Assembly of a hybrid mangrove, Bruguiera hainesii, and its two ancestral contributors, Bruguiera cylindrica and Bruguiera gymnorhiza.

Mangroves are plants that live in tropical and subtropical coastal regions of the world, they are adapted to high salt environments and cyclic tidal flooding. Mangroves play important ecological roles, including acting as breeding grounds for many fish species and to prevent coastal erosion. The genomes of three mangrove species, Bruguiera gymnorhiza, Bruguiera cylindrica, and a hybrid of the two, Bruguiera hainesii were sequenced, assembled and annotated. The two progenitor species, B. gymnorhiza and B. cylindrica, were found to be highly similar to each other and sufficiently similar to B. parviflora to allow it to be used for reference based scaffolding to generate chromosome level scaffolds. The two subgenomes of B. hainesii were independently assembled and scaffolded. Analysis of B. hainesii confirms that it is a hybrid and the hybridisation event was estimated at 2.4 to 3.5 million years ago using a Bayesian Relaxed Molecular Clock approach.

Genome-wide association mapping for grain manganese in rice (Oryza sativa L.) using a multi-experiment approach.

Manganese (Mn) is an essential trace element for plants and commonly contributes to human health; however, the understanding of the genes controlling natural variation in Mn in crop plants is limited. Here, the integration of two of genome-wide association study approaches was used to increase the identification of valuable quantitative trait loci (QTL) and candidate genes responsible for the concentration of grain Mn across 389 diverse rice cultivars grown in Arkansas and Texas, USA, in multiple years. Single-trait analysis was initially performed using three different SNP datasets. As a result, significant loci could be detected using the high-density SNP dataset. Based on the 5.2 M SNP dataset, major QTLs were located on chromosomes 3 and 7 for Mn containing six candidate genes. In addition, the phenotypic data of grain Mn concentration were combined from three flooded-field experiments from the two sites and 3 years using multi-experiment analysis based on the 5.2 M SNP dataset. Two previous QTLs on chromosome 3 were identified across experiments, whereas new Mn QTLs were identified that were not found in individual experiments, on chromosomes 3, 4, 9 and 11. OsMTP8.1 was identified in both approaches and is a good candidate gene that could be controlling grain Mn concentration. This work demonstrates the utilisation of multi-experiment analysis to identify constitutive QTLs and candidate genes associated with the grain Mn concentration. Hence, the approach should be advantageous to facilitate genomic breeding programmes in rice and other crops considering QTLs and genes associated with complex traits in natural populations.

Genome-wide SNP discovery and identification of QTL associated with agronomic traits in oil palm using genotyping-by-sequencing (GBS).

Oil palm has become one of the most important oil crops in the world. Marker-assisted selections have played a pivotal role in oil palm breeding programs. Here, we report the use of genotyping-by-sequencing (GBS) approach for a large-scale SNP discovery and genotyping of a mapping population. Reduced representation libraries of 108 F2 progeny were sequenced and a total of 524 million reads were obtained. We detected 21,471 single nucleotide substitutions, most of which (62.6%) represented transition events. Of 3417 fully informative SNP markers, we were able to place 1085 on a linkage map, which spanned 1429.6 cM and had an average of one marker every 1.26 cM. Three QTL affecting trunk height were detected on LG 10, 14 and 15, whereas a single QTL associated with fruit bunch weight was identified on LG 3. The use of GBS approach proved to be rapid, cost-effective and highly reproducible in this species.

The AKR gene family and modifying sex ratios in palms through abiotic stress responsiveness.

