Complete genome characterization of cacao leafroll virus, a newly described cacao-infecting polerovirus.

The complete genome sequence of cacao leafroll virus (CaLRV; family Solemoviridae, genus Polerovirus) was determined by high-throughput sequencing of total RNA isolated from symptomatic cacao Theobroma cacao L. plants (n = 4). The CaLRV genome sequences ranged from 5,976 to 5,997 nucleotides (nt) in length and contained seven open reading frames (ORFs). Nucleotide and amino acid (aa) sequence comparisons showed that, among selected well-characterized poleroviruses, the CaLRV genome shared the highest nt sequence identity of 62% with that of potato leafroll virus (PLRV, NC_076505). A comparison of the predicted aa sequence of the CaLRV coat protein indicated that cotton leafroll dwarf virus (CLRDV, NC_014545) and melon aphid-borne yellows virus (MABYV, NC_010809) were the closest relatives, sharing 57% aa sequence identity. Bayesian phylogenetic analysis based on complete genome sequences showed that CaLRV grouped with well-characterized poleroviruses that cause diseases of cereal and vegetable crops. During the course of publishing this work, the nearly complete genome sequence of a member of the same polerovirus species, referred to as "cacao polerovirus" (OR605721), with which CaLRV shares 99% nt sequence identity, was reported.

A Previously Undescribed Polerovirus (Solemoviridae) Infecting Theobroma cacao Germplasm.

Cacao Theobroma cacao L. (Malvaceae) is an economically important crop cultivated in tropical climates for the bean from which chocolate and other products are made (Zarrillo et al., 2018). Virus-like symptoms consisting of discoloration, leaf distortion with downward rolling of leaves, and yellow speckling or mottling (Fig. S1), were observed in imported cacao germplasm at the USDA-ARS-SHRS cacao quarantine facilities in the fall of 2020. Total RNA was isolated from leaves collected from four symptomatic plants using silica RNA extraction method (Rott and Jelkmann, 2001). Ribosomal RNA (rRNA)-depleted RNA samples were used for cDNA library construction, followed by high throughput sequencing on Illumina® NovaSeq 6000 platform (Novogene Corp., Sacramento, CA). Quality-filtered, 150-bp paired-ended reads (2,601,293-3,104,474) were assembled de novo using SPAdes v.3.14.1 (Nurk et al., 2013). The contigs (200,799 to 276,851) were queried against the NCBI virus reference sequence (RefSeq) database using the discontiguous megablast algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). The resultant contigs (n=1,344) were 150-nt to 1463-nt in length (k-mer coverage from 6.3x to 26,721.7x) and shared their highest nucleotide (nt) identity with potato leafroll virus (PLRV; NC_001747; genus Polerovirus; family Solemoviridae), at 69.1%-72.8%. The contigs pooled from the four samples were assembled into 15 scaffolds. BLASTn analyses of the 15 scaffolds against the RefSeq database indicated the best matches were to thirteen other polerovirus species, with top hits to cereal yellow dwarf virus-RPV (D10206) and pepper vein yellows virus (LC528383), having similarity scores of 66.2% and 100% respectively. The 15 scaffolds matched to the 5' terminal end, ORF1-2, ORF3, ORF4 and ORF3-5 of the different polerovirus genomes. For confirmatory sequencing, total RNA was subjected to reverse transcription using SuperScript IV (Invitrogen, Carlsbad, CA), followed by RT-PCR amplification with general polerovirus primers PoconF/PoconcpR (Xiang et al., 2008) expected to yield an amplicon of ~1,400-bp located at the 3' end of the RNA-dependent, RNA polymerase (RdRp), including the complete coat protein (CP) and movement protein (MP) genes. Amplicons were ligated to pGEM-T Easy vector (Promega, Madison, WI) and sequenced bi-directionally by Sanger sequencing (Eton Bio, Research Triangle Park, NC). BLASTn analysis of the polerovirus-like nt sequences (GenBank accession nos. (ON745771-ON745774) indicated the closest relatives were potato leafroll virus (OK058524) and cucumber aphid-borne yellows virus (FJ460218), at 71% and 73%, respectively. The CP amino acid (aa) sequence shared the greatest similarity to cereal yellow dwarf virus RPV (NP_840023), at 53%, and the MP aa sequence shared the greatest aa similarity to wheat yellow leaf dwarf virus-GPV (YP_003029842), at 38%. These results provide robust support for the association of a previously undescribed polerovirus with symptomatic cacao trees, herein named, cacao leafroll virus (Solemoviridae; Polerovirus). Although Koch's postulates have not been completed to confirm causality, the presence of this virus in cacao germplasm undermine efforts to distribute pathogen-free germplasm and may pose a risk to cacao production in trees established from virus-infected plant material. To our knowledge, this is the first report of a polerovirus infecting cacao trees. All trees of these accessions at the quarantined facility in Miami, FL have been destroyed.

