Cacao domestication II: progenitor germplasm of the Trinitario cacao cultivar

Cacao (Theobroma cacao L.) has been cultivated in Central America since pre-Columbian times. The type of cacao cultivated in this region was called Criollo; cacao populations from the Amazon basin were called Forastero. The type of Forastero most commonly cultivated until 1950 was named Amelonado. Historical data show Trinitario cacao to have originated in Trinidad, resulting from natural hybridisation between Criollo and Amelonado Forastero. Doubts persist on the source of the Amelonado Forastero involved in the origin of Trinitario; the Amelonado parent may have come from the Lower Amazon, the Orinoco or the Guyanas. Most of the cacao cultivated worldwide until 1950 consisted of Criollo, Trinitario and Amelonado. From the early 1950s, Forastero material collected in the Upper Amazon region during the 1930s and 1940s began to be employed in breeding programmes. To gain a better understanding of the origin and the genetic basis of the cacao cultivars exploited before the utilisation of germplasm collected in the Upper Amazon, a study was carried out using restriction fragment length polymorphism and microsatellite markers. Trinitario samples from 17 countries were analysed. With molecular markers, it was possible to clearly identify three main genotypes (represented by clones SP1, MAT1-6 and SIAL70) implicated in the origin of most Trinitario clones.     

Genetic diversity of naturalized cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) in Puerto Rico

Identification of genetically diverse cacao with disease resistance, high productivity, and desirable organoleptic traits is vitally important to the agricultural crop’s long-term sustainability. Environmental changes, pests, and diseases as well as nation’s sovereign property rights have led to a decrease in accessibility and exchange of germplasm of interest. Having been introduced during colonial times, naturalized cacao in Puerto Rico could serve as an unexplored source of genetic diversity in improvement programs. An island-wide survey was carried out to identify naturalized trees and to determine their genetic associations to reference cacao accessions. Samples were genotyped with Expressed Sequence Tag-derived single nucleotide polymorphism (SNP) markers. Principal coordinate, cluster, and population structure analysis using the genotype data for both local and reference samples assigned individuals into five distinct genetic backgrounds: Criollo, Trinitario, Amelonado, Upper Amazon Forastero (UAF), and Nacional. Puerto Rican cacao fit into four (Criollo, Trinitario, Amelonado and UAF) of the five genetic backgrounds, being mainly composed of individuals of Criollo ancestry. Based on historical evidence, cacao of Criollo background was probably brought to Puerto Rico from Venezuela and/or Central America during colonial times. Trinitario, Amelonado, and UAF genetic backgrounds are most likely products of more modern introductions. Genotyping cacao in Puerto Rico provides information on the history and possible origin of the naturalized trees on the island. In addition, the assessment has allowed the targeting of material for incorporation and long-term conservation filling gaps in the existing collection and providing new germplasm to be evaluated for agronomic performance.     

FURTHER OBSERVATIONS ON THE PATHOGENICITY OF CALONECTRIA RIGIDIUSCULA (BERK. & BR.) SACC. TO THEOBROMA CACAO L

Quantitative data are provided for the occurrence of Calonectria rigidiuscula and other fungi in occluded Mirid lesions and other sites in shoots of cacao plants. Mirid lesions appear to be particularly favourable for the development of C. rigidiuscula. The fungus also occurs as a saprophyte on cacao pods, and as a wound parasite in woody plants other than cacao; it was successfully introduced into plants of varied affinities by inoculation. C. rigidiuscula spreads from inoculations in cacao stems much more rapidly than other fungi. The results confirm that it is the most important fungus infecting Mirid lesions and causing dieback of cacao in West Africa.
Inoculation tests with a range of Amelonado and Trinitario clones suggested that the clones vary in their susceptibility to C. rigidiuscula , but the plant-to-plant variation was too great to conclude that any one is highly resistant. Various types of introduced cacao were also tested; preliminary experiments indicate that certain types of Upper Amazon cacao may be resistant, but they need further investigation.     