Sex ratio (SR), the ratio of female inflorescences to total inflorescences, is one of the main yield components of oil palm (Elaeis guineensis Jacq). The SR quantitative trait locus (QTL) was recently identified on linkage (LG) 8 with a phenotype variance explained (PVE) of 11.3 %. The use of both genetic and physical mapping is one strategy for uncovering the genetic basis of the traits. Here, we report the construction of bacterial artificial chromosome (BAC) and fosmid libraries, and their use for physical mapping in oil palm. Combined, the libraries consist of more than 200,000 clones, representing 6.35 genome equivalents. Physical mapping at the SR locus was implemented by incorporating the published oil palm genome sequence and positive BAC/fosmid clones as identified by colony PCR screening. Based on the previously published sequences, the interval (about 184 kb) was comprised of 19 contigs of the known sequences (~117 kb, 64 %). After, combining the 454 pyrosequences of 15 positive clones and the previously published sequences, the known sequences were revealed to cover about 82 % of the interval (~150 kb), and were used for identifying the new markers by designing 35 gene-based and 23 simple sequence repeat (SSR)-amplified primers. As a result, a putative aldo-keto reductase gene (named EgAKR1) was revealed to be a promising candidate for sex ratio determination, via controlling female inflorescence number (11 % of PVE). This was predicted from the two newly identified polymorphic marker loci (mEgSSRsr8-21LB and mEgAKR1-9) designing from EgAKR1. The functions of AKR gene families in other plant species and our promoter analysis suggested that EgAKR1 may contribute to the sex ratio through abiotic stress responsiveness.

Assembly and analysis of a male sterile rubber tree mitochondrial genome reveals DNA rearrangement events and a novel transcript.

BACKGROUND: The rubber tree, Hevea brasiliensis, is an important plant species that is commercially grown to produce latex rubber in many countries. The rubber tree variety BPM 24 exhibits cytoplasmic male sterility, inherited from the variety GT 1. RESULTS: We constructed the rubber tree mitochondrial genome of a cytoplasmic male sterile variety, BPM 24, using 454 sequencing, including 8 kb paired-end libraries, plus Illumina paired-end sequencing. We annotated this mitochondrial genome with the aid of Illumina RNA-seq data and performed comparative analysis. We then compared the sequence of BPM 24 to the contigs of the published rubber tree, variety RRIM 600, and identified a rearrangement that is unique to BPM 24 resulting in a novel transcript containing a portion of atp9. CONCLUSIONS: The novel transcript is consistent with changes that cause cytoplasmic male sterility through a slight reduction to ATP production efficiency. The exhaustive nature of the search rules out alternative causes and supports previous findings of novel transcripts causing cytoplasmic male sterility.

Characterization of rubber tree microRNA in phytohormone response using large genomic DNA libraries, promoter sequence and gene expression analysis.

The para rubber tree is the most widely cultivated tree species for producing natural rubber (NR) latex. Unfortunately, rubber tree characteristics such as a long life cycle, heterozygous genetic backgrounds, and poorly understood genetic profiles are the obstacles to breeding new rubber tree varieties, such as those with improved NR yields. Recent evidence has revealed the potential importance of controlling microRNA (miRNA) decay in some aspects of NR regulation. To gain a better understanding of miRNAs and their relationship with rubber tree gene regulation networks, large genomic DNA insert-containing libraries were generated to complement the incomplete draft genome sequence and applied as a new powerful tool to predict a function of interested genes. Bacterial artificial chromosome and fosmid libraries, containing a total of 120,576 clones with an average insert size of 43.35 kb, provided approximately 2.42 haploid genome equivalents of coverage based on the estimated 2.15 gb rubber tree genome. Based on these library sequences, the precursors of 1 member of rubber tree-specific miRNAs and 12 members of conserved miRNAs were successfully identified. A panel of miRNAs was characterized for phytohormone response by precisely identifying phytohormone-responsive motifs in their promoter sequences. Furthermore, the quantitative real-time PCR on ethylene stimulation of rubber trees was performed to demonstrate that the miR2118, miR159, miR164 and miR166 are responsive to ethylene, thus confirmed the prediction by genomic DNA analysis. The cis-regulatory elements identified in the promoter regions of these miRNA genes help augment our understanding of miRNA gene regulation and provide a foundation for further investigation of the regulation of rubber tree miRNAs.

Genetic variation and geographic distribution of Leishmania orientalis and Leishmania martiniquensis among Leishmania/HIV co-infection in Thailand.