Complete genome sequences of three newly discovered cacao mild mosaic virus isolates from Theobroma cacao L. in Brazil and Puerto Rico and evidence for recombination.

To analyze the DNA virome associated with cacao (Theobroma cacao L.) trees showing virus-like symptoms in Brazil (BR) and Puerto Rico (PR) during 2018-2019, total DNA was isolated from symptomatic leaves and subjected to high-throughput Illumina sequencing. The assembled complete badnaviral genome sequences were verified by PCR amplification, cloning, and DNA sequencing. Based on pairwise distances and phylogenetic analysis, three badnaviral genomes were identified, and these viruses were found to be isolates of the previously described cacao mild mosaic virus (CaMMV). The three genomes were 7,520, 7,524, and 7,514 bp in size for the isolates CaMMV-BR321, CaMMV-BR322, and CaMMV-PR3, respectively. Each genome contained four predicted open reading frames: ORFs 1-3 and ORFY. The CaMMV-PR3 isolate was identified as a probable recombinant, with a CaMMV-BR-like virus as the major parent.

The Previously Unidentified, Divergent Badnavirus Species Cacao red vein-banding virus is Associated with Cacao Swollen Shoot Disease in Nigeria.

Cacao swollen shoot disease (CSSD) of Theobroma cacao was reported in Nigeria in 1944; however, no badnaviral genome sequences have been found associated with the symptomatic trees. In 2017, leaf samples (n = 18) were collected from cacao trees from Osun and Oyo, Nigeria showing foliar symptoms that included red vein-banding and shoot swelling, and variable secondary mosaic, mottling, and fern-like pattern symptoms. Abutting primers designed around previously determined 500-bp intergenic region sequences were used for polymerase chain reaction (PCR) amplification. Of the 18 samples, 9 yielded an approximately 7,000-bp, apparently genome-size product. The nine genomes were sequenced and found to encode four open reading frames, and to share 86 to 99% nucleotide identity. Pairwise analysis of the Nigerian genomes with 21 previously reported CSSD badnaviruses, at the complete genome and reverse-transcription ribonuclease H (1,230 bp) sequence levels, indicated 71 to 75 and 72 to 76% nucleotide identity, respectively. Phylogenetic analysis of the nine complete genomes indicated that the closest relatives of the divergent Nigerian isolates were previously described West African CSSD badnaviruses. Based on pairwise comparisons and phylogenetic analyses, the Nigerian CSSD isolates constitute a previously unrecognized Badnavirus sp., herein named Cacao red vein-banding virus (CRVBV). Primers designed based on the CRVBV genome sequences amplified a 1,068-bp fragment from 16 of 18 field samples tested by PCR, suggesting the possible existence of additional CRVBV variants.

The proposed new species, cacao red vein virus, and three previously recognized badnavirus species are associated with cacao swollen shoot disease.