Genetic diversity and structure of farm and GenBank accessions of cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) in Cameroon revealed by microsatellite markers

The genetic diversity of 400 accessions collected in cacao farms, 95 GenBank, and 31 reference accessions was analyzed using the 12 microsatellite markers. The GenBank and reference accessions were subdivided into 12 accession groups (AG) that belong to the traditional cacao genetic groups (GG) Lower Amazon Forastero (LA), Upper Amazon Forastero (UA), Trinitario, and Criollo (Cr). The 12-microsatellite loci revealed a total of 125 alleles, 113 of which were present in the farm accession group (FA). The within and between group variation for all AGs accounted respectively for 81% and 19% of the total molecular variation. The average F _is for the FA was 0.15 suggesting a moderate level of inbreeding. Significant differences for the level of gene diversity were found between the farm (0.50), GenBank (0.42 to 0.62), and reference (0.10 to 0.60) AGs. Genetic differentiation among AGs was variable with F _st values varying between 0.14 and 0.57 for the different AGs. Analysis using a Bayesian model-based method showed the existence of a high level of admixture for the farm accessions group. The LA genes were most represented in the FA (54%), followed by UA (33%) and Cr (7%). The genes of LA were also the most represented in the GenBank (48%), followed by UA (24%) and Cr (14%). Only 14% and 6% of the genes of the GenBank and farm accessions, respectively, could not be attributed to any of the reference GGs. The results suggest the predominating presence of LA genes in the Cameroon farm accessions and a high level of admixture, with apparent presence of genes of more than three GGs in most accessions. The traditional Trinitario types appear to have almost disappeared from farmers fields. The admixture must be the result of hybridization and recombination of these genes from the different GGs in seed gardens and in farmers’ fields. The use of selected farm accessions will depend on the GG that it belongs to and also on their level of heterozygosity. Further implications of the results for breeding and for introduction of new germplasm into the Cameroon GenBank are discussed.     

Complex origin of Trinitario-type Theobroma cacao (Malvaceae) from Trinidad and Tobago revealed using plastid genomics

Trinidad and Tobago has a long history of producing high-quality cacao (Theobroma cacao L.). Cacao genotypes in Trinidad and Tobago are of a highly distinctive kind, the so-called “Trinitario” cultivar group, widely considered to be of elite quality. The origin of Trinitario cacao is unclear, although it is generally considered to be of hybrid origin. We used massive parallel sequencing to identify polymorphic plastidic single nucleotide polymorphisms (cpSNPs) and polymorphic plastidic simple sequence repeats (cpSSRs) in order to determine the origin of the Trinitario cultivar group by comparing patterns of polymorphism to a reference set of ten completely sequenced chloroplast genomes (nine T. cacao and one outgroup, T. grandiflorum (Willd. ex Spreng.) Schum). Only three cpSNP haplotypes were present in the Trinitario cultivars sampled, each highly distinctive and corresponding to reference genotypes for the Criollo (CRI), Upper Amazon Forastero (UAF) and Lower Amazon Forastero (LAF) varietal groups. These three cpSNP haplotypes likely represent the founding lineages of cacao to Trinidad and Tobago. The cpSSRs were more variable with eight haplotypes, but these clustered into three groups corresponding to the three cpSNP haplotypes. The most common haplotype found in farms of Trinidad and Tobago was LAF, followed by UAF and then CRI. We conclude that the Trinitario cultivar group is of complex hybrid origin and has derived from at least three original introduction events.     

Field experiments on the resistance of cocoa to cocoa swollen-shoot virus

Progenies from crosses between different Upper Amazon cocoa types and between Upper Amazon and Amelonado were compared for their field resistance to infection with cocoa swollen-shoot virus (CSSV) virulent strain A. Among the intra-Amazon progenies, those from crosses of Scavina, Iquitos and Nanay groups showed most resistance. Progenies from crosses within these groups may have sufficient resistance to be of immediate practical value in reducing crop losses in areas where CSSV is widespread. Some Nanay progenies were more resistant than others and this provides scope for improvement by breeding. Progeny of crosses between Upper Amazon and Amelonado parents were less resistant than those from intra-Amazon crosses. The present results confirm those previously obtained in gauze-house tests on young plants.     