Since 1999, the number of asymptomatic leishmaniasis cases has increased continuously in Thailand, particularly among patients with HIV who are prone to develop symptoms of cutaneous and visceral leishmaniasis further. The asymptomatic infection could play a key role in Leishmania transmission and distribution. Understanding population structure and phylogeographic patterns could be crucially needed to develop effective diagnoses and appropriate guidelines for therapy. In this study, genetic variation and geographic distribution of the Leishmania/HIV co-infected population were investigated in endemic northern and southern Thailand. Interestingly, Leishmania orientalis was common and predominant in these two regions with common regional haplotype distribution but not for the others. Recent population expansion was estimated, probably due to the movement and migration of asymptomatic individuals; therefore, the transmission and prevalence of Leishmania infection could be underestimated. These findings of imbalanced population structure and phylogeographic distribution patterns provide valuable, insightful population structure and geographic distribution of Leishmania/HIV co-infection to empower prevention and control of transmission and expansion of asymptomatic leishmaniasis.

Validation of quantitative loop-mediated isothermal amplification assay using a fluorescent distance-based paper device for detection of Escherichia coli in urine.

Uropathogenic Escherichia coli (UPEC) causes up to 90% of urinary tract infections (UTI) which is more prevalent among females than males. In urine, patients with symptomatic UTI usually have a high concentration of bacterial infection, ≥ 105 colony-forming units (CFU) per mL, in which the culture method is regularly the gold standard diagnosis. In this study, a simple and inexpensive distance-based paper device (dPAD) combined with the fluorescent closed tube LAMP assay was validated for simultaneously screening and semi-quantifying the infection level of E. coli in 440 urine samples of patients with UTI. The dPAD could measure the LAMP amplicons and semi-quantify the levels of E. coli infection in heavy (≥ 104 CFU/mL), light (≤ 103 CFU/mL) and no infection. The sensitivity and specificity had reliable performances, achieving as high as 100 and 92.7%, respectively. The one step LAMP assay could be performed within 3 h, which was 7.5 times faster than the culture method. To empower early UTI diagnosis and fast treatment, this inexpensive dPAD tool combined with the fluorescent closed tube LAMP assay is simple, reliably fast and practically portable for point-of-care settings, particularly in resource-limited areas, which can be set up in all levels of healthcare facilities.

Comparative Analysis and Phylogenetic Relationships of Ceriops Species (Rhizophoraceae) and Avicennia lanata (Acanthaceae): Insight into the Chloroplast Genome Evolution between Middle and Seaward Zones of Mangrove Forests.

Ceriops and Avicennia are true mangroves in the middle and seaward zones of mangrove forests, respectively. The chloroplast genomes of Ceriops decandra, Ceriops zippeliana, and Ceriops tagal were assembled into lengths of 166,650, 166,083 and 164,432 bp, respectively, whereas Avicennia lanata was 148,264 bp in length. The gene content and gene order are highly conserved among these species. The chloroplast genome contains 125 genes in A. lanata and 129 genes in Ceriops species. Three duplicate genes (rpl2, rpl23, and trnM-CAU) were found in the IR regions of the three Ceriops species, resulting in expansion of the IR regions. The rpl32 gene was lost in C. zippeliana, whereas the infA gene was present in A. lanata. Short repeats (<40 bp) and a lower number of SSRs were found in A. lanata but not in Ceriops species. The phylogenetic analysis supports that all Ceriops species are clustered in Rhizophoraceae and A. lanata is in Acanthaceae. In a search for genes under selective pressures of coastal environments, the rps7 gene was under positive selection compared with non-mangrove species. Finally, two specific primer sets were developed for species identification of the three Ceriops species. Thus, this finding provides insightful genetic information for evolutionary relationships and molecular markers in Ceriops and Avicennia species.

Distance-based paper device using combined SYBR safe and gold nanoparticle probe LAMP assay to detect Leishmania among patients with HIV.