BACKGROUND: Cacao swollen shoot virus (CSSV), Cacao swollen shoot CD virus (CSSCDV), and Cacao swollen shoot Togo A virus (CSSTAV) cause cacao swollen shoot disease (CSSD) in West Africa. During 2000-2003, leaf and shoot-swelling symptoms and rapid tree death were observed in cacao in Cote d'Ivoire and Ghana. Molecular tests showed positive infection in only ~50-60% of symptomatic trees, suggesting the possible emergence of an unknown badnavirus. METHODS: The DNA virome was determined from symptomatic cacao samples using Illumina-Hi Seq, and sequence accuracy was verified by Sanger sequencing. The resultant 14, and seven previously known, full-length badnaviral genomic and RT-RNase H sequences were analyzed by pairwise distance analysis to resolve species relationships, and by Maximum likelihood (ML) to reconstruct phylogenetic relationships. The viral coding and non-coding sequences, genome organization, and predicted conserved protein domains (CPDs) were identified and characterized at the species level. RESULTS: The 21 CSSD-badnaviral genomes and RT-RNase H sequences shared 70-100% and 72-100% identity, respectively. The RT-RNase H analysis predicted four species, based on an ≥80% species cutoff. The ML genome sequence tree resolved three well-supported clades, with ≥70% bootstrap, whereas, the RT-RNase H phylogeny was poorly resolved, however, both trees grouped CSSD isolates within one large clade, including the newly discovered Cacao red vein virus (CRVV) proposed species. The genome arrangement of the four species consists of four, five, or six predicted open reading frames (ORFs), and the CPDs have similar architectures. By comparison, two New World cacao-infecting badnaviruses encode four ORFs, and harbor CPDs like the West African species. CONCLUSIONS: Three previously recognized West African cacao-infecting badnaviral species were identified, and a fourth, previously unidentified species, CRVV, is described for the first time. The CRVV is a suspect causal agent of the rapid decline phenotype, however Koch's Postulates have not been proven. To reconcile viral evolutionary with epidemiology considerations, more detailed information about CSSD-genomic variability is essential. Also, the functional basis for the multiple genome arrangements and subtly distinct CPD architectures among cacao-infecting badnaviruses is poorly understood. New knowledge about functional relationships may help explain the diverse symptomatologies observed in affected cacao trees.

Molecular characterization of previously elusive badnaviruses associated with symptomatic cacao in the New World.

Suspected virus-like symptoms were observed in cacao plants in Trinidad during 1943, and the viruses associated with these symptoms were designated as strains A and B of cacao Trinidad virus (CTV). However, viral etiology has not been demonstrated for either phenotype. Total DNA was isolated from symptomatic cacao leaves exhibiting the CTV A and B phenotypes and subjected to Illumina HiSeq and Sanger DNA sequencing. Based on de novo assembly, two apparently full-length badnavirus genomes of 7,533 and 7,454 nucleotides (nt) were associated with CTV strain A and B, respectively. The Trinidad badnaviral genomes contained four open reading frames, three of which are characteristic of other known badnaviruses, and a fourth that is present in only some badnaviruses. Both badnaviral genomes harbored hallmark caulimovirus-like features, including a tRNAMet priming site, a TATA box, and a polyadenylation-like signal. Pairwise comparisons of the RT-RNase H region indicated that the Trinidad isolates share 57-71% nt sequence identity with other known badnaviruses. Based on the system for badnavirus species demarcation in which viruses with less than 80% nt sequence identity in the RT-RNase gene are considered members of separate species, these isolates represent two previously unidentified badnaviruses, herein named cacao mild mosaic virus and cacao yellow vein banding virus, making them the first cacao-infecting badnaviruses identified thus far in the Western Hemisphere.

Three de novo assembled wild cacao genomes from the Upper Amazon.

Theobroma cacao, the chocolate tree, is indigenous to the Amazon basin, the greatest biodiversity hotspot on earth. Recent advancement in plant genomics highlights the importance of de novo sequencing of multiple reference genomes to capture the genome diversity present in different cacao populations. In this study, three high-quality chromosome-level genomes of wild cacao were constructed, de novo assembled with HiFi long reads sequencing, and scaffolded using a reference-free strategy. These genomes represent the three most important genetic clusters of cacao trees from the Upper Amazon region. The three wild cacao genomes were compared with two reference genomes of domesticated cacao. The five cacao genetic clusters were inferred to have diverged in the early and middle Pleistocene period, approximately 1.83-0.69 million years ago. The results shown here serve as an example of understanding how the Amazonian biodiversity was developed. The three wild cacao genomes provide valuable resources for studying genetic diversity and advancing genetic improvement of this species.

3D pollination biology using micro-computed tomography and geometric morphometrics in Theobroma cacao.