Tracing the native ancestors of the modern Theobroma cacao L. population in Ecuador

The native Theobroma cacao L. population from Ecuador, known as Nacional, is famous for its fine cocoa flavour. From the beginning of the twentieth century, however, it has been subjected to genetic erosion due principally to successive introductions of foreign germplasm whose hybrid descendants gradually replaced the native plantations, implying a decrease in cocoa quality. We attempted to trace this native cacao within a wide pool of modern Ecuadorian cacao population. Three hundred and twenty-two cacao accessions collected from different geographical areas along the pacific coast of Ecuador and maintained in two living collections were analysed using 40 simple-sequence repeat markers. Most of Ecuadorian cacao accessions displayed a high diversity and heterozygosity level. A factorial analysis of correspondence (FAC) showed a continuous variation among them, with a few ones, grouped at an extreme side of the FAC cloud, showing higher levels of homozygosity and lower introgression level by foreign cacaos. A paternity analysis revealed that these highly homozygous individuals are the most probable ancestors of the modern Nacional hybrid pool. These particular accessions studied could represent the native Nacional cacao present in Ecuador before the foreign introductions. Their identification will help to conserve valuable genetic material and to improve cocoa quality in new cacao varieties.     

Molecular characterization of an international cacao collection using microsatellite markers

Plant germplasm collections invariably contain varying levels of genetic redundancy, which hinders the efficient conservation and utilization of plant germplasm. Reduction of genetic redundancies is an essential step to improve the accuracy and efficiency of genebank management. The present study targeted the assessment of genetic redundancy and genetic structure in an international cacao (Theobroma cacao L.) collection maintained in Costa Rica. A total of 688 cacao accessions maintained in this collection were genotyped with 15 simple sequence repeat (SSR) loci, using a capillary electrophoresis genotyping system. The SSR markers provided a high resolution among the accessions. Thirty-six synonymously labeled sets, involving 135 accessions were identified based on the matching of multilocus SSR profiles. After the elimination of synonymous sets, the level of redundancy caused by closely related accessions in the collection was assessed using a simulated sampling scheme that compared allelic diversity in different sample sizes. The result of the simulation suggested that a random sample of 113 accessions could capture 90% of the total allelic diversity in this collection. Principal Coordinate Analysis revealed that the Trinitario hybrids from Costa Rica shared a high similarity among groups as well as among individual accessions. The analysis of the genetic structure illustrated that the within-country/within-region difference accounted for 84.6% of the total molecular variation whereas the among-country/among-region difference accounted for 15.4%. The Brazilian germplasm contributed most to this collection in terms of total alleles and private alleles. The intercountry/interregion relationship by cluster analysis largely agreed with the geographical origin of each germplasm group and supported the hypothesis that the Upper Amazon region is the center of diversity for cacao. The results of the present study indicated that the CATIE International Cacao Collection contains a high level of genetic redundancy. It should be possible to rationalize this collection by reducing redundancy and ensuring optimal representation of the genetic diversity from distinct germplasm groups. The results also demonstrated that SSR markers, together with the statistical tools for individual identification and redundancy assessment, are technically practical and sufficiently informative to assist the management of a tropical plant germplasm collection.     

A new cacao linkage map based on codominant markers: development and integration of 201 new microsatellite markers

A linkage map of cacao based on codominant markers has been constructed by integrating 201 new simple sequence repeats (SSR) developed in this study with a number of isoenzymes, restriction fragment length polymorphisms (RFLP), microsatellite markers and resistance and defence gene analogs (Rgenes-RFLP) previously mapped in cacao. A genomic library enriched for (GA)_n and (CA)_n was constructed, and 201 new microsatellite loci were mapped on 135 individuals from the same mapping population used to establish the first reference maps. This progeny resulted from a cross between two heterozygous cacao clones: an Upper-Amazon Forastero (UPA 402) and a Trinitario (UF 676). The new map contains 465 markers (268 SSRs, 176 RFLPs, five isoenzymes and 16 Rgenes-RFLP) arranged in ten linkage groups corresponding to the haploid chromosome number of cacao. Its length is 782.8 cM, with an average interval distance between markers of 1.7 cM. The new microsatellite markers were distributed throughout all linkage groups of the map, but their distribution was not random. The length of the map established with only SSRs was 769.6 cM, representing 94.8% of the total map. The current level of genome coverage is approximately one microsatellite every 3 cM. This new reference map provides a set of useful markers that is transferable across different mapping populations and will allow the identification and comparison of the most important regions involved in the variation of the traits of interest and the development of marker-assisted selection strategies.Communicated by H. Nybom     

