Male Buds Research Articles

Transcriptome Analysis of Litsea cubeba Floral Buds Reveals the Role of Hormones and Transcription Factors in the Differentiation Process.

Background:Litsea cubeba (Lour.) Pers. is an important economic plant that is rich in valuable essential oil. The essential oil is often used as a raw material for perfumes, food additives, insecticides and bacteriostats. Most of the essential oil is contained in the fruit, and the quantity and quality of fruit are dependent on the flowers. To explore the molecular mechanism of floral bud differentiation, high-throughput RNA sequencing was used to detect differences in the gene expression of L. cubeba female and male floral buds at three differentiation stages.Results:This study obtained 160.88 Gbp of clean data that were assembled into 100,072 unigenes, and a total of 38,658 unigenes were annotated. A total of 27,521 simple sequence repeats (SSRs) were identified after scanning the assembled transcriptome, and the mono-nucleotide repeats were predominant, followed by di-nucleotide and tri-nucleotide repeats. A total of 12,559 differentially expressed genes (DEGs) were detected from the female (F) and male (M) floral bud comparisons. The gene ontology (GO) databases revealed that these DEGs were primarily contained in “metabolic processes”, “cellular processes”, and “single-organism processes”. The Kyoto Encyclopedia of Genes and Genomes (KEGG) databases suggested that the DEGs belonged to “plant hormone signal transduction” and accounted for a relatively large portion in all of these comparisons. We analyzed the expression level of plant hormone-related genes and detected the contents of several relevant plant hormones in different stages. The results revealed that the dynamic changes in each hormone content were almost consistent with the expression levels of relevant genes. The transcription factors selected from the DEGs were analyzed. Most DEGs of MADS-box were upregulated and most DEGs of bZIP were downregulated. The expression trends of the DEGs were nearly identical in female and male floral buds, and qRT-PCR analysis revealed consistency with the transcriptome data.Conclusions:We sequenced and assembled a high-quality L. cubeba floral bud transcriptome, and the data appeared to be well replicated (n = 3) over three developmental time points during flower development. Our study explored the changes in the contents of several plant hormones during floral bud differentiation using biochemical and molecular biology techniques, and the changes in expression levels of several flower development related transcription factors. These results revealed the role of these factors (i.e., hormones and transcription factors) and may advance our understanding of their functions in flower development in L. cubeba.

English

Crop diseases significantly suppress plant yields and in extreme cases wipe out entire crop species threatening food security and eroding rural livelihoods. It is therefore critical to estimate the extent to which shocks like disease epidemics can affect food availability and the capacity of smallholder farmers to mitigate and reverse the effects of such shocks. This study utilizes sex-disaggregated data from 341 households in Uganda to analyze: first, gender and access to agricultural resources and their control; second, whether men and women in the targeted banana-farming communities share similar perceptions toward the effectiveness of the banana Xanthomonas wilt (BXW) control technologies and their respective information dissemination pathways; third, whether gender and farmer perceptions influence on farm adoption of BXW management practices. Lastly, it determines the impact of adoption of BXW control practices on food security. Results show that whereas most household assets are jointly owned, men have more individual ownership, control, and decision-making on income from household assets than women. Perceptions on effectiveness of BXW control practices and communication channels also differed between men and women. Men rated cutting down of infected plants to be more effective than women, but tissue culture, removal of male buds and disinfecting of farm tools were perceived to be equally effective by both men and women. In addition, apart from newspapers which were more effective in delivering BXW information to men, we found no differences in the effectiveness of other BXW information sources. More importantly, the study finds both gender and farmer perceptions on BXW control to significantly affect adoption of BXW control practices and household food security. For better and sustainable management of plant epidemics in Uganda, it is therefore critical that existing gender-based and underlying perception constraints are addressed. Key words: Gender-based constraints, food security, perceptions, technology adoption, Xanthomonas wilt.