Asymptomatic visceral leishmaniasis cases increase continuously, particularly among patients with HIV who are at risk to develop further symptoms of leishmaniasis. A simple, sensitive and reliable diagnosis is crucially needed due to risk populations mostly residing in rural communities with limited resources of laboratory equipment. In this study, a highly sensitive and selective determination of Leishmania among asymptomatic patients with Leishmania/HIV co-infection was achieved to simultaneously interpret and semi-quantify using colorimetric precipitates (gold-nanoparticle probe; AuNP-probe) and fluorescence (SYBR safe dye and distance-based paper device; dPAD) in one-step loop-mediated isothermal amplification (LAMP) assay. The sensitivities and specificities of 3 detection methods were equivalent and had reliable performances achieving as high as 95.5%. Detection limits were 102 parasites/mL (0.0147 ng/µL) which were 10 times more sensitive than other related studies. To empower leishmaniasis surveillance as well as prevention and control, this dPAD combined with SYBR safe and gold nanoparticle probe LAMP assay is reliably fast, simple, inexpensive and practical for field diagnostics to point-of-care settings in resource-limited areas which can be set up in all levels of healthcare facilities, especially in low to middle income countries.

Assessment of the Genetic Diversity and Population Structure of Rhizophora apiculata Blume (Rhizophoraceae) in Thailand.

Rhizophora apiculata is one of the most widespread and economically important mangrove trees in the Indo-West Pacific region. Knowledge of the genetic variation of R. apiculata in Thailand is limited. Here, we generated a whole-genome sequence of R. apiculata using the 10× Genomics technology. R. apiculata genome assembly was 230.47 Mb. Based on its genome, 2640 loci of high-quality biallelic SNPs were identified from 82 R. apiculata accessions collected from 17 natural mangrove forests in Thailand to assess the genetic diversity and population structure among them. A moderate level of genetic diversity of R. apiculata was observed. The average observed heterozygosity (Ho = 0.48) was higher than the average expected heterozygosity (He = 0.36). Two subpopulations were observed and confirmed from three approaches: population structure, PCA, and phylogenetic analyses. They corresponded to the Gulf of Thailand and the Andaman Sea separated by the Malay Peninsula. AMOVA analyses indicated that genetic variation was attributable to 76.22% within populations and 23.78% among populations. A high level of genetic differentiation between the two subpopulations (FST = 0.24, p < 0.001) was observed. This study evaluated the genetic diversity and population structure of R. apiculata, providing useful information for sustainable mangrove management in Thailand.

Zoonotic Transmission of Blastocystis Subtype 1 among People in Eastern Communities of Thailand: Organic Fertilizer from Pig Feces as a Potential Source.

Blastocystis sp., the most common intestinal protozoa, remains a public health problem among people in many countries, particularly in rural areas of developing countries. The infection usually reflects poor sanitation in communities by waterborne, zoonotic, and person-to-person transmission. Interestingly, at least 17 subtypes (STs) have been reported and are associated with a broad range of animal hosts, including humans. In this study, we reported potential evidence of zoonotic transmission of Blastocystis ST1 in rural communities of eastern Thailand where the overall prevalence of Blastocystis infection was 15.7%. Two major and three minor subtypes were found to be distributed unequally in this region. Of 5 STs, only ST1 was found to be associated with pig feces in an open farm system that produced organic fertilizer for agriculture uses in the community. This finding suggests that properly protective contact and standard production of organic fertilizer from pig feces by-products could be key factors for reducing the prevalence of Blastocystis infection and prevent Blastocystis reinfection among people in the community. IMPORTANCEBlastocystis sp. remains a public health problem among people, particularly in rural areas of many developing countries. The infection usually reflects poor sanitation in communities by waterborne, zoonotic, and person-to-person transmission. In this study, we reported potential evidence of zoonotic transmission of Blastocystis subtype 1 (ST1) in rural communities of eastern Thailand. Two major and three minor subtypes were found to be unequally distributed in this region. Interestingly, only ST1 was found to be associated with pig feces in an open farm system that produced organic fertilizer for agriculture uses in the community. The finding makes significant contributions to genetic and molecular investigations of microbial topics of practical value and suggest that properly protective contact and standard production of organic fertilizer from pig feces by-products could be key factors for reducing the prevalence of Blastocystis infection and prevent Blastocystis reinfection among people in the community.