Chloroplast microsatellite primers for cacao (Theobroma cacao) and other Malvaceae

? Premise of the study: Chloroplast microsatellites were developed in Theobroma cacao to examine the genetic diversity of cacao cultivars in Trinidad and Tobago. ? Methods and Results: Nine polymorphic microsatellites were designed from the chloroplast genomes of two T. cacao accessions. These microsatellites were tested in 95 hybrid accessions from Trinidad and Tobago. An average of 2.9 alleles per locus was found. ? Conclusions: These chloroplast microsatellites, particularly the highly polymorphic pentameric repeat, were useful in assessing genetic variation in T. cacao. In addition, these markers should also prove to be useful for population genetic studies in other species of Malvaceae.     

Development of EST-SSR markers through de novo RNA sequencing and application for biomass productivity in kenaf (<Emphasis Type="Italic">Hibiscus cannabinus</Emphasis> L.)

Kenaf is a multipurpose crop, but a lack of genetic information hinders genetic and molecular research. In this study, we aimed to develop EST-SSR markers from mutant and wild-type cultivars, and to assess the genetic diversity of the kenaf resources. A total of 33 Gb of sequence data comprising 130,480 unigenes was assembled by de novo RNA-sequencing of six kenaf cultivars, and 5619 SSRs were identified. Tri-nucleotide motifs occurred most frequently (82.67%) followed by di-, tetra-, and penta-motifs. In total, 515 polymorphic EST-SSRs were derived by pairwise comparisons of the cultivars based on in silico analyses. Of these, 70 markers were successfully validated among six cultivars. We used the six cultivars, together with 39 kenaf accessions from worldwide to assess genetic diversity and to characterize the EST-SSRs. The number of alleles per locus ranged from 2 to 8. PIC and genetic diversity values ranged from 0.08 to 0.79 and 0.08–0.82, respectively. The phylogenetic and population structure showed that the 45 accessions could be clearly divided into three groups based on different days to flowering (DTF). Genetic differentiation among the DTF groups showed a proportionally high level of variance. Association analysis between the DTF and the markers revealed three significant associations. Furthermore, using a multiplex PCR with three markers, DTF could be perfectly discriminated. These markers will be useful in marker-assisted selection after further validation with segregating populations of kenaf.     

Molecular Characterization of Cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis>) Germplasm from Jamaica Using Single Nucleotide Polymorphism (SNP) Markers

Cacao is an economically important commodity in Jamaica. Knowledge of the genetic diversity of Jamaican cacao germplasm is essential for their conservation and management. In spite of cacao’s economic importance in Jamaica, the crop is under studied, therefore limiting sound decisions toward improving productivity. Assessment of germplasm and on-farm genetic diversity is required to assist selecting superior genotypes to propagate and distribute across the island, as well as to use them as parental clones in breeding programs. Using 94 single nucleotide polymorphism (SNP) markers, 140 Jamaican cacao samples from two germplasm collections and a farmer’s estate along with 150 reference samples were analyzed. The principal coordinate analysis demonstrated that the majority of the Jamaican cacao selections were hybrids derived from five original germplasm groups, including Criollo, Amelonado and three Upper Amazon Forastero groups. Among the Upper Amazon groups, the Bayesian clustering analysis revealed that the Parinari (PA) ancestral lineage contributed the most (29.9%) to the Jamaican cacao germplasm. The germplasm collections showed greater diversity in terms of ancestral contributions compared to the farmer’s estate. However, the genetic differentiation between the three collecting sites was small (Fst?=?0.036), indicating that samples collected from the three sites were derived from a common pool of germplasm. The current study supports the historical records and clarified the ancestry of Jamaican cacao. Although the majority of the cacao genetic groups were observed in the Jamaican cacao collections, several diversity gaps were found in both germplasm collections and in the farmer’s estate, especially germplasm with disease resistance to cacao frosty pod rot that was recently found in Jamaica.     