Complementary transcriptome and proteome profiling in cabbage buds of a recessive male sterile mutant provides new insights into male reproductive development

Plant male reproductive development is a very complex biological process that involves multiple metabolic pathways. To reveal novel insights into male reproductive development, we conducted an integrated profiling of gene activity in the developing buds of a cabbage recessive genetic male sterile mutant. Using RNA-Seq and label-free quantitative proteomics, 2881 transcripts and 1245 protein species were identified with significant differential abundance between the male sterile line 83121A and its isogenic maintainer line 83121B. Analyses of function annotations and correlations between transcriptome and proteome and protein interaction networks were also conducted, which suggested that the male sterility involves a complex regulatory pattern. Moreover, several key biological processes, such as fatty acid metabolism, tapetosome biosynthesis, amino acid metabolism and protein synthesis and degradation were identified as being of relevance to male reproductive development. A large number of protein species involved in sporopollenin synthesis, amino acid synthesis, ribosome assembly, protein processing in endoplasmic reticulum and lipid transfer were observed to be significantly down-accumulated in 83121A buds, indicating their potential roles in the regulation of cabbage microspore abortion. In summary, the conjoint analysis of the transcriptome and proteome provided a global picture regarding the molecular dynamics in male sterile buds of 83121A. Biological significanceMale sterile mutants are excellent materials for the study of plant male reproductive development. This study revealed the molecular dynamics of recessive male sterility in cabbage at the transcriptome and proteome levels, which deepens our understanding of the metabolic pathways involved in male development. Moreover, the male sterility-related genes identified in this study could provide a reference for the artificial regulation of cabbage fertility by using genetic engineering technology, which may result in potential applications in agriculture such as production of hybrid seeds using male sterility.

Comparative de novo transcriptome analysis of male and female Sea buckthorn.

Sea buckthorn is a dioecious medicinal plant found at high altitude. The plant has both male and female reproductive organs in separate individuals. In this article, whole transcriptome de novo assemblies of male and female flower bud samples were carried out using Illumina NextSeq500 platform to determine the role of the genes involved in sex determination. Moreover, genes with differential expression in male and female transcriptomes were identified to understand the underlying sex determination mechanism. The current study showed 63,904 and 62,272 coding sequences (CDS) in female and male transcriptome data sets, respectively. 16,831 common CDS were screened out from both transcriptomes, out of which 625 were upregulated and 491 were found to be downregulated. To understand the potential regulatory roles of differentially expressed genes in metabolic networks and biosynthetic pathways: KEGG mapping, gene ontology, and co-expression network analysis were performed. Comparison with Flowering Interactive Database (FLOR-ID) resulted in eight differentially expressed genes viz. CHD3-type chromatin-remodeling factor PICKLE (PKL), phytochrome-associated serine/threonine-protein phosphatase (FYPP), protein TOPLESS (TPL), sensitive to freezing 6 (SFR6), lysine-specific histone demethylase 1 homolog 1 (LDL1), pre-mRNA-processing-splicing factor 8A (PRP8A), sucrose synthase 4 (SUS4), ubiquitin carboxyl-terminal hydrolase 12 (UBP12), known to be broadly involved in flowering, photoperiodism, embryo development, and cold response pathways. Male and female flower bud transcriptome data of Sea buckthorn may provide comprehensive information at genomic level for the identification of genetic regulation involved in sex determination.

A Global View of Transcriptome Dynamics During Male Floral Bud Development in Populus tomentosa

To obtain a comprehensive overview of the dynamic transcriptome during male floral bud development in Populus tomentosa, high-throughput RNA-seq was conducted during eight flowering-related stages. Among the 109,212 de novo assembled unigenes, 6,959 were differentially expressed during the eight stages. The overrepresented classed of genes identified by Gene Ontology (GO) enrichment included ‘response to environmental stimuli’ and ‘plant-type spore development’. One-third of the differentially expressed genes were transcription factors (TFs). Several genes and gene families were analyzed in depth, including MADS-box TFs, Squamosa promoter binding protein-like family, receptor-like kinases, FLOWERING LOCUS T/TERMINAL-FLOWER-LIKE 1 family, key genes involved in anther and tapetum development, as well as LEAFY, WUSCHEL and CONSTANS. The results provided new insights into the roles of these and other well known gene families during the annual flowering cycle. To explore the mechanisms regulating poplar flowering, a weighted gene co-expression network was constructed using 98 floral-related genes involved in flower meristem identity and flower development. Many modules of co-expressed genes and hub genes were identified, such as APETALA1 and HUA1. This work provides many new insights on the annual flowering cycle in a perennial plant, and a major new resource for plant biology and biotechnology.

