Morphological and genetic characterization of jackfruit (Artocarpus heterophyllus) in the Kayunga and Luwero districts of Uganda.

BACKGROUND: Jackfruit (Artocarpus heterophyllus) is an economically valuable fruit tree in Uganda. However, the production of jackfruit in Uganda is low. Additionally, because of deforestation, genetic erosion of the resource is predicted before its exploitation for crop improvement and conservation. As a prerequisite for crop improvement and conservation, 100 A. heterophyllus tree isolates from the Kayunga and Luwero districts in Uganda were characterized using 16 morphological and 10 microsatellite markers. RESULTS: The results from the morphological analysis revealed variations in tree height, diameter at breast height (DBH), and crown diameter, with coefficient of variation (CV) values of 20%, 41%, and 33%, respectively. Apart from the pulp taste, variation was also observed in qualitative traits, including tree vigor, trunk surface, branching density, tree growth habit, crown shape, leaf blade shape, fruit shape, fruit surface, flake shape, flake color, flake flavor and pulp consistency/texture. Genotyping revealed that the number of alleles amplified per microsatellite locus ranged from 2 to 5, with an average of 2.90 and a total of 29. The mean observed (Ho) and expected (He) heterozygosity were 0.71 and 0.57, respectively. Analysis of molecular variance (AMOVA) indicated that 81% of the variation occurred within individual trees, 19% among trees within populations and 0% between the two populations. The gene flow (Nm) in the two populations was 88.72. The results from the 'partitioning around medoids' (PAM), principal coordinate analysis (PCoA) and genetic cluster analysis further revealed no differentiation of the jackfruit populations. The Mantel test revealed a negligible correlation between the morphological and genetic distances. CONCLUSIONS: Both morphological and genetic analyses revealed variation in jackfruit within a single interbreeding population. This diversity can be exploited to establish breeding and conservation strategies to increase the production of jackfruit and hence boost farmers' incomes. However, selecting germplasm based on morphology alone may be misleading.

Genome-wide identification, expression analysis, and abiotic stress response of the CBL and CIPK gene families in Artocarpus nanchuanensis.

Artocarpus nanchuanensis, the northernmost species in the jackfruit genus, has great economic and horticultural value due to its nutritious fruit and beautiful tree shape. Calcineurin B-like proteins (CBLs) act as plant-specific Ca2+ sensors and participate in regulating plant responses to various abiotic stresses by interacting with CBL-interacting protein kinases (CIPKs). However, the characteristics and functions of the CBL and CIPK genes in A. nanchuanensis are still unclear. Here, we identified 14 CBL and 33 CIPK genes from the A. nanchuanensis genome, and based on phylogenetic analysis, they were divided into 4 and 7 clades, respectively. Gene structure and motif analysis indicated that the AnCBL and AnCIPK genes were relatively conserved. Colinear analysis showed that segmental duplication contributed to the expansion of the AnCBL and AnCIPK gene families. Expression analysis showed that AnCBL and AnCIPK genes were widely expressed in various tissues of A. nanchuanensis and exhibited tissue-specific expression. In addition, three genes (AnCBL6, AnCIPK7/8) may play important roles in response to salt, cold, and drought stresses. In summary, this study lays an important foundation for the improvement of stress resistance in A. nanchuanensis and provides new insight for the functional research on CBL and CIPK gene families.

Linking breadfruit cultivar names across the globe connects histories after 230 years of separation.

Every crop has a story. The story of breadfruit (Artocarpus altilis), an increasingly valued staple crop in tropical agroforestry systems, is filled with intrigue, oppression, and remains incomplete. The Caribbean is a major producer and consumer of breadfruit, yet most breadfruit there came from a single 1793 introduction aimed at providing a cheap food source for slaves forced to work on British plantations. St. Vincent was the first significant point of Caribbean introduction and played a vital role in subsequent breadfruit distribution throughout the region. Hundreds of cultivars are documented in breadfruit's native Oceania. It remains a mystery, however, which ones were introduced to the Caribbean 230 years ago-still comprising the vast diversity found there today. Integrating local knowledge, historical documents and specimens, morphological data, and DNA, we identify eight major global breadfruit lineages-five of which are found in the Caribbean and likely represent the original 1793 introduction. Genetic data were able to match two Caribbean cultivar names confidently to their Oceania counterparts. Genetics and morphology together enabled additional possible matches. Many other named cultivars within lineages are too genetically similar to differentiate, highlighting difficulties of defining and identifying variation among clonally propagated triploid crops. Breadfruit is important in resilient agroforestry in tropical islands predicted to be especially affected by climate change. Findings reveal global links, building upon collective knowledge that can be used to inform breadfruit management. Results are also summarized in a brochure about breadfruit history and diversity in St. Vincent, and the Caribbean more broadly.