Evaluation and use of a screening method to aid selection of cocoa (Theobroma cacao) with field resistance to cocoa swollen-shoot virus in Ghana

Seeds of cocoa progenies were inoculated with cocoa swollen-shoot virus manually or by vector transfer techniques to assess the relative resistance of the progenies to infection. Assessment of progeny included in both inoculation tests and in field trials were positively correlated, indicating that the inoculation technique is suitable for the selection of progenies for testing in the field. Pure Upper Amazon progenies were more resistant than selfed Amelonado, with hybrids between Amelonado and Upper Amazons usually intermediate. Among the main Upper Amazon populations, Iquitos and Nanay clones were better sources of resistance than Parinari and Scavina clones. A survey of these populations indicated that, within populations, resistance levels do not vary greatly. A range of progenies based on the Upper Amazon female parents in existing seed gardens was screened for resistance and some were consistently more resistant than the equivalent Amazon x Amelonado hybrid now being distributed to farmers.     

Geographic and genetic population differentiation of the Amazonian chocolate tree (Theobroma cacao L)

Numerous collecting expeditions of Theobroma cacao L. germplasm have been undertaken in Latin-America. However, most of this germplasm has not contributed to cacao improvement because its relationship to cultivated selections was poorly understood. Germplasm labeling errors have impeded breeding and confounded the interpretation of diversity analyses. To improve the understanding of the origin, classification, and population differentiation within the species, 1241 accessions covering a large geographic sampling were genotyped with 106 microsatellite markers. After discarding mislabeled samples, 10 genetic clusters, as opposed to the two genetic groups traditionally recognized within T. cacao, were found by applying Bayesian statistics. This leads us to propose a new classification of the cacao germplasm that will enhance its management. The results also provide new insights into the diversification of Amazon species in general, with the pattern of differentiation of the populations studied supporting the palaeoarches hypothesis of species diversification. The origin of the traditional cacao cultivars is also enlightened in this study.     

The origin of cultivated <Emphasis Type="Italic">Coffea arabica</Emphasis> L. varieties revealed by AFLP and SSR markers

Molecular markers were used to assess polymorphism between and within the genetic bases of coffee (i.e. Typica and Bourbon) spread from Yemen since the early 18th century that have given rise to most arabica cultivars grown world-wide. Eleven Coffea arabica accessions derived from the disseminated bases were evaluated by amplified fragment length polymorphism (AFLP) using 37 primer combinations and simple-sequence repeats (SSRs) produced by six microsatellites. Four cultivars growing in Yemen and 11 subspontaneous accessions collected in the primary centre of diversity of the species were included in the study in order to define their relationship with the accessions derived from the genetic bases of cultivars. One hundred and seven AFLP markers were used to calculate genetic distances and construct a dendrogram. The accessions derived from the disseminated bases were grouped separately, according to their genetic origin, and were distinguished from the subspontaneous accessions. The Yemen cultivars were classified with the Typica-derived accessions. Except for one AFLP marker, all AFLP and SSR markers present in the cultivated accessions were also detected in the subspontaneous accessions. Polymorphism among the subspontaneous accessions was much higher than among the cultivated accessions. It was very low within the genetic bases, confirming the historical documentation on their dissemination. The results enabled a discussion of the genetic diversity reductions that successively occurred during the dissemination of C. arabica from its primary centre of diversity.     