Premise: Imaging technologies that capture three-dimensional (3D) variation in floral morphology at micro- and nano-resolutions are increasingly accessible. In herkogamous flowers, such as those of Theobroma cacao, structural barriers between anthers and stigmas represent bottlenecks that restrict pollinator size and access to reproductive organs. To study the unresolved pollination biology of cacao, we present a novel application of micro-computed tomography (micro-CT) using floral dimensions to quantify pollinator functional size limits. Methods: We generated micro-CT data sets from field-collected flowers and museum specimens of potential pollinators. To compare floral variation, we used 3D Slicer to place landmarks on the surface models and performed a geometric morphometric (GMM) analysis using geomorph R. We identified the petal side door (an opening between the petal hoods and filament) as the main bottleneck for pollinator access. We compared its mean dimensions with proposed pollinators to identify viable candidates. Results: We identified three levels of likelihood for putative pollinators based on the number of morphological (body) dimensions that fit through the petal side door. We also found floral reward microstructures whose presence and location were previously unclear. Discussion: Using micro-CT and GMM to study the 3D pollination biology of cacao provides new evidence for predicting unknown pollinators. Incorporating geometry and floral rewards will strengthen plant-pollinator trait matching models for cacao and other species.

Identification of QTL regions and SSR markers associated with resistance to reniform nematode in Gossypium barbadense L. accession GB713.

The identification of molecular markers that are closely linked to gene(s) in Gossypium barbadense L. accession GB713 that confer a high level of resistance to reniform nematode (RN), Rotylenchulus reniformis Linford & Oliveira, would be very useful in cotton breeding programs. Our objectives were to determine the inheritance of RN resistance in the accession GB713, to identify SSR markers linked with RN resistance QTLs, and to map these linked markers to specific chromosomes. We grew and scored plants for RN reproduction in the P(1), P(2), F(1), F(2), BC(1)P(1), and BC(1)P(2) generations from the cross of GB713 × Acala Nem-X. The generation means analysis using the six generations indicated that one or more genes were involved in the RN resistance of GB713. The interspecific F(2) population of 300 plants was genotyped with SSR molecular markers that covered most of the chromosomes of Upland cotton (G. hirsutum L.). Results showed two QTLs on chromosome 21 and one QTL on chromosome 18. One QTL on chromosome 21 was at map position 168.6 (LOD 28.0) flanked by SSR markers, BNL 1551_162 and GH 132_199 at positions 154.2 and 177.3, respectively. A second QTL on chromosome 21 was at map position 182.7 (LOD 24.6) flanked by SSR markers BNL 4011_155 and BNL 3279_106 at positions 180.6 and 184.5, respectively. Our chromosome 21 map had 61 SSR markers covering 219 cM. One QTL with smaller genetic effects was localized to chromosome 18 at map position 39.6 (LOD 4.0) and flanked by SSR markers BNL 1721_178 and BNL 569_131 at positions 27.6 and 42.9, respectively. The two QTLs on chromosome 21 had significant additive and dominance effects, which were about equal for each QTL. The QTL on chromosome 18 showed larger additive than dominance effects. Following the precedent set by the naming of the G. longicalyx Hutchinson & Lee and G. aridum [(Rose & Standley) Skovsted] sources of resistance, we suggest the usage of Ren (barb1) and Ren (barb2) to designate these QTLs on chromosome 21 and Ren (barb3) on chromosome 18.

SSR markers closely associated with genes for resistance to root-knot nematode on chromosomes 11 and 14 of Upland cotton.

Molecular markers closely linked to genes that confer a high level of resistance to root-knot nematode (RKN) [Meloidogyne incognita (Kofoid & White) Chitwood] in cotton (Gossypium hirsutum L.) germplasm derived from Auburn 623 RNR would greatly facilitate cotton breeding programs. Our objectives were to identify simple sequence repeat (SSR) markers linked to RKN resistance quantitative trait loci (QTL) and map these markers to specific chromosomes. We developed three recombinant inbred line (RIL) populations by single seed descent from the crosses of RKN-resistant parents M-240 RNR (M240), developed from the Auburn 623 RNR source, moderately resistant Clevewilt 6 (CLW6), one of the parents of Auburn 623 RNR, and susceptible parent Stoneville 213 (ST213). These crosses were CLW6 × ST213, M240 × CLW6, and M240 × ST213. RILs from these populations were grown under greenhouse conditions, inoculated with RKN eggs, scored for root gall index, eggs plant(-1), and eggs g(-1) root. Plants were also genotyped with SSR markers. Results indicated that a minimum of two major genes were involved in the RKN resistance of M240. One gene was localized to chromosome 11 and linked to the marker CIR 316-201. This CIR 316-201 allele was also present in CLW6 but not in Mexico Wild (MW) (PI593649), both of which are parents of Auburn 623 RNR. A second RKN resistance gene was localized to the short arm of chromosome 14 and was linked to the SSR markers BNL3545-118 and BNL3661-185. These two marker alleles were not present in CLW6 but were present in MW. Our data also suggest that the chromosome 11 resistance QTL primarily affects root galling while the QTL on chromosome 14 mediates reduced RKN egg production. The SSRs identified in this study should be useful to select plants with high levels of RKN resistance in segregating populations derived from Auburn 623 RNR.