Folk nomenclature and traditional knowledge of breadfruit [Artocarpus altilis (Parkinson) Fosberg] diversity in four Anglophone Caribbean countries.

BACKGROUND: Since its introduction to the Anglophone Caribbean in 1793, breadfruit has had a diverse history in the region, and there is a considerable repository of traditional knowledge about the crop, that is undocumented. Consequently, it remains underutilized as a food source, despite recognition of its potential to contribute to food and nutrition security. Understanding the folk taxonomy and traditional knowledge associated with its diversity and uses is a prerequisite to develop programs for its commercial production and utilization. METHOD: This study was conducted among 170 respondents who were selected across four Anglophone Caribbean countries and provided information on the ethnobotany and traditional knowledge associated with breadfruit biodiversity, including systems of naming, identification and classification of breadfruit cultivars or types. RESULTS: Breadfruit has socio-cultural and economic value and is produced for both home use and sale by most respondents (68%). The genetic diversity of breadfruit managed by the respondents is also important, as a total of 51 vernacular names were identified, with nine of those names recorded for the first time in this study. Breadfruit types were identified by morphological and agronomical characteristics, with other important traits relating to use and cooking quality. Classification of breadfruit cultivars or types was based on eating-quality, most suitable methods of preparation and ease of cooking. CONCLUSION: The ethnobotanical and traditional knowledge obtained from this study may be useful in assessing the genetic diversity of breadfruit and guiding future community-based conservation and classification studies of this important crop resource in the Caribbean. This is crucial to support the commercialization of breadfruit to improve its contribution to food and nutrition security.

Amis Pacilo and Yami Cipoho are not the same as the Pacific breadfruit starch crop-Target enrichment phylogenomics of a long-misidentified Artocarpus species sheds light on the northward Austronesian migration from the Philippines to Taiwan.

'Breadfruit' is a common tree species in Taiwan. In the indigenous Austronesian Amis culture of eastern Taiwan, 'breadfruit' is known as Pacilo, and its fruits are consumed as food. On Lanyu (Botel Tobago) where the indigenous Yami people live, 'breadfruit' is called Cipoho and used for constructing houses and plank-boats. Elsewhere in Taiwan, 'breadfruit' is also a common ornamental tree. As an essential component of traditional Yami culture, Cipoho has long been assumed to have been transported from the Batanes Island of the Philippines to Lanyu. As such, it represents a commensal species that potentially can be used to test the hypothesis of the northward Austronesian migration 'into' Taiwan. However, recent phylogenomic studies using target enrichment show that Taiwanese 'breadfruit' might not be the same as the Pacific breadfruit (Artocarpus altilis), which was domesticated in Oceania and widely cultivated throughout the tropics. To resolve persistent misidentification of this culturally and economically important tree species of Taiwan, we sampled 36 trees of Taiwanese Artocarpus and used the Moraceae probe set to enrich 529 nuclear genes. Along with 28 archived Artocarpus sequence datasets (representing a dozen taxa from all subgenera), phylogenomic analyses showed that all Taiwanese 'breadfruit' samples, together with a cultivated ornamental tree from Hawaii, form a fully supported clade within the A. treculianus complex, which is composed only of endemic Philippine species. Morphologically, the Taiwanese 'breadfruit' matches the characters of A. treculianus. Within the Taiwanese samples of A. treculianus, Amis samples form a fully supported clade derived from within the paraphyletic grade composed of Yami samples, suggesting a Lanyu origin. Results of our target enrichment phylogenomics are consistent with the scenario that Cipoho was transported northward from the Philippines to Lanyu by Yami ancestors, though the possibility that A. treculianus is native to Lanyu cannot be ruled out completely.

