly reduced following exposure to liquid nitrogen, but then increased in parallel with embryo recovery. In contrast, the production of methane was cyclic during recovery, with the first cycle occurring earlier for embryos recovered from liquid nitrogen and desiccation than those recovered from earlier steps in the protocol. These results suggest that loss of somatic embryo viability during cryopreservation may be related to the oxidative status of the tissue, and its capacity to produce ethylene. This study has demonstrated that headspace volatile analysis provides a robust non-destructive analytical approach for assessing the survival and recovery of plant somatic embryos following cryopreservation.     

The genome of the tropical tree Theobroma cacao L.

Summary 2C values for angiosperms vary over 2500-fold and a positive correlation exists between C-value and latitude in herbaceous plants. Woody plants differ from herbaceous plants in chromosome size and C-value. In addition, tropical hardwoods have smaller chromosomes than other tropical plants and do not share the correlation of minimum generation time with genome size seen in herbaceous plants. Theobroma cacao is a tropical hardwood cultivated for its beans, which are used to make chocolate and cocoa butter. Its cytology is typical of the pantropical and subtropical family Sterculiaceae. Its small chromosomes, single secondary constriction, and lack of C-banding suggest a small genome. The genome size of T. cacao, measured by reassociation kinetics, is 2.01 x 10⁸, which is small compared to both temperate and tropical plants previously studied. We also provide data on the melting point, base composition, and relative extent of methylation (at sites most commonly methylated in higher plants), of T. cacao DNA.     

Isolation and characterization of first microsatellite markers for the noble crayfish, <Emphasis Type=Italic>Astacus astacus</Emphasis>

Noble crayfish (Astacus astacus) is listed as vulnerable by the IUCN Red List of Threatened Species in Europe. However, very little is known about genetic diversity and structuring of noble crayfish populations, mainly because of the lack of informative genetic markers. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4,000 recombinant clones. Eight loci revealed polymorphisms in a panel of 172 individuals from seven populations in Northern Europe. Number of alleles per locus ranged from two to 10 (average 4.4) and heterozygosity levels among populations varied from 0 to 0.80 for H _o and from 0 to 0.72 for H _e.     

Assessment of genetic diversity and population structure of Chinese wild almond, Amygdalus nana, using EST- and genomic SSRs

Amygdalus nana L., commonly known as wild almond, is an endangered wild relative of cultivated almond, which has great potential in almond crop breeding. In this study, we used microsatellite (SSR) loci derived from both expressed sequence tag (EST) and anonymous genomic sequence to explore the genetic diversity and population structure of A. nana in Xinjiang of China. Seven natural populations were collected across the whole distribution of A. nana in China, including populations from both inside (four populations) and outside (three populations) the established protected areas. A total of 22 and 19 alleles were detected from the seven pairs of EST and genomic SSR loci, respectively. Generally, the genomic SSRs showed lower levels of variation than EST-SSRs, which may partially due to the higher cross-species transferability in EST-SSRs than in genomic SSRs. The population-level genetic diversity (A = 1.84, P = 50.00%, H_o = 0.3491, H_E = 0.2271) was lower than cultivated almond and several wild fruit species with similar breeding system. Most of the genetic variation (82.16%) was partitioned within populations. In particular, the population collected from Tacheng County (outside the protected areas) had the highest levels of genetic diversity and had significantly different genetic constitution from other populations.     

Genetic diversity in the northernmost<Emphasis Type="Italic"> Oryza rufipogon</Emphasis> populations estimated by SSR markers

To estimate genetic diversity of the residual northern populations of Oryza rufipogon, a total of 232 individuals from six populations were analyzed using microsatellites (SSRs). The O. rufipogon populations with different status included three from Dongxiang (Jiangxi Province) and three from Chaling (Hunan Province) in China. The 23 rice SSR primer pairs selected from the RiceGenes Database detected a total of 115 alleles, indicating that all the SSR loci were polymorphic in this study. The total gene diversity was 0.919 in the six O. rufipogon populations, and the Donxiang populations showed higher diversity than the Chaling populations. More significant genetic differentiation and less gene flow were found among the Dongxiang populations than those from Chaling. The two putative introgressed populations showed relatively high genetic variation. One in situ conserved population from Dongxiang had the lowest level of genetic diversity. The re-introduced population from Chaling restored about 90% of the genetic variation, compared with the original source population. It is concluded from these results that a relatively high level of genetic variation resided in the northern O. rufipogon populations and continued efforts of conservation of these populations are needed; and that the conservation of some Chaling and Dongxiang populations has been effective in preventing gene flow from cultivated rice. Introgression of cultivated rice demonstrated significant impacts on genetic variability of the O. rufipogon populations, and should be carefully considered in conserving this wild rice. This study also suggested that re-introduction to its original habitats is an effective approach to restore O. rufipogon populations.Communicated by J.S. Heslop-Harrison     

Development and characterization of new microsatellite markers for ginseng (<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer)

Panax ginseng C.A. Meyer, commonly known as Korean or Asian ginseng, is a perennial herb native to Korea and China. Its roots are highly prized for several medicinal properties. The present study describes development and characterization of twenty-two polymorphic microsatellite markers for this species. A total of 99 alleles were detected with an average of 4.5 alleles per locus across 20 accessions. Values for observed (H _O ) and expected (H _E ) heterozygosities ranged from 0.05 to 1.00 and from 0.18 to 0.73, respectively. Eleven loci deviated from Hardy–Weinberg equilibrium (P < 0.001). Significant (P < 0.05) heterozygote deficiency was observed at 13 loci. Exact test for linkage disequilibrium showed significant values (P < 0.05) between 12 pairs of loci. These microsatellite markers provide powerful tools for understanding population and conservation genetics of this species and also for genetic differentiation and authentication of different Panax species being used in commercial ginseng products.     

Isolation of DNA from plants high in polyphenolics

DNA suitable for digestion with restriction endonucleases has been isolated fromTheobroma cacao, a plant high in polyphenolic compounds, and several other plant taxa using the method presented in this paper. The method relies on concentrating nuclei away from most of the cytoplasmic components and inhibiting the formation of oxidized polyphenolic compounds and their interaction with the DNA fraction in the remaining steps.     

Patterns of Allozyme Variation at Two Stages of the Life History in Wild Rice <Emphasis Type="Italic">Oryza rufipogon</Emphasis> and Conservation Genetic Implications

Oryza rufipogon Griff. occurs widely in aquatic ecosystem of tropics and subtropics of monsoon Asia as well as Southern China. It is a vital gene source for rice breeding programs. Many populations of the species, unfortunately, have drastically diminished because of the disappearance of aquatic habitats as a result of human disturbance. In order to determine patterns of genetic variation at two stages of the life-cycle in the wild rice species, we investigated allozyme variation of four natural populations in China. Two southern populations have significant asexual reproduction while two other northern marginal populations show a mixed reproduction in China. At 22 allozyme loci, a significantly lower genetic diversity was observed in the ratoons than in the seeds of the two southern populations, whereas a significantly higher genetic diversity was found in the ratoons than in the seeds of the two northern marginal populations. The results suggest that the variation of reproductive system is probably associated with their patterns of genetic variation in the species. Moreover, a significantly higher genetic differentiation among populations found in the ratoons than in the seeds may stem from pollen-mediated gene flow among them. Finally, we propose suggestions for conservation management of the endangered species.