Molecular characterization of an earliest cacao (Theobroma cacao L.) collection from Upper Amazon using microsatellite DNA markers

Cacao (Theobroma cacao L.) is indigenous to the Amazon region of South America. The river basins in the Upper Amazon harbor a large number of diverse cacao populations. Since the 1930s, several numbers of populations have been collected from the present-day Peruvian Amazon and maintained as ex situ germplasm repositories in various countries, with the largest held in the International Cacao Genebank in Trinidad. The lack of information on population structure and pedigree relationship and the incorrect labeling of accessions are major concerns for efficient conservation and use of cacao germplasm. In the present study, we assessed the individual identity, sibship, and population structure in cacao populations collected from the present-day Loreto Region, Peru in the 1930–1940s. Using a capillary electrophoresis genotyping system, we analyzed the simple sequence repeat variation of 612 cacao accessions collected from the Marañon, Nanay, and Ucayali river systems. A total of 180 cases of mislabeling were identified using a Bayesian clustering method for admixture detection. Using maximum likelihood-based methods, we reconstructed 78 full-sib families nested in 48 half-sib families, indicating that the pods collected in the 1930s were from 48 mother trees, maximum. Likelihood simulation also identified eight probable parents that are responsible for 117 pairs of mother–offspring relationships in this collection. Principal coordinate analysis (PCoA) and the Bayesian clustering method cohesively demonstrated a pronounced structure of genetic diversity, stratified by the river systems of the Peruvian Amazon. Our results also show that, in spite of the high level of allelic diversity in this collection, it was composed of a large number of related family members collected from a relatively small area, including a couple of sites in the Ucayali and Nanay rivers, as well as the lower Marañon river near Iquitos. The vast majority of the Peruvian Amazon, especially the upper Marañon River and its tributaries, have not been sampled by collecting expeditions. The improved understanding of the individual identities, genealogical relationships, and geographical origin of cacao germplasm in this collection will contribute to more efficient conservation and utilization of these germplasm. Additionally, this study also provides more baseline information to help guide future collecting expeditions in the Peruvian Amazon.     

The effects of cocoa swollen-shoot virus on the growth and yield of Amelonado and Amazon cocoa (Theobroma cacao) in Ghana

The virulent strain A of cocoa swollen-shoot virus (CSSV) severely decreased the growth and yield of Amelonado cocoa (Theobroma cacao) trees kept free of capsids (Distantiella theobroma and Sahlbergella singularis) and the dieback fungus (Calonectria rigidiuscula) in Ghana. Fifteen per cent of graft-inoculated Amelonado trees showed symptoms within 4 months, and 48, 80 and 100% within 6, 12 and 20 months, respectively. Infected trees, whether shaded or unshaded, began to decline 6 months after infection, and deteriorated rapidly during the next 27 months by which time 16 % had died and most others were moribund; fertilizer applications had no significant effect on the rate at which infected trees deteriorated. Yields of pods and dry cocoa were greatly reduced 2 yr after infection and were very low after 3 yr; yields were significantly reduced by virus infection but there were no significant further effects of applying fertilizer. These results confirm that CSSV strain A alone is very damaging and often eventually lethal to Amelonado trees in Ghana, and indicate that the conflicting results obtained previously in Ghana and Nigeria were probably due to differences in the virulence of the CSSV strains tested. In contrast, the virus had much less effect on cocoa trees of the Amazon type; only 3% of graft-inoculated Amazon trees showed symptoms within 4 months, and 43, 84 and 97% after 1, 2 and 3 yr, respectively. Slight deterioration of tree canopies was first detected c. 15 months after infection and, although it continued slowly during the next 21 months, the decline was much less severe than that of Amelonado trees. Yields of both unshaded and shaded trees were apparently reduced by virus infection, but yield losses were much smaller than those of Amelonado trees. These results support the present objectives of controlling the spread of CSSV in Ghana by roguing infected trees, and selecting cultivars with greater tolerance to infection for future use.     

Molecular genetic diversity and association mapping of nut and kernel traits in Slovenian hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis>) germplasm