A Complex of Badnavirus Species Infecting Cacao Reveals Mixed Infections, Extensive Genomic Variability, and Interspecific Recombination.

The incidence of cacao swollen shoot disease (CSSD) in cacao (Theobroma cacao L.) has increased in West Africa since ~2000. To investigate the genomic and species diversity of the CSSD-badnaviruses infecting cacao in Côte d'Ivoire and Ghana, symptomatic leaves were subjected to high-throughput sequencing. Among the 30 newly determined genomes, three badnaviruses were identified, Cacao swollen shoot Togo B virus (CSSTBV), Cacao swollen shoot CD virus, and Cacao swollen shoot CE virus (CSSCEV). The phylogenetic trees reconstructed for the reverse transcriptase (RT) and ribonuclease H (RNase H) sequences were incongruent with the complete viral genomes, which had the most robust statistical support. Recombination seems to be involved in the CSSD-badnavirus diversification. The genomic diversity varied among different CSSD-badnaviruses, with CSSTBV showing the lowest nucleotide diversity (π = 0.06236), and CSSCEV exhibiting the greatest variability (π = 0.21911). Evidence of strong purifying selection was found in the coding regions of the CSSTBV isolates.

Identification of associations between SSR markers and fiber traits in an exotic germplasm derived from multiple crosses among Gossypium tetraploid species.

Genetic improvement in yield and fiber quality is needed for worldwide cotton production. Identification of molecular markers associated with fiber-related traits can facilitate selection for these traits in breeding. This study was designed to identify associations between SSR markers and fiber traits using an exotic germplasm population, species polycross (SP), derived from multiple crosses among Gossypium tetraploid species. The SP population underwent 11 generations of mixed random mating and selfing followed by 12 generations of selfing. A total of 260 lines were evaluated for fiber-related traits under three environments in 2005 and 2006. Large genotypic variance components in traits were identified relative to components of genotype x environment. Eighty-six primer pairs amplified a total of 314 polymorphic fragments among 260 lines. A total of 202 fragments with above 6% allele frequency were analyzed for associations. Fifty-nine markers were found to have a significant (P < 0.05, 0.01, or 0.001) association with six fiber traits. There were six groups identified within the population using structure analysis. Allele frequency divergence among six groups ranged from 0.11 to 0.27. Of the 59 marker-trait associations, 39 remained significant after correction for population structure and kinship using a mixed linear model. The effect of population sub-structure on associations was most significant in boll weight among the traits analyzed. The sub-structure among the SP lines may be caused by natural selection, the breeding method applied during development of inbred lines, and unknown factors. The identified marker-trait associations can be useful in breeding and help determine genetic mechanisms underlying interrelationships among fiber traits.

Identification and genomic location of a reniform nematode (Rotylenchulus reniformis) resistance locus (Ren ari) introgressed from Gossypium aridum into upland cotton (G. hirsutum).

In this association mapping study, a tri-species hybrid, [Gossypium arboreum x (G. hirsutum x G. aridum)(2)], was crossed with MD51ne (G. hirsutum) and progeny from the cross were used to identify and map SSR markers associated with reniform nematode (Rotylenchulus reniformis) resistance. Seventy-six progeny (the 50 most resistant and 26 most susceptible) plants were genotyped with 104 markers. Twenty-five markers were associated with a resistance locus that we designated Ren(ari) and two markers, BNL3279_132 and BNL2662_090, mapped within 1 cM of Ren(ari). Because the SSR fragments associated with resistance were found in G. aridum and the bridging line G 371, G. aridum is the likely source of this resistance. The resistance is simply inherited, possibly controlled by a single dominant gene. The markers identified in this project are a valuable resource to breeders and geneticists in the quest to produce cotton cultivars with a high level of resistance to reniform nematode.