Assessing nutrient uptake pattern with respect to dry matter accumulation in Ney Poovan (AB) banana at critical growth stages

A field experiment was conducted to assess the nutrient uptake pattern and partitioning of nutrients in Ney Poovan banana (AB) at critical growth stages like 10-leaf stage, 20-leaf stage, shooting stage and harvesting stage. From 10-leaf stage to harvesting, the per cent dry matter (DM) accumulation decreased from 42 to 16 in underground segment and increased from 58 to 84 in above ground segment. The sample was partitioned into root, corm, pseudostem, leaf lamina, petiole, peduncle, bunch and male bud, processed and analysed for macro and micronutrients at different critical growth stages. The nutrient accumulation pattern, with respect to dry matter production was worked out. To produce 37.5 t banana ha−1, about 444 kg N, 69 kg P, 933 kg K, 2.4 kg Cu, 7.3 kg Mn, 3.4 kg Fe and 5.9 kg Zn were taken up by Ney Poovan banana. About 10 to 52% removal occurred through bunch harvest from the total nutrient uptake from the soil and remaining nutrients accumulated in the residual tissues were available for in situ recycling. The data provided opportunity for proper fertiliser scheduling to achieve optimum yield with higher nutrient use efficiency and nutrient budgeting in banana ecosystem was also done.

In Vitro Proliferation of Female Buds for Induction of Somatic Embryogenesis from False Horn Plantain (AAB, cv. Curraré).

Most cultivated bananas (Musa spp.) are polyploids, and their fruits are seedless and propagated exclusively vegetatively; however, they can also be cloned by micropropagation techniques, viz., direct organogenesis (DO) or somatic embryogenesis (SE). Banana indirect SE (ISE), with an embryogenic callus phase, is possible using young male or female flowers as direct explant depending on the genotype or shoot tips (scalps). For the False Horn Plantain, cv. Curraré (AAB, plantain subgroup), which has a degenerating male bud, female flowers are used to regenerate plants through ISE. Here, a protocol for increasing the number of initial explant material from a single mother plant and its embryogenic response is described. For those purposes, hands with young female buds are in vitro proliferated in the presence of 1 μM indole-3-acetic acid and 2.5 μM thidiazuron. Friable embryogenic cultures, here called ISE-2, obtained from the new proliferative secondary female bud clusters are initiated on medium containing auxins. Embryogenic suspensions are then established from the ISE-2 cultures. Regeneration of plants is achieved from embryogenic suspensions after plating on semisolid medium free of plant growth regulators; greenhouse acclimatized plantlets are ready for banana farming. This study demonstrates that proliferative female buds are a proper choice for ISE.

De novo transcriptome assembly and comparative analysis between male and benzyladenine-induced female inflorescence buds of Plukenetia volubilis

Plukenetia volubilis is a promising oilseed crop due to its seeds being rich in unsaturated fatty acids, especially alpha-linolenic acid. P. volubilis is monoecious, with separate male and female flowers on the same inflorescence. We previously reported that male flowers were converted to female flowers by exogenous cytokinin (6-benzyladenine, 6-BA) treatment in P. volubilis. To identify candidate genes associated with floral sex differentiation of P. volubilis, we performed de novo transcriptome assembly and comparative analysis on control male inflorescence buds (MIB) and female inflorescence buds (FIB) induced by 6-BA using Illumina sequencing technology. A total of 57,664 unigenes with an average length of 979 bp were assembled from 104.1 million clean reads, and 45,235 (78.45%) unigenes were successfully annotated in the public databases. Notably, Gene Ontology analyses revealed that 4193 and 3880 unigenes were enriched in the categories of reproduction and reproductive processes, respectively. Differential expression analysis identified 1385 differentially expressed unigenes between MIB and FIB, of which six unigenes related to cytokinin and auxin signaling pathways and 16 important transcription factor (TF) genes including MADS-box family members were identified. In particular, several unigenes encoding important TFs, such as homologs of CRABS CLAW, RADIALIS-like 1, RADIALIS-like 2, HECATE 2, WUSCHEL-related homeobox 9, and SUPERMAN, were expressed at higher levels in FIB than in MIB. The expression patterns of the 36 selected unigenes revealed by transcriptome analysis were successfully validated by quantitative real-time PCR. This study not only provides comprehensive gene expression profiles of P. volubilis inflorescence buds, but also lays the foundation for research on the molecular mechanism of floral sex determination in P. volubilis and other monoecious plants.