A chromosome-level genome assembly of Artocarpus nanchuanensis (Moraceae), an extremely endangered fruit tree.

Artocarpus nanchuanensis (Moraceae), which is naturally distributed in China, is a representative and extremely endangered tree species. In this study, we obtained a high-quality chromosome-scale genome assembly and annotation information for A. nanchuanensis using integrated approaches, including Illumina, Nanopore sequencing platform, and Hi-C. A total of 128.71 Gb of raw Nanopore reads were generated from 20-kb libraries, and 123.38 Gb of clean reads were obtained after filtration with 160.34× coverage depth and a 17.48-kb average read length. The final assembled A. nanchuanensis genome was 769.44 Mb with a 2.09 Mb contig N50, and 99.62% (766.50 Mb) of the assembled data was assigned to 28 pseudochromosomes. In total, 39,596 genes (95.10%, 39,596/41,636) were successfully annotated, and 129 metabolic pathways were detected. Plants disease resistance/insect resistance genes, plant-pathogen interaction metabolic pathways, and abundant biosynthesis pathways of vitamins, flavonoid, and gingerol were detected. Unigene reveals the basis of species-specific functions, and gene family in contraction and expansion generally implies strong functional differences in the evolution. Compared with other related species, a total of 512 unigenes, 309 gene families in contraction, and 559 gene families in expansion were detected in A. nanchuanensis. This A. nanchuanensis genome information provides an important resource to expand our understanding of the unique biological processes, nutritional and medicinal benefits, and evolutionary relationship of this species. The study of gene function and metabolic pathway in A. nanchuanensis may reveal the theoretical basis of a special trait in A. nanchuanensis and promote the study and utilization of its rare medicinal value.

Sequencing and de novo transcriptome assembly for discovering regulators of gene expression in Jack (Artocarpus heterophyllus).

Jack (Artocarpus heterophyllus) is a multipurpose fruit-tree species with minimal genomic resources. The study reports developing comprehensive transcriptome data containing 80,411 unigenes with an N50 value of 1265 bp. We predicted 64,215 CDSs from the unigenes and annotated and functionally categorized them into the biological process (23,230), molecular function (27,149), and cellular components (17,284). From 80,411 unigenes, we discovered 16,853 perfect SSRs with 192 distinct repeat motif types reiterating 4 to 22 times. Besides, we identified 2741 TFs from 69 TF families, 53 miRNAs from 19 conserved miRNA families, 25,953 potential lncRNAs, and placed three functional eTMs in different lncRNA-miRNA pairs. The regulatory networks involving genes, TFs, and miRNAs identified several regulatory and regulated nodes providing insight into miRNAs' gene associations and transcription factor-mediated regulation. The comparison of expression patterns of some selected miRNAs vis-à-vis their corresponding target genes showed an inverse relationship indicating the possible miRNA-mediated regulation of the genes.

cDNA cloning of a novel lectin that induce cell apoptosis from Artocarpus hypargyreus.

We isolated a novel lectin (AHL) from Artocarpus hypargyreusHance and showed its immunomodulatory activities. In this study, the amino acid sequence of AHL was determined by cDNA sequencing. AHL cDNA (875bp) contains a 456-bp open reading frame (ORF), which encodes a protein with 151 amino acids. AHL is a new member of jacalin-related lectin family (JRLs), which share high sequence similarities to KM+ and Morniga M, and contain the conserved carbohydrate binding domains. The antitumor activity of AHL was also explored using Jurkat T cell lines. AHL exhibits a strong binding affinity to cell membrane, which can be effectively inhibited by methyl-α-D-galactose. AHL inhibits cell proliferation in a time- and dose-dependent manner through apoptosis, evidenced by morphological changes, phosphatidylserine externalization, poly ADP-ribose polymerase (PARP) cleavage, Bad and Bax up-regulation, and caspase-3 activation. We further showed that the activation of ERK and p38 signaling pathways is involved for the pro-apoptotic effect of AHL.

Cultivation potential projections of breadfruit (Artocarpus altilis) under climate change scenarios using an empirically validated suitability model calibrated in Hawai'i.