Complete chloroplast genome sequences of five Bruguiera species (Rhizophoraceae): comparative analysis and phylogenetic relationships.

Bruguiera is a genus of true mangroves that are mostly distributed in the Indo-West Pacific region. However, the number of published whole chloroplast genome sequences of Bruguiera species are limited. Here, the complete chloroplast sequences of five Bruguiera species were sequenced and assembled using Illumina data. The chloroplast genomes of B. gymnorhiza, B. hainesii, B. cylindrica, B. parviflora and B. sexangula were assembled into 161,195, 164,295, 164,297, 163,228 and 164,170 bp, respectively. All chloroplast genomes contain 37 tRNA and eight rRNA genes, with either 84 or 85 protein-coding genes. A comparative analysis of these genomes revealed high similarity in gene structure, gene order and boundary position of the LSC, SSC and two IR regions. Interestingly, B. gymnorhiza lost a rpl32 gene in the SSC region. In addition, a ndhF gene in B. parviflora straddles both the SSC and IRB boundary regions. These genes reveal differences in chloroplast evolution among Bruguiera species. Repeats and SSRs in the chloroplast genome sequences were found to be highly conserved between B. cylindrica and B. hainesii as well as B. gymnorhiza and B. sexangula indicating close genetic relationships based on maternal inheritance. Notably, B. hainesii, which is considered a hybrid between B. gymnorhiza and B. cylindrica, appears to have inherited the chloroplast from B. cylindrica. Investigating the effects of selection events on shared protein-coding genes showed a positive selection in rps7 and rpl36 genes in all species compared to land-plant species. A phylogenetic analysis, based on 59 conserved chloroplast protein-coding genes, showed strong support that all Bruguiera species are in the clade Rhizophoraceae. This study provides valuable genetic information for the study of evolutionary relationships and population genetics in Bruguiera and other mangrove species.

Genetic loci regulating cadmium content in rice grains.

It has been estimated that up to 90% of human exposure to cadmium is through food, and that cadmium within rice grains can be a major contributor to that dietary source. In this study genome wide association mapping was conducted on the Bengal and Assam Aus Panel (BAAP) of rice to identify quantitative trait loci and candidate genes for lowering grain cadmium. Field experiments were conducted over two years under two different irrigation systems: continually flooded and alternate wetting and drying (AWD). There was significant effects of water treatment, genotype, and genotype by water treatment interaction. Importantly, AWD increased grain cadmium, on average, by 49.6% and 108.8% in year 1 and 2 respectively. There was between 4.6 and 28 fold variation in cadmium concentration. A total of 58 QTLs were detected but no loci are clearly specific to one water regime despite approximately 20% of variation attributable to genotype by water regime interaction. A number of QTLs were consistent across most water treatments and years. These included QTLs on chromosome 7 (7.23-7.61, 8.93-9.04, and 29.12-29.14 Mbp), chromosome 5 (8.66-8.72 Mbp), and chromosome 9 (11.46-11.64 Mbp). Further analysis of the loci on chromosome 7 (8.93-9.04 Mbp), identified the candidate gene OsNRAMP1, where cultivars with a deletion upstream of the gene had higher concentrations of cadmium compared to the cultivars that did not have the deletion. The distribution of alleles within the BAAP suggest this QTL is easily detected in this population because it is composed of aus cultivars. Local genome cluster analysis suggest high Cd alleles are uncommon, but should be avoided in breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s10681-020-02752-1).

The complete mitochondrial genome of Luffa acutangula.