De novo transcriptome assembly from flower buds of dioecious, gynomonoecious and chemically masculinized female Coccinia grandis reveals genes associated with sex expression and modification

BackgroundCoccinia grandis (ivy gourd), is a dioecious member of Cucurbitaceae having heteromorphic sex chromosomes. Chromosome constitution of male and female plants of C. grandis is 22A + XY and 22A + XX respectively. Earlier we showed that a unique gynomonoecious form of C. grandis (22A + XX) also exists in nature bearing morphologically hermaphrodite flowers (GyM-H). Additionally, application of silver nitrate (AgNO3) on female plants induces stamen development leading to the formation of morphologically hermaphrodite flowers (Ag-H) despite the absence of Y-chromosome. Due to the unavailability of genome sequence and the slow pace at which sex-linked genes are identified, sex expression and modification in C. grandis are not well understood.ResultsWe have carried out a comprehensive RNA-Seq study from early-staged male, female, GyM-H, and Ag-H as well as middle-staged male and GyM-H flower buds. A de novo transcriptome was assembled using Trinity and annotated by BLAST2GO and Trinotate pipelines. The assembled transcriptome consisted of 467,233 ‘Trinity Transcripts’ clustering into 378,860 ‘Trinity Genes’. Female_Early_vs_Male_Early, Ag_Early_vs_Female_Early, and GyM-H_Middle_vs_Male_Middle comparisons exhibited 35,694, 3574, and 14,954 differentially expressed transcripts respectively. Further, qRT-PCR analysis of selected candidate genes validated digital gene expression profiling results. Interestingly, ethylene response-related genes were found to be upregulated in female buds compared to male buds. Also, we observed that AgNO3 treatment suppressed ethylene responses in Ag-H flowers by downregulation of ethylene-responsive transcription factors leading to stamen development. Further, GO terms related to stamen development were enriched in early-staged male, GyM-H, and Ag-H buds compared to female buds supporting the fact that stamen growth gets arrested in female flowers.ConclusionsSuppression of ethylene responses in both male and Ag-H compared to female buds suggests a probable role of ethylene in stamen suppression similar to monoecious cucurbits such as melon and cucumber. Also, pollen fertility associated GO terms were depleted in middle-staged GyM-H buds compared to male buds indicating the necessity of Y-chromosome for pollen fertility. Overall, this study would enable identification of new sex-biased genes for further investigation of stamen arrest, pollen fertility, and AgNO3-mediated sex modification.

Molecular characterization of Pistacia atlantica Desf. subsp. atlantica (Anacardiaceae) in Algeria: Genome size determination, chromosome count and genetic diversity analysis using SSR markers

Abstract The current study aims at the molecular characterization of Pistacia atlantica Desf. Subsp atlantica in Northwestern Algeria. The genome size estimation was performed for the first time for the species using flow cytometry on 97 samples. The flow cytometry analysis led to estimate a very small genome (2C = 1.21 ± 0.02 pg). Meristematic somatic cells of male floral buds showed the best prometaphase which on (2 n = 2x = 30) chromosome complement was observed. Two chromosomes bearing satellites showed evident and large secondary constrictions. sixty-one genotypes were characterized using 6 SSR microsatellites. A total of 26 alleles were amplified. Allele frequencies ranged from 0.008 to 0.68. Eight rare alleles (30.8%) were observed. The observed heterozygosity (Ho) ranged from 0.35 to 0.67, while the expected heterozygosity (He) ranged from 0.48 to 0.77. The loci Pislen 526 and Pislen 114 were the most polymorphic with 7 and 6 observed alleles respectively. Pislen 526 and PKATG014 were the most informative markers. Cluster analysis based on the euclidean distance using (UPGMA) led to observe divergences between genotypes within site and between sites. The current study is the first molecular characterization of the subspecies; it indicated an evident diversity due to a genetic flow between populations. More genetic diversity studies are required to learn about the structure and the evolution of natural P. atlantica subsp. atlantica using more SSR markers and more accessions from other areas.

Comparative transcriptome analysis reveals differentially expressed genes associated with sex expression in garden asparagus (Asparagus officinalis)

