A SNP variation in the Sucrose synthase (SoSUS) gene associated with sugar-related traits in sugarcane.

Background: Sugarcane (Saccharum spp.) is an economically significant crop for both the sugar and biofuel industries. Breeding sugarcane cultivars with high-performance agronomic traits is the most effective approach for meeting the rising demand for sugar and biofuels. Molecular markers associated with relevant agronomic traits could drastically reduce the time and resources required to develop new sugarcane varieties. Previous sugarcane candidate gene association analyses have found single nucleotide polymorphism (SNP) markers associated with sugar-related traits. This study aims to validate these associated SNP markers of six genes, including Lesion simulating disease 1 (LSD), Calreticulin (CALR), Sucrose synthase 1 (SUS1), DEAD-box ATP-dependent RNA helicase (RH), KANADI1 (KAN1), and Sodium/hydrogen exchanger 7 (NHX7), in a diverse population in 2-year and two-location evaluations. Methods: After genotyping of seven targeted SNP markers was performed by PCR Allelic Competitive Extension (PACE) SNP genotyping, the association with sugar-related traits and important cane yield component traits was determined on a set of 159 sugarcane genotypes. The marker-trait relationships were validated and identified by both t-test analysis and an association analysis based on the general linear model. Results: The mSoSUS1_SNPCh10.T/C and mSoKAN1_SNPCh7.T/C markers that were designed from the SUS1 and KAN1 genes, respectively, showed significant associations with different amounts of sugar-related traits and yield components. The mSoSUS1_SNPCh10.T/C marker was found to have more significant association with sugar-related traits, including pol, CCS, brix, fiber and sugar yield, with p values of 6.08 × 10-6 to 4.35 × 10-2, as well as some cane yield component traits with p values of 1.61 × 10-4 to 3.35 × 10-2. The significant association is consistent across four environments. Conclusion: Sucrose synthase (SUS) is considered a crucial enzyme involved in sucrose metabolism. This marker is a high potential functional marker that may be used in sugarcane breeding programs to select superior sugarcane with good fiber and high sugar contents.

A SNP variation in an expansin (EgExp4) gene affects height in oil palm.

Oil palm (Elaeis guineensis Jacq.), an Aracaceae family plant, is utilized for both consumable and non-consumable products, including cooking oil, cosmetics and biodiesel production. Oil palm is a perennial tree with 25 years of optimal harvesting time and a height of up to 18 m. However, harvesting of oil palm fruit bunches with heights of more than 2-3 meters is challenging for oil palm farmers. Thus, understanding the genetic control of height would be beneficial for using gene-based markers to speed up oil palm breeding programs to select semi-dwarf oil palm varieties. This study aims to identify Insertion/Deletions (InDels) and single nucleotide polymorphisms (SNPs) of five height-related genes, including EgDELLA1, EgGRF1, EgGA20ox1, EgAPG1 and EgExp4, in short and tall oil palm groups by PacBio SMRT sequencing technology. Then, the SNP variation's association with height was validated in the Golden Tenera (GT) population. All targeted genes were successfully amplified by two rounds of PCR amplification with expected sizes that ranged from 2,516 to 3,015 base pair (bp), covering 5' UTR, gene sequences and 3' UTR from 20 short and 20 tall oil palm trees. As a result, 1,166, 909, 1,494, 387 and 5,384 full-length genomic DNA sequences were revealed by PacBio SMRT sequencing technology, from EgDELLA1, EgGRF1, EgGA20ox1, EgAPG1 and EgExp4 genes, respectively. Twelve variations, including eight InDels and four SNPs, were identified from EgDELLA1, EgGRF1, EgGA20ox1 and EgExp4. No variation was found for EgAPG1. After SNP through-put genotyping of 4 targeted SNP markers was done by PACE™ SNP genotyping, the association with height was determined in the GT population. Only the mEgExp4_SNP118 marker, designed from EgExp4 gene, was found to associate with height in 2 of 4 height-recordings, with p values of 0.0383 for height (HT)-1 and 0.0263 for HT-4. In conclusion, this marker is a potential gene-based marker that may be used in oil palm breeding programs for selecting semi-dwarf oil palm varieties in the near future.

Characterization of centromeric satellite DNAs (MALREP) in the Asian swamp eel (Monopterus albus) suggests the possible origin of repeats from transposable elements.

Centromeric satellite DNA (cen-satDNA) sequences of the Asian swamp eel (Monopterus albus) were characterized. Three GC-rich cen-satDNA sequences were detected as a 233 bp MALREP-A and a 293 bp MALREP-B localized to all chromosomes, and a 293 bp MALREP-C distributed on eight chromosome pairs. Sequence lengths of MALREP-B and MALREP-C were 60 bp larger than that of MALREP-A, showing partial homology with core sequences (233 bp). Size differences between MALREP-A and MALREP-B/C suggest the possible occurrence of two satDNA families. The presence of an additional 60 bp in MALREP-B/C resulted from an ancient dimer of 233 bp monomers and subsequent mutation and homogenization between the two monomers. All MALREPs showed partial homology with transposable elements (TEs), suggesting that the MALREPs originated from the TEs. The MALREPs might have been acquired in the Asian swamp eel, thereby promoting fixation in the species.