Based on PacBio de novo assembly, we report the first complete mitochondrial genome of Luffa acutangula (460,333 bp) containing nine large chloroplast-derived sequences (1.9-17.3 kb) across the mitogenome. The base composition of the mitogenome in descending order is A: 28.02%, C: 22.04%, G: 21.83% and T: 28.10%, and the G + C content is 43.87%. There are 63 mitochondrial genes including 40 protein-coding genes, 3 rRNA genes and 20 tRNA genes. Additionally, a total of 288 repeats ranging from 31 to 5,301 bp were identified, accounting for 5.7% of the mitogenome. Two large direct repeats (5,301 and 405 bp) within the mitogenome were found for the formation of four subgenomic molecules. A phylogenetic analysis showed that L. acutangula was closely related to other species in Cucurbiaceae. This mitogenome provides useful genetic information for evolutionary studies.

Genome Wide Association Mapping of Grain and Straw Biomass Traits in the Rice Bengal and Assam Aus Panel (BAAP) Grown Under Alternate Wetting and Drying and Permanently Flooded Irrigation.

Growing demand for staple crops like rice will need to be achieved predominately through agricultural intensification and more efficient use of inputs. To meet this demand it is essential that the genetic diversity within rice is fully utilized. The aus subpopulation is considered an underappreciated resource within that diversity. A new rice panel, the Bengal and Assam Aus Panel (BAAP) of 266 aus accessions was generated with ∼2 million informative SNPs obtained using skim sequencing at ∼4× depth. The BAAP was grown in the field in Bangladesh in the 'boro' season under both continuously flooded and Alternate Wetting and Drying (AWD) irrigation during 2013 and 2014 in Mymensingh and during 2014 in Madhupur. Heading date, grain mass, straw biomass and harvest index were measured. The majority (94%) of BAAP accessions flowered within a relatively small window of 10 days. The AWD irrigation treatment generally caused an increase in grain mass, but no significant genotype by treatment interactions were detected for this trait. Shoot biomass was the only trait that showed evidence of genotype by treatment interaction. The average LD (Linkage Disequilibrium) decay across the genome was 243 Kbp. Genome wide association mapping revealed 115 quantitative trait loci (QTLs). There was little evidence of QTLs specific to the irrigation treatment, and only a few QTLs co-localized with known genes. However, some QTLs were detected across multiple sites and years. These QTLs should be targets for breeding, and include a region around 2.2 Mbp on chromosome 1, a large region in the middle of chromosome 7 and two regions on chromosome 11 (∼10 Mbp and ∼29 Mbp). The BAAP appears to be a valuable addition to the growing collection of GWA mapping populations of rice.

De novo hybrid assembly of the rubber tree genome reveals evidence of paleotetraploidy in Hevea species.

Para rubber tree (Hevea brasiliensis) is an important economic species as it is the sole commercial producer of high-quality natural rubber. Here, we report a de novo hybrid assembly of BPM24 accession, which exhibits resistance to major fungal pathogens in Southeast Asia. Deep-coverage 454/Illumina short-read and Pacific Biosciences (PacBio) long-read sequence data were acquired to generate a preliminary draft, which was subsequently scaffolded using a long-range "Chicago" technique to obtain a final assembly of 1.26 Gb (N50 = 96.8 kb). The assembled genome contains 69.2% repetitive sequences and has a GC content of 34.31%. Using a high-density SNP-based genetic map, we were able to anchor 28.9% of the genome assembly (363 Mb) associated with over two thirds of the predicted protein-coding genes into rubber tree's 18 linkage groups. These genetically anchored sequences allowed comparative analyses of the intragenomic homeologous synteny, providing the first concrete evidence to demonstrate the presence of paleotetraploidy in Hevea species. Additionally, the degree of macrosynteny conservation observed between rubber tree and cassava strongly supports the hypothesis that the paleotetraploidization event took place prior to the divergence of the Hevea and Manihot species.

Comparative mitochondrial genome analysis of Pythium insidiosum and related oomycete species provides new insights into genetic variation and phylogenetic relationships.