BackgroundGarden asparagus (Asparagus officinalis) is a highly valuable vegetable crop of commercial and nutritional interest. It is also commonly used to investigate the mechanisms of sex determination and differentiation in plants. However, the sex expression mechanisms in asparagus remain poorly understood.ResultsDe novo transcriptome sequencing via Illumina paired-end sequencing revealed more than 26 billion bases of high-quality sequence data from male and female asparagus flower buds. A total of 72,626 unigenes with an average length of 979 bp were assembled. In comparative transcriptome analysis, 4876 differentially expressed genes (DEGs) were identified in the possible sex-determining stage of female and male/supermale flower buds. Of these DEGs, 433, including 285 male/supermale-biased and 149 female-biased genes, were annotated as flower related. Of the male/supermale-biased flower-related genes, 102 were probably involved in anther development. In addition, 43 DEGs implicated in hormone response and biosynthesis putatively associated with sex expression and reproduction were discovered. Moreover, 128 transcription factor (TF)-related genes belonging to various families were found to be differentially expressed, and this finding implied the essential roles of TF in sex determination or differentiation in asparagus. Correlation analysis indicated that miRNA-DEG pairs were also implicated in asparagus sexual development.ConclusionsOur study identified a large number of DEGs involved in the sex expression and reproduction of asparagus, including known genes participating in plant reproduction, plant hormone signaling, TF encoding, and genes with unclear functions. We also found that miRNAs might be involved in the sex differentiation process. Our study could provide a valuable basis for further investigations on the regulatory networks of sex determination and differentiation in asparagus and facilitate further genetic and genomic studies on this dioecious species.

Rapid alkalinization factor (RALF) genes are related to genic male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Male sterility is commonly and widely used for hybrid seed production in Brassica rapa L. ssp. pekinensis (B. rapa), and some genes related to anther and pollen development may play significant roles in pollen fertility. Hence, in this study, we performed whole-genome scanning and identified 38 genes from the RALF gene family in B. rapa, which were grouped into four clades according to the phylogenetic tree. Thirty-six of the sequences contained only exons; the other two contained both exonic and intronic sequences. Conserved motifs of the 38 RALF genes were identified. The Ka/Ks ratios of duplicated RALF gene pairs were less than 1, indicating that they had gone through purifying selection. Moreover, a total of 14 RALF-like differentially expressed genes (DEGs) were identified by RNA-Seq between male fertile and male sterile buds, and all were highly or specifically expressed in male fertile buds of the AB line ‘AB01’. A qRT-PCR analysis confirmed that the expression patterns of the 14 RALF DEGs in the AB line ‘AB01’ were similar to those in the other three genic male sterility (GMS) AB lines ‘AB02’, ‘AB03’, and ‘AB04’, indicating that the RALF genes may be related to pollen development in B. rapa. Meanwhile, the results of qRT-PCR validated the RNA-Seq transcriptome as accurate and reliable. This study will provide valuable information for exploring the molecular mechanism underlying sterility in GMS AB lines of B. rapa.

Transcriptome analysis of female and male flower buds of Idesia polycarpa Maxim. var. vestita Diels

Idesia polycarpa Maxim. var. vestita Diels, a dioecious plant, is widely used for biodiesel due to the high oil content of its fruits. However, it is hard to distinguish its sex in the seedling stage, which makes breeding and production problematic as only the female tree can produce fruits, and the mechanisms underlying sex determination and differentiation remain unknown due to the lack of available genomic and transcriptomic information. To begin addressing this issue, we performed the transcriptome analysis of its female and male flower. 28,668,977 and 22,227,992 clean reads were obtained from the female and male cDNA libraries, respectively. After quality checks and de novo assembly, a total of 84,213 unigenes with an average length of 1179 bp were generated and 65,972 unigenes (78.34%) could be matched in at least one of the NR, NT, Swiss-Prot, COG, KEGG and GO databases. Functional annotation of the unigenes uncovered diverse biological functions and processes, including reproduction and developmental process, which may play roles in sex determination and differentiation. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed many unigenes annotated as metabolic pathways, biosynthesis of secondary metabolites pathways, plant–pathogen interaction, and plant hormone signal transduction. Moreover, 29,953 simple sequence repeats were identified using the microsatellite software. This work provides the first detailed transcriptome analysis of female and male flower of I. polycarpa and lays foundations for future studies on the molecular mechanisms underlying flower bud development of I. polycarpa.