Humanity faces significant challenges to agriculture and human nutrition, and changes in climate are predicted to make such challenges greater in the future. Neglected and underutilized crops may play a role in mitigating and addressing such challenges. Breadfruit is a long-lived tree crop that is a nutritious, carbohydrate-rich staple, which is a priority crop in this regard. A fuzzy-set modeling approach was applied, refined, and validated for breadfruit to determine its current and future potential productivity. Hawai'i was used as a model system, with over 1,200 naturalized trees utilized to calibrate a habitat suitability model and 56 producer sites used to validate the model. The parameters were then applied globally on 17 global climate models at the RCP 4.5 and RCP 8.5 global climate projections for 2070. Overall, breadfruit suitability increases in area and in quality, with larger increases occurring in the RCP 8.5 projection. Current producing regions largely remain unchanged in both projections, indicating relative stability of production potential in current growing regions. Breadfruit, and other tropical indigenous food crops present strong opportunities for cultivation and food security risk management strategies moving forward.

Draft Genomes of Two Artocarpus Plants, Jackfruit (A. heterophyllus) and Breadfruit (A. altilis).

Two of the most economically important plants in the Artocarpus genus are jackfruit (A. heterophyllus Lam.) and breadfruit (A. altilis (Parkinson) Fosberg). Both species are long-lived trees that have been cultivated for thousands of years in their native regions. Today they are grown throughout tropical to subtropical areas as an important source of starch and other valuable nutrients. There are hundreds of breadfruit varieties that are native to Oceania, of which the most commonly distributed types are seedless triploids. Jackfruit is likely native to the Western Ghats of India and produces one of the largest tree-borne fruit structures (reaching up to 45 kg). To-date, there is limited genomic information for these two economically important species. Here, we generated 273 Gb and 227 Gb of raw data from jackfruit and breadfruit, respectively. The high-quality reads from jackfruit were assembled into 162,440 scaffolds totaling 982 Mb with 35,858 genes. Similarly, the breadfruit reads were assembled into 180,971 scaffolds totaling 833 Mb with 34,010 genes. A total of 2822 and 2034 expanded gene families were found in jackfruit and breadfruit, respectively, enriched in pathways including starch and sucrose metabolism, photosynthesis, and others. The copy number of several starch synthesis-related genes were found to be increased in jackfruit and breadfruit compared to closely-related species, and the tissue-specific expression might imply their sugar-rich and starch-rich characteristics. Overall, the publication of high-quality genomes for jackfruit and breadfruit provides information about their specific composition and the underlying genes involved in sugar and starch metabolism.

A transcriptome screen for positive selection in domesticated breadfruit and its wild relatives (Artocarpus spp.).

PREMISE OF THE STUDY: Underutilized crops, such as breadfruit (Artocarpus altilis, Moraceae) have the potential to improve global food security. Humans have artificially selected many cultivars of breadfruit since its domestication began approximately 3500 years ago. The goal of this research was to identify transcriptomic signals of positive selection and to develop genomic resources that may facilitate the development of improved breadfruit cultivars in the future. METHODS: A reference transcriptome of breadfruit was assembled de novo and annotated. Twenty-four transcriptomes of breadfruit and its wild relatives were generated and analyzed to reveal signals of positive selection that may have resulted from local adaptation or natural selection. Emphasis was placed on MADS-box genes, which are important because they often regulate fruiting timing and structures, and on carotenoid biosynthesis genes, which can impact the nutritional quality of the fruit. KEY RESULTS: Over 1000 genes showed signals of positive selection, and these genes were enriched for localization to plastids. Nucleotide sites and individuals under positive selection were discovered in MADS-box genes and carotenoid biosynthesis genes, with several sites located in cofactor or DNA-binding domains. A McDonald-Kreitman test comparing wild to cultivated samples revealed selection in one of the carotenoid biosynthesis genes, abscisic acid 8'-hydroxylase 3. CONCLUSIONS: This research highlights some of the many genes that may have been intentionally or unintentionally selected for during the human-mediated dispersal of breadfruit and stresses the importance of conserving a varied germplasm collection. It has revealed candidate genes for further study and produced new genomic resources for breadfruit.