European hazelnut (Corylus avellana L.), cultivated in several areas of the world including Europe, Anatolia, and the USA, is an economically important nut crop due to its high mineral, oleic acid, amino acid, and phenolic compound content and pleasant flavor. This study examined molecular genetic diversity and population structure of 54 wild accessions and 48 cultivars from the Slovenian national hazelnut collection using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Eleven AFLP primer combinations and 49 SSR markers yielded 532 and 504 polymorphic fragments, respectively. As expected for a wind-pollinated, self-incompatible species, levels of genetic diversity were high with cultivars and wild accessions having mean dissimilarity values of 0.50 and 0.60, respectively. In general, cultivars and wild accessions clustered separately in dendrogram, principal coordinate, and population structure analyses with regional clustering of the wild material. The accessions were also characterized for ten nut and seven kernel traits and some wild accessions were shown to have breeding potential. Morphological principal component analysis showed distinct clustering of cultivars and wild accessions. An association mapping panel composed of 64 hazelnut cultivars and wild accessions had considerable variation for the nut and kernel quality traits. Morphological and molecular data were associated to identify markers controlling the traits. In all, 49 SSR markers were significantly associated with nut and kernel traits [P < 0.0001 and LD value (r ²) = 0.15–0.50]. This work is the first use of association mapping in hazelnut and has identified molecular markers associated with important quality parameters in this important nut crop.     

Polymorphism of DNA sequences of cryptochrome genes is not associated with the photoperiodic flowering of wild soybean along a latitudinal cline

Both cultivated soybean and its wild relative Glycine soja exhibit strong photoperiodic sensitivity at different latitudes. Recent studies have demonstrated that the blue light-absorbing cryptochrome gene, CRY1a, is involved in the photoperiodic flowering of soybeans. However, no sequence variation was found in the cDNA among cultivars at different latitudinal clines. In the present study, we examined whether positive selection due to polymorphisms in the cryptochrome genes of G. soja occurs. Partial DNA sequences, mainly exons, of cryptochrome genes CRY1a-1d and CRY2a-2c were analyzed for 18 accessions in the Japanese archipelago. The neutral evolutionary pattern of the polymorphisms for all cryptochrome genes except for CRY1a was summarized using Tajima's D test and low nucleotide diversity was shown for all genes. Although CRY1a did not show neutral evolution, balancing selection was recognized in the intron while not in the exon. No geographical pattern of polymorphisms was observed in the cryptochrome genes. These results reject the possibility of cryptochrome genes being involved in the photoperiodic flowering of wild soybeans along a latitudinal cline.     

Differential effects of temperature on fruit development and bean quality of contrasting genotypes of cacao (Theobroma cacao)

The effects of temperature and light integral on fruit growth and development of five cacao genotypes (Amelonado, AMAZ 15/15, SCA 6, SPEC 54/1 and UF 676) were studied in semi‐controlled environment glasshouses in which the thermal regimes of cacao‐growing regions of Brazil, Ghana and Malaysia were simulated. Fruit losses because of physiological wilt (cherelle wilt) were greater at higher temperatures and also differed significantly between genotypes, reflecting genetic differences in competition for assimilates between vegetative and reproductive components. Short‐term measurements of fruit growth indicated faster growth rates at higher temperatures. In addition, a significant negative linear relationship between temperature and development time was observed. There was an effect of genotype on this relationship, such that time to fruit maturation at a given temperature was greatest for the clone UF 676 and least for AMAZ 15/15. Analysis of base temperatures, derived from these relationships indicated genetic variability in sensitivity of cacao fruit growth to temperature (base temperatures ranged from 7.5°C for Amelonado and AMAZ 15/15 to 12.9 for SPEC 54/1). Final fruit size was a positive function of bean number for all genotypes and a positive function of light integral for Amelonado in the Malaysia simulated environment (where the temperature was almost constant). In simulated environments where temperature was the main variable (Brazil and Ghana) increases in temperature resulted in a significant decrease in final pod size for one genotype (Amelonado) in Brazil and for two genotypes (SPEC 54/1 and UF 676) in Ghana. It was hypothesised that pod growth duration (mediated by temperature), assimilation and bean number are all determinants of final pod size but that under specific conditions one of these factors may override the others. There was variability between genotypes in the response of bean size and bean lipid content to temperature. Negative relationships between temperature and bean size were found for Amelonado and UF 676. Lipid concentration was a curvilinear function of temperature for Amelonado and UF 676, with optimal temperatures of 23°C and 24°C, respectively. The variability observed here of different cacao genotypes to temperature highlights the need and opportunities for appropriate matching of planting material with local environments.