English

Diverse cultural practices have been communicated to and/or applied by resource-poor households over the past two decades to improve the overall farm health and crop tolerance to biotic/abiotic factors. This study assessed the current diversity and use of cultural practices on banana fields in Ituri and North Kivu provinces, eastern Democratic Republic of Congo (DR Congo). Interview schedules coupled with farm diagnosis were used to take stock of cultural practices. Farmers’ choice of banana cultivars was mainly influenced by bunch size, demand/price, pulp flavor/taste, and juice quality. Availability of planting materials and the lifespan of mats were also important. In contrast, drought tolerance, soil fertility conditions, length of production cycle, and pest and disease resistance were not highly considered. Suckers were the predominant type of planting material used. Banana-bean (in 15 to 39% of farms) and banana-taro-bean (18 to 30%) were the dominant intercrops. Staking of banana plants with bunches was applied by 94 to 95% of the respondents, possibly due to the perceived immediate benefits from bunches. Cutting of green leaves to among other things improve sunlight penetration for legume intercrops which was practiced by 74 to 85% of the farmers. This practice has greatly contributed to the perpetuation of banana Xanthomonas wilt disease (XW) in the region. Male bud removal was applied in 55 to 66% of farms to control XW and improve bunch size. However, 38 to 51% of these farmers de-budded after the recommended time. Other common cultural practices included de-suckering, pseudostem use for mulch and weeding. Strategies for safe application of some of the practices such as weeding, leaf cutting, de-trashing and in light of diseases such as XW are recommended. In addition, some practices such as mulching and male bud removal are knowledge intensive, while others such de-suckering have no immediate perceived benefits to farmers, thus the need to strengthen knowledge extension to enhance their adoption.   Key words: Cropping, cultivars, cultural practices, de-budding, de-leafing, de-suckering. &nbsp

Transcriptome profiling of the floral buds and discovery of genes related to sex-differentiation in the dioecious cucurbit Coccinia grandis (L.) Voigt

Dioecious species offer an inclusive structure to study the molecular basis of sexual dimorphism in angiosperms. Despite having a small genome and heteromorphic sex chromosomes, Coccinia grandis is a highly neglected dioecious species with little information available on its physical state, genetic orientation and key sex-defining elements. In the present study, we performed RNA-Seq and DGE analysis of male (MB) and female (FB) buds in C. grandis to gain insights into the molecular basis of sex determination in this plant. De novo assembly of 75 million clean reads resulted in 72,479 unigenes for male library and 63,308 unigenes for female library with a mean length of 736bp. 61,458 (85.57%) unigenes displayed significant similarity with protein sequences from publicly available databases. Comparative transcriptome analyses revealed 1410 unigenes as differentially expressed (DEGs) between MB and FB samples. A consistent correlation between the expression levels of DEGs was observed for the RNA-Seq pattern and qRT-PCR validation. Functional annotation showed high enrichment of DEGs involved in phytohormone biosynthesis, hormone signaling and transduction, transcriptional regulation and methyltransferase activity. High induction of hormone responsive genes such as ARF6, ACC synthase1, SNRK2 and BRI1-associated receptor kinase 1 (BAK1) suggest that multiple phytohormones and their signaling crosstalk play crucial role in sex determination in this species. Beside, the transcription factors such as zinc fingers, homeodomain leucine zippers and MYBs were identified as major determinants of male specific expression. Moreover, the detection of multiple DEGs as the miRNA target site implies that a small RNA mediated gene silencing cascade may also be regulating gender differentiation in C. grandis. Overall, the present transcriptome resources provide us a large number of DEGs involved in sex expression and could form the groundwork for unravelling the molecular mechanism of sex determination in C. grandis.

Microsporogenesis and Induction of Unreduced Pollen with High Temperatures in Rubber Tree Clone RRIM 600

In order to induce unreduced pollens, microsporogenesis and male flower bud (MFB) development were compared in rubber tree clone RRIM 600. We observed strong asynchronism in different MFBs in an inflorescence. Asynchronism of microsporogenesis in different microsporangiums from a MFB was also observed. The relationship between microsporogenesis and external morphology was examined, which was used to estimate microsporogenesis stages of MFBs. Unreduced pollen was successfully induced by high temperature exposure in this study, with the highest production ratio of about 20.17% at 44 °C. Our findings showed that diplotene to metaphase I may be the most effective stage for unreduced pollen induction, and 42–44 °C may be the suitable treatment temperature in rubber trees. Thus, microsporogenesis of MFBs has been elucidated in detail in the rubber tree clone RRIM 600 and will provide a reference for future breeding studies of rubber trees.