Origin and diversity of an underutilized fruit tree crop, cempedak (Artocarpus integer, Moraceae).

PREMISE OF THE STUDY: Underutilized crops and their wild relatives are important resources for crop improvement and food security. Cempedak [Artocarpus integer (Thunb). Merr.] is a significant crop in Malaysia but underutilized elsewhere. Here we performed molecular characterization of cempedak and its putative wild relative bangkong (Artocarpus integer (Thunb). Merr. var. silvestris Corner) to address questions regarding the origin and diversity of cempedak. METHODS: Using data from 12 microsatellite loci, we assessed the genetic diversity and genetic/geographic structure for 353 cempedak and 175 bangkong accessions from Malaysia and neighboring countries and employed clonal analysis to characterize cempedak cultivars. We conducted haplotype network analyses on the trnH-psbA region in a subset of these samples. We also analyzed key vegetative characters that reportedly differentiate cempedak and bangkong. KEY RESULTS: We show that cempedak and bangkong are sister taxa and distinct genetically and morphologically, but the directionality of domestication origin is unclear. Genetic diversity was generally higher in bangkong than in cempedak. We found a distinct genetic cluster for cempedak from Borneo as compared to cempedak from Peninsular Malaysia. Finally, cempedak cultivars with the same names did not always share the same genetic fingerprint. CONCLUSIONS: Cempedak origins are complex, with likely admixture and hybridization with bangkong, warranting further investigation. We provide a baseline of genetic diversity of cempedak and bangkong in Malaysia and found that germplasm collections in Malaysia represent diverse coverage of the four cempedak genetic clusters detected.

Allele phasing has minimal impact on phylogenetic reconstruction from targeted nuclear gene sequences in a case study of Artocarpus.

PREMISE OF THE STUDY: Untapped information about allele diversity within populations and individuals (i.e., heterozygosity) could improve phylogenetic resolution and accuracy. Many phylogenetic reconstructions ignore heterozygosity because it is difficult to assemble allele sequences and combine allele data across unlinked loci, and it is unclear how reconstruction methods accommodate variable sequences. We review the common methods of including heterozygosity in phylogenetic studies and present a novel method for assembling allele sequences from target-enriched Illumina sequencing libraries. METHODS: We performed supermatrix phylogeny reconstruction and species tree estimation of Artocarpus based on three methods of accounting for heterozygous sequences: a consensus method based on de novo sequence assembly, the use of ambiguity characters, and a novel method for incorporating read information to phase alleles. We characterize the extent to which highly heterozygous sequences impeded phylogeny reconstruction and determine whether the use of allele sequences improves phylogenetic resolution or decreases topological uncertainty. KEY RESULTS: We show here that it is possible to infer phased alleles from target-enriched Illumina libraries. We find that highly heterozygous sequences do not contribute disproportionately to poor phylogenetic resolution and that the use of allele sequences for phylogeny reconstruction does not have a clear effect on phylogenetic resolution or topological consistency. CONCLUSIONS: We provide a framework for inferring phased alleles from target enrichment data and for assessing the contribution of allelic diversity to phylogenetic reconstruction. In our data set, the impact of allele phasing on phylogeny is minimal compared to the impact of using phylogenetic reconstruction methods that account for gene tree incongruence.

Cloning of a novel lectin from Artocarpus lingnanensis that induces apoptosis in human B-lymphoma cells.

We isolated a novel lectin (Artocarpus nitidus subsp. lingnanensis lectin, ALL) from Artocarpus nitidus subsp. lingnanensis and showed its mitogenic activities. In this study, we determined the amino acid sequence of ALL by cDNA sequencing. ALL cDNA (933 bp) contains a 657-bp open reading frame (ORF), which encodes a protein with 218 amino acids. ALL shares high sequence similarities with Jacalin and Morniga G and belongs to jacalin-related lectin family. We also examined the antitumor activity of ALL using Raji, a human B-lymphoma cell line. ALL exhibits a strong binding affinity to cell membrane, which can be effectively inhibited by N-acetyl-D-galactosamine (GalNAc). ALL inhibits Raji cell proliferation in a time- and dose-dependent manner through apoptosis, evidenced by morphological changes, phosphatidylserine externalization, poly ADP-ribose polymerase (PARP) cleavage, Bcl-2 down-regulation, and caspase-3 activation. We further showed that the activation of p38 mitogen-activated protein kinase (MAPK) signaling pathways is required for the pro-apoptotic activity of ALL.

Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights.

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin (FTP) and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. FTP is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically active recombinant FTP in Escherichia coli BL21, optimizing conditions with the best set yielding >40 mg/l culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/ml of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP-mannose and FTP-glucose crystals were obtained, and they diffracted X-rays to a resolution of 1.58 (P212121), 1.70 (P3121) and 1.60 (P3121) Å respectively. The best solution showed four monomers per asymmetric unit. Molecular dynamics (MD) simulation suggested that FTP displays higher affinity for mannose than glucose. Cell studies revealed that FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/ml and was also capable of stimulating cell migration at 50 µg/ml. In conclusion, our optimized expression system allowed high amounts of correctly folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example in wound healing and tissue regeneration.

Transcriptome and selected metabolite analyses reveal points of sugar metabolism in jackfruit (Artocarpus heterophyllus Lam.).

Artocarpus heterophyllus Lam., commonly known as jackfruit, produces the largest tree-borne fruit known thus far. The edible part of the fruit develops from the perianths, and contains many sugar-derived compounds. However, its sugar metabolism is poorly understood. A fruit perianth transcriptome was sequenced on an Illumina HiSeq 2500 platform, producing 32,459 unigenes with an average length of 1345nt. Sugar metabolism was characterized by comparing expression patterns of genes related to sugar metabolism and evaluating correlations with enzyme activity and sugar accumulation during fruit perianth development. During early development, high expression levels of acid invertases and corresponding enzyme activities were responsible for the rapid utilization of imported sucrose for fruit growth. The differential expression of starch metabolism-related genes and corresponding enzyme activities were responsible for starch accumulated before fruit ripening but decreased during ripening. Sucrose accumulated during ripening, when the expression levels of genes for sucrose synthesis were elevated and high enzyme activity was observed. The comprehensive transcriptome analysis presents fundamental information on sugar metabolism and will be a useful reference for further research on fruit perianth development in jackfruit.

cDNA cloning and functional expression of the alpha-D-galactose-binding lectin frutalin in Escherichia coli.

cDNA clones encoding frutalin, the alpha-D-galactose-binding lectin expressed in breadfruit seeds (Artocarpus incisa), were isolated and sequenced. The deduced amino acid sequences indicated that frutalin may be encoded by a family of genes. The NCBI database searches revealed that the frutalin sequence is highly homologous with jacalin and mornigaG sequences. Frutalin cDNA was re-amplified and cloned into the commercial expression vector pET-25b(+) for frutalin production in Escherichia coli. An experimental factorial design was employed to maximise the soluble expression of the recombinant lectin. The results indicated that temperature, time of induction, concentration of IPTG and the interaction between the concentration of IPTG and the time of induction had the most significant effects on the soluble expression level of recombinant frutalin. The optimal culture conditions were as follows: induction with 1 mM IPTG at 22 degrees C for 20 h, yielding 16 mg/l of soluble recombinant frutalin. SDS-PAGE and Western blot analysis revealed that recombinant frutalin was successfully expressed by bacteria with the expected molecular weight (17 kDa). These analyses also showed that recombinant frutalin was mainly produced as insoluble protein. Recombinant frutalin produced by bacteria revealed agglutination properties and carbohydrate-binding specificity similar to the native breadfruit lectin.

Evaluation of genetic diversity in jackfruit (Artocarpus heterophyllus Lam.) based on amplified fragment length polymorphism markers.