Transcriptome analysis of oil palm inflorescences revealed candidate genes for an auxin signaling pathway involved in parthenocarpy.

Oil palm parthenocarpic fruits, which are produced without fertilization, can be targeted to increase oil content because the majority of the fruit is occupied by mesocarp, the part in which palm oil is stored. Consequently, gaining an understanding of the parthenocarpic mechanism would be instrumental for producing parthenocarpic oil palm. This study aims to determine effects of auxin treatment and analyze differentially expressed genes in oil palm pistils at the pollination/anthesis stage, using an RNA sequencing (RNA seq) approach. The auxin treatment caused 100% parthenocarpy when auxin was sprayed before stigmas opened. The parthenocarpy decreased to 55%, 8% and 5% when the auxin was sprayed 1, 2 and 3 days after the opening of stigmas, respectively. Oil palm plants used for RNA seq were plants untreated with auxin as controls and auxin-treated plants on the day before pollination and 1 day after pollination. The number of raw reads ranged from 8,425,859 to 11,811,166 reads, with an average size ranging from 99 to 137 base pairs (bp). When compared with the oil palm transcriptome, the mapped reads ranged from 8,179,948 to 11,320,799 reads, representing 95.85-98.01% of the oil palm matching. Based on five comparisons between RNA seq of treatments and controls, and confirmation using reverse transcription polymerase chain reaction and quantitative real-time RT-PCR expression, five candidate genes, including probable indole-3-acetic acid (IAA)-amido synthetase GH3.8 (EgGH3.8), IAA-amido synthetase GH3.1 (EgGH3.1), IAA induced ARG7 like (EgARG7), tryptophan amino transferase-related protein 3-like (EgTAA3) and flavin-containing monooxygenase 1 (EgFMO1), were differentially expressed between auxin-treated and untreated samples. This evidence suggests a pathway of parthenocarpic fruit development at the beginning of fruit development. However, more research is needed to identify which genes are definitely involved in parthenocarpy.

Karyological characterization and identification of four repetitive element groups (the 18S - 28S rRNA gene, telomeric sequences, microsatellite repeat motifs, Rex retroelements) of the Asian swamp eel (Monopterus albus).

Among teleost fishes, Asian swamp eel (Monopterus albus Zuiew, 1793) possesses the lowest chromosome number, 2n = 24. To characterize the chromosome constitution and investigate the genome organization of repetitive sequences in M. albus, karyotyping and chromosome mapping were performed with the 18S - 28S rRNA gene, telomeric repeats, microsatellite repeat motifs, and Rex retroelements. The 18S - 28S rRNA genes were observed to the pericentromeric region of chromosome 4 at the same position with large propidium iodide and C-positive bands, suggesting that the molecular structure of the pericentromeric regions of chromosome 4 has evolved in a concerted manner with amplification of the 18S - 28S rRNA genes. (TTAGGG)n sequences were found at the telomeric ends of all chromosomes. Eight of 19 microsatellite repeat motifs were dispersedly mapped on different chromosomes suggesting the independent amplification of microsatellite repeat motifs in M. albus. Monopterus albus Rex1 (MALRex1) was observed at interstitial sites of all chromosomes and in the pericentromeric regions of most chromosomes whereas MALRex3 was scattered and localized to all chromosomes and MALRex6 to several chromosomes. This suggests that these retroelements were independently amplified or lost in M. albus. Among MALRexs (MALRex1, MALRex3, and MALRex6), MALRex6 showed higher interspecific sequence divergences from other teleost species in comparison. This suggests that the divergence of Rex6 sequences of M. albus might have occurred a relatively long time ago.

ACC oxidase and miRNA 159a, and their involvement in fresh fruit bunch yield (FFB) via sex ratio determination in oil palm.

Oil palm (Elaeis guineesis Jacq.) is the most productive oil-bearing crop, yielding more oil per area than any other oil-bearing crops. However, there are still efforts to improve oil palm yield, in order to serve consumer and manufacturer demand. Oil palm produces female and male inflorescences in an alternating cycle. So, high sex ratio (SR), the ratio of female inflorescences to the total inflorescences, is a favorable trait in term of increasing yields in oil palm. This study aims to understand the genetic control for SR related traits, such as fresh fruit bunch yield (FFB), by characterizing genes at FFB quantitative trait loci (QTLs) on linkage 10 (chromosome 6) and linkage 15 (chromosome 10). Published oil palm sequences at the FFB QTLs were used to develop gene-based and simple sequence repeat (SSR) markers. We used the multiple QTL analysis model (MQM) to characterize the relationship of new markers with the SR traits in the oil palm population. The RNA expression of the most linked QTL genes was also evaluated in various tissues of oil palm. We identified EgACCO1 (encoding aminocyclopropane carboxylate (ACC) oxidase) at chromosome 10 and EgmiR159a (microRNA 159a) at chromosome 6 to be the most linked QTL genes or determinants for FFB yield and/or female inflorescence number with a phenotype variance explained (PVE) from 10.4 to 15 % and suggest that these play the important roles in sex determination and differentiation in oil palm. The strongest expression of EgACCO1 and the predicted precursor of EgmiR159a was found in ovaries and, to a lesser extent, fruit development. In addition, highly normalized expression of EgmiR159a was found in female flowers. In summary, the QTL analysis and the RNA expression reveal that EgACCO1 and EgmiR159a are the potential genetic factors involved in female flower determination and hence would affect yield in oil palm. However, to clarify how these genetic factors regulate female flower determination, more investigation of their down regulation or target may be essential. Additionally, if more sex determination genes controlled by plant hormones are identified, it may possible to reveal a crosstalk of sex determination genes with hormones and environment factors.