Cadmium accumulation and allocation in different cacao cultivars.

Cadmium (Cd) is a biologically non-essential heavy metal that can cause toxic effects in plants, animals and humans already at low concentrations compared to other metals. After Cd concentrations in cacao beans of various provenances, particularly from Latin America, were found to exceed the new regulations enforced by the European Union in 2019, there is an urgent need to find measures to lower Cd accumulation in cacao beans to acceptable values. In this research, the long-term cacao cultivar trial CEDEC-JAS in northern Honduras was used to investigate differences between 11 cultivars in Cd uptake and translocation. Sampling of various plant parts, including rootstocks, scions, leaves and beans, from three replicate trees per cultivar and the soil around each tree was conducted at this site. Results indicate that concentrations of available soil Cd were more closely correlated with Cd concentrations of the rootstocks (R2 = 0.56), scions (R2 = 0.59) and leaves (R2 = 0.46) than with bean Cd concentrations (R2 = 0.26). In addition, Cd concentrations of rootstocks, scions and leaves showed close relationships to available soil Cd concentrations, with no significant differences between the cultivars. In contrast, bean Cd concentrations showed only weak correlations to available soil Cd and Cd concentrations in the vegetative plant parts, but significant variation among cultivars. Three cultivars, which were analysed in more detail, showed significant differences in Cd concentrations of mature beans, but not of immature beans. These results suggest that cultivar-related differences in bean Cd concentrations primarily result from differences in Cd loading during bean maturation, possibly due to cultivar-specific differences in the xylem-to-phloem transfer of Cd. The results show that selection of cultivars with low Cd transfer from vegetative parts into the beans has high potential to keep Cd accumulation in cacao beans at levels that are safe for consumption.

Population genomic analyses of the chocolate tree, Theobroma cacao L., provide insights into its domestication process.

Domestication has had a strong impact on the development of modern societies. We sequenced 200 genomes of the chocolate plant Theobroma cacao L. to show for the first time to our knowledge that a single population, the Criollo population, underwent strong domestication ~3600 years ago (95% CI: 2481-13,806 years ago). We also show that during the process of domestication, there was strong selection for genes involved in the metabolism of the colored protectants anthocyanins and the stimulant theobromine, as well as disease resistance genes. Our analyses show that domesticated populations of T. cacao (Criollo) maintain a higher proportion of high-frequency deleterious mutations. We also show for the first time the negative consequences of the increased accumulation of deleterious mutations during domestication on the fitness of individuals (significant reduction in kilograms of beans per hectare per year as Criollo ancestry increases, as estimated from a GLM, P = 0.000425).

Effects of chromosome-specific introgression in upland cotton on fiber and agronomic traits.

Interspecific chromosome substitution is among the most powerful means of introgression and steps toward quantitative trait locus (QTL) identification. By reducing the genetic "noise" from other chromosomes, it greatly empowers the detection of genetic effects by specific chromosomes on quantitative traits. Here, we report on such results for 14 cotton lines (CS-B) with specific chromosomes or chromosome arms from G. barbadense L. substituted into G. hirsutum and chromosome-specific F2 families. Boll size, lint percentage, micronaire, 2.5% span length, elongation, strength, and yield were measured by replicated field experiments in five diverse environments and analyzed under an additive-dominance (AD) genetic model with genotype and environment interaction. Additive effects were significant for all traits and dominance effects were significant for all traits except 2.5% span length. CS-B25 had additive effects increasing fiber strength and fiber length and decreasing micronaire. CS-B16 and CS-B18 had additive effects related to reduced yields. The results point toward specific chromosomes of G. barbadense 3-79 as the probable locations of the genes that significantly affect quantitative traits of importance. Our results provided a scope to analyze individual chromosomes of the genome in homozygous and heterozygous conditions and thus detected novel effects of alleles controlling important QTL.