Artocarpus heterophyllus Lam., commonly called jackfruit, is a medium-sized evergreen tree that bears high yields of the largest known edible fruit. Yet, it has been little explored commercially due to wide variation in fruit quality. The genetic diversity and genetic relatedness of 50 jackfruit accessions were studied using amplified fragment length polymorphism markers. Of 16 primer pairs evaluated, eight were selected for screening of genotypes based on the number and quality of polymorphic fragments produced. These primer combinations produced 5976 bands, 1267 (22%) of which were polymorphic. Among the jackfruit accessions, the similarity coefficient ranged from 0.137 to 0.978; the accessions also shared a large number of monomorphic fragments (78%). Cluster analysis and principal component analysis grouped all jackfruit genotypes into three major clusters. Cluster I included the genotypes grown in a jackfruit region of Karnataka, called Tamaka, with very dry conditions; cluster II contained the genotypes collected from locations having medium to heavy rainfall in Karnataka; cluster III grouped the genotypes in distant locations with different environmental conditions. Strong coincidence of these amplified fragment length polymorphism-based groupings with geographical localities as well as morphological characters was observed. We found moderate genetic diversity in these jackfruit accessions. This information should be useful for tree breeding programs, as part of our effort to popularize jackfruit as a commercial crop.

Antiproliferative effect of T/Tn specific Artocarpus lakoocha agglutinin (ALA) on human leukemic cells (Jurkat, U937, K562) and their imaging by QD-ALA nanoconjugate.

T/Tn specificity of Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha (Moraceae) fruit and a heterodimer (16 kD and 12 kD) of molecular mass 28 kD, was further confirmed by SPR analysis using T/Tn glycan containing mammalian glycoproteins. N-terminal amino acid sequence analysis of ALA showed homology at 15, 19-21, 24-27, and 29 residues with other lectin members of Moraceae family viz., Artocarpus integrifolia (jacalin) lectin, Artocarpus hirsuta lectin, and Maclura pomifera agglutinin. It is mitogenic to human PBMC and the maximum proliferation was observed at 1 ng/ml. It showed an antiproliferative effect on leukemic cells, with the highest effect toward Jurkat cells (IC(50) 13.15 ng/ml). Synthesized CdS quantum dot-ALA nanoconjugate was employed to detect the expression of T/Tn glycans on Jurkat, U937, and K562 leukemic cells surfaces as well as normal lymphocytes by fluorescence microscopy. No green fluorescence was observed with normal lymphocytes indicating that T/Tn determinants, which are recognized as human tumor associated structures were cryptic on normal lymphocyte surfaces, whereas intense green fluorescent dots appeared during imaging of leukemic cells, where such determinants were present in unmasked form. The above results indicated that QD-ALA nanoconjugate is an efficient fluorescent marker for identification of leukemic cell lines that gives rise to high quality images.

cDNA cloning and functional expression of KM+, the mannose-binding lectin from Artocarpus integrifolia seeds.

KM+, a mannose-binding lectin present in the seeds of Artocarpus integrifolia, has interesting biological properties and potential pharmaceutical use [A. Panunto-Castelo, M.A. Souza, M.C. Roque-Barreira, J.S. Silva, KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection, Glycobiology 11 (2001) 1035-1042. ; L.L.P. daSilva, A. Panunto-Castelo, M.H.S. Goldman, M.C. Roque-Barreira, R.S. Oliveira, M.D. Baruffi, J.B. Molfetta-Machado, Composition for preventing or treating appearance of epithelia wounds such as skin and corneal wounds or for immunomodulating, comprises lectin, Patent number WO20041008.]. Here, we have isolated clones encoding the full-length KM+ primary sequence from a cDNA library, through matrix PCR-based screening methodology. Analysis of KM+ nucleotide and deduced amino acid sequences provided strong evidence that it neither enters the secretory pathway nor undergoes post-translational modifications, which is in sharp contrast with jacalin, the more abundant lectin from A. integrifolia seeds. Current investigations into the KM+ properties are often impaired by the difficulty in obtaining sufficient quantities of jacalin-free KM+ through direct seed extraction. To obtain active recombinant protein (rKM+) in larger amounts, we tested three different expression systems. Expression vectors were constructed to produce: (a) rKM+ in E. coli in its native form, (b) rKM+ with GST as an N-terminal tag and (c) native rKM+ in Saccharomyces cerevisiae. The presence of the GST-tag significantly improved the overall rKM+ yield; however, most of the obtained rGST-KM+ was insoluble. Production of rKM+ in the yeast host yielded the highest quantities of soluble lectin that retained the typical high-mannose oligosaccharide-binding properties of the natural protein. The possible biotechnological applications of recombinant KM+ are discussed.