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.

Fine mapping of a preharvest sprouting QTL interval on chromosome 2B in white wheat.

KEY MESSAGE: Fine mapping by recombinant backcross populations revealed that a preharvest sprouting QTL on 2B contained two QTLs linked in coupling with different effects on the phenotype. Wheat preharvest sprouting (PHS) occurs when grain germinates on the plant before harvest, resulting in reduced grain quality. Previous mapping of quantitative trait locus (QTL) revealed a major PHS QTL, QPhs.cnl-2B.1, located on chromosome 2B significant in 16 environments that explained from 5 to 31 % of the phenotypic variation. The objective of this project was to fine map the QPhs.cnl-2B.1 interval. Fine mapping was carried out in recombinant backcross populations (BC1F4 and BC1F5) that were developed by backcrossing selected doubled haploids to a recurrent parent and self-pollinating the BC1F4 and BC1F5 generations. In each generation, three markers in the QPhs.cnl-2B.1 interval were used to screen for recombinants. Fine mapping revealed that the QPhs.cnl-2B.1 interval contained two PHS QTLs linked in coupling. The distal PHS QTL, located between Wmc453c and Barc55, contributed 8 % of the phenotypic variation and also co-located with a major seed dormancy QTL determined by germination index. The proximal PHS QTL, between Wmc474 and CNL415-rCDPK, contributed 16 % of the variation. Several candidate genes including Mg-chelatase H subunit family protein, GTP-binding protein and calmodulin/Ca(2+)-dependent protein kinase were linked to the PHS QTL. Although many recombinant lines were identified, the lack of polymorphism for markers in the QTL interval prevented the localization of the recombination breakpoints and identification of the gene underlying the phenotype.

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.

Comparative genetic analysis of a wheat seed dormancy QTL with rice and Brachypodium identifies candidate genes for ABA perception and calcium signaling.

Wheat preharvest sprouting (PHS) occurs when seed germinates on the plant before harvest resulting in reduced grain quality. In wheat, PHS susceptibility is correlated with low levels of seed dormancy. A previous mapping of quantitative trait loci (QTL) revealed a major PHS/seed dormancy QTL, QPhs.cnl-2B.1, located on wheat chromosome 2B. A comparative genetic study with the related grass species rice (Oryza sativa L.) and Brachypodium distachyon at the homologous region to the QPhs.cnl-2B.1 interval was used to identify the candidate genes for marker development and subsequent fine mapping. Expressed sequence tags and a comparative mapping were used to design 278 primer pairs, of which 22 produced polymorphic amplicons that mapped to the group 2 chromosomes. Fourteen mapped to chromosome 2B, and ten were located in the QTL interval. A comparative analysis revealed good macrocollinearity between the PHS interval and 3 million base pair (mb) region on rice chromosomes 7 and 3, and a 2.7-mb region on Brachypodium Bd1. The comparative intervals in rice were found to contain three previously identified rice seed dormancy QTL. Further analyses of the interval in rice identified genes that are known to play a role in seed dormancy, including a homologue for the putative Arabidopsis ABA receptor ABAR/GUN5. Additional candidate genes involved in calcium signaling were identified and were placed in a functional protein association network that includes additional proteins critical for ABA signaling and germination. This study provides promising candidate genes for seed dormancy in both wheat and rice as well as excellent molecular markers for further comparative and fine mapping.

Molecular cloning and functional expression of the Penaeus monodon 5-HT receptor.

Serotonin (5-HT) mediates a number of diverse physiological functions in crustaceans by interacting with various 5-HT receptor subtypes. A putative 5-HT receptor cloned from the ovary of the black tiger prawn (Penaeus monodon) consisted of 2291 nucleotides, encoding a putative 5-HT(1Pem) receptor protein of 591 amino acids. Transient expression of 5-HT(1Pem) in HEK293 cells demonstrated a saturable [3H]-5-HT binding with a Kd of 10.43+/-1.13 nM and Bmax of 1.53+/-0.06 pmol/mg. The putative 5-HT(1Pem) receptor is expressed in all tissues examined and is constitutively expressed in the ovary during ovarian maturation and spent phase. Polyclonal antibodies against the third intracellular loop (i3 loop) of the 5-HT receptor showed that the 5-HT(1Pem) receptor protein was expressed in the trabeculae of ovarian stages 1 and 2 but on the cortical rod and surrounding the oocyte membrane of stages 3 and 4, suggesting that receptor localization plays a critical role in regulating ovarian maturation and spawning in penaeus shrimp.