Oomycetes are eukaryotic microorganisms, which are phylogenetically distinct from the true-fungi, which they resemble morphologically. While many oomycetes are pathogenic to plants, Pythium insidiosum is capable of infecting humans and animals. Mitochondrial (mt) genomes are valuable genetic resources for exploring the evolution of eukaryotes. During the course of 454-based nuclear genome sequencing, we identified a complete 54.9 kb mt genome sequence, containing 2 large inverted repeats, from P. insidiosum. It contains 65 different genes (including 2 ribosomal RNA genes, 25 transfer RNA genes and 38 genes encoding NADH dehydrogenases, cytochrome b, cytochrome c oxidases, ATP synthases, and ribosomal proteins). Thirty-nine of the 65 genes have two copies, giving a total of 104 genes. A set of 30 conserved protein-coding genes from the mt genomes of P. insidiosum, 11 other oomycetes, and 2 diatoms (outgroup) were used for phylogenetic analyses. The oomycetes can be classified into 2 phylogenetic groups, in relation to their taxonomic lineages: Saprolegnialean and Peronosporalean. P. insidiosum is more closely related to Pythium ultimum than other oomycetes. In conclusion, the complete mt genome of P. insidiosum was successfully sequenced, assembled, and annotated, providing a useful genetic resource for exploring the biology and evolution of P. insidiosum and other oomycetes.

Construction of a high-density integrated genetic linkage map of rubber tree (Hevea brasiliensis) using genotyping-by-sequencing (GBS).

Construction of linkage maps is crucial for genetic studies and marker-assisted breeding programs. Recent advances in next generation sequencing technologies allow for the generation of high-density linkage maps, especially in non-model species lacking extensive genomic resources. Here, we constructed a high-density integrated genetic linkage map of rubber tree (Hevea brasiliensis), the sole commercial producer of high-quality natural rubber. We applied a genotyping-by-sequencing (GBS) technique to simultaneously discover and genotype single nucleotide polymorphism (SNP) markers in two rubber tree populations. A total of 21,353 single nucleotide substitutions were identified, 55% of which represented transition events. GBS-based genetic maps of populations P and C comprised 1704 and 1719 markers and encompassed 2041 cM and 1874 cM, respectively. The average marker densities of these two maps were one SNP in 1.23-1.25 cM. A total of 1114 shared SNP markers were used to merge the two component maps. An integrated linkage map consisted of 2321 markers and spanned the cumulative length of 2052 cM. The composite map showed a substantial improvement in marker density, with one SNP marker in every 0.89 cM. To our knowledge, this is the most saturated genetic map in rubber tree to date. This integrated map allowed us to anchor 28,965 contigs, covering 135 Mb or 12% of the published rubber tree genome. We demonstrated that GBS is a robust and cost-effective approach for generating a common set of genome-wide SNP data suitable for constructing integrated linkage maps from multiple populations in a highly heterozygous agricultural species.

SNP identification from RNA sequencing and linkage map construction of rubber tree for anchoring the draft genome.

Hevea brasiliensis, or rubber tree, is an important crop species that accounts for the majority of natural latex production. The rubber tree nuclear genome consists of 18 chromosomes and is roughly 2.15 Gb. The current rubber tree reference genome assembly consists of 1,150,326 scaffolds ranging from 200 to 531,465 bp and totalling 1.1 Gb. Only 143 scaffolds, totalling 7.6 Mb, have been placed into linkage groups. We have performed RNA-seq on 6 varieties of rubber tree to identify SNPs and InDels and used this information to perform target sequence enrichment and high throughput sequencing to genotype a set of SNPs in 149 rubber tree offspring from a cross between RRIM 600 and RRII 105 rubber tree varieties. We used this information to generate a linkage map allowing for the anchoring of 24,424 contigs from 3,009 scaffolds, totalling 115 Mb or 10.4% of the published sequence, into 18 linkage groups. Each linkage group contains between 319 and 1367 SNPs, or 60 to 194 non-redundant marker positions, and ranges from 156 to 336 cM in length. This linkage map includes 20,143 of the 69,300 predicted genes from rubber tree and will be useful for mapping studies and improving the reference genome assembly.