De novo sequencing and comparative transcriptome analysis of the male and hermaphroditic flowers provide insights into the regulation of flower formation in andromonoecious Taihangia rupestris

BackgroundTaihangia rupestris, an andromonoecious plant species, bears both male and hermaphroditic flowers within the same individual. However, the establishment and development of male and hermaphroditic flowers in andromonoecious Taihangia remain poorly understood, due to the limited genetic and sequence information. To investigate the potential molecular mechanism in the regulation of Taihangia flower formation, we used de novo RNA sequencing to compare the transcriptome profiles of male and hermaphroditic flowers at early and late developmental stages.ResultsFour cDNA libraries, including male floral bud, hermaphroditic floral bud, male flower, and hermaphroditic flower, were constructed and sequenced by using the Illumina RNA-Seq method. Totally, 84,596,426 qualified Illumina reads were obtained and then assembled into 59,064 unigenes, of which 24,753 unigenes were annotated in the NCBI non-redundant protein database. In addition, 12,214, 7,153, and 8,115 unigenes were assigned into 53 Gene Ontology (GO) functional groups, 25 Clusters of Orthologous Group (COG) categories, and 126 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. By pairwise comparison of unigene abundance between the samples, we identified 1,668 differential expressed genes (DEGs), including 176 transcription factors (TFs) between the male and hermaphroditic flowers. At the early developmental stage, we found 263 up-regulated genes and 436 down-regulated genes expressed in hermaphroditic floral buds, while 844 up-regulated genes and 314 down-regulated genes were detected in hermaphroditic flowers at the late developmental stage. GO and KEGG enrichment analyses showed that a large number of DEGs were associated with a wide range of functions, including cell cycle, epigenetic processes, flower development, and biosynthesis of unsaturated fatty acid pathway. Finally, real-time quantitative PCR was conducted to validate the DEGs identified in the present study.ConclusionIn this study, transcriptome data of this rare andromonoecious Taihangia were reported for the first time. Comparative transcriptome analysis revealed the significant differences in gene expression profiles between male and hermaphroditic flowers at early and late developmental stages. The transcriptome data of Taihangia would be helpful to improve the understanding of the underlying molecular mechanisms in regulation of flower formation and unisexual flower establishment in andromonoecious plants.

Endogenous phytohormone profiles in male and female floral buds of the persimmons (Diospyros kaki Thunb.) during development

Despite great interest from both the basic science community and from economic stakeholders, research on sex differentiation in persimmons (Diospyros kaki Thunb.) remains limited. Our previous study found that the developmental processes of both male and female floral buds were divided morphologically into 11 stages, of which stage 2 (on June 17) and stage 8 (on April 17 of the following year) were the two crucial morphological stages for sex differentiation. In this study, we used high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry to measure the amounts of indole-3-acetic acid (IAA), gibberellin 3 (GA3), zeatin (ZT), abscisic acid (ABA), and jasmonic acid (JA) in both male and female floral buds at stages 1, 1.5, 2, 6, 7, 8, 10, and 11 to uncover the regulatory effects of endogenous phytohormones on sex differentiation. The results showed that low levels of IAA, ABA, as well as the ratio of ABA to IAA, and high levels of GA3 in male floral buds in the middle of June were related to the formation of three-flower cymes. Moreover, high levels of GA3 in male floral buds at stage 8 may accelerate the arrest of pistil primordia and the development of stamen primordia in the middle of the following April. In addition, high levels of the ratio of ABA to ZT from stages 6–11 might promote the differentiation of male floral buds. However, high levels of IAA from stages 7–10 as well as ZT and ABA from stages 6–10 in female floral buds may stimulate the abortion of stamen primordia and promote the development of pistil primordia, leading to female unisexual flowers. These results are relevant for future research on the regulation of controlled sex differentiation and pollination with exogenous phytohormones.

In vitro Response of Asparagus breslerianus to NaCl

Asparagus breslerianus a wild species in Iran, exhibited tolerance to salt in dry gypsum hills and dry lands. In order to check for salt tolerance thresholdvia in vitro conditions, the A. breslerianus callus was subjected to NaCl (sodium chloride) treatments. Six weeks old calli derived from male spear bud, were exposed to 0, 21.88, 43.76, 65.64, 76.58, 87.52, 109.40, 131.28, 153.16 and 175.04 mM NaCl in MS (Murashige and Skoog) basal medium supplemented with 0.88 µM BA (6-Benzylaminopurine) and 1.07 µM NAA (1- Naphthalene acetic acid). According to results, friable and compact type, green and white green calli were obtained up to 87.52 mM NaCl. Soft type, yellow and cream calli were observed with increasing salinity further 109.4 mM NaCl. Calli growth and plantlets regeneration were high in media up to 109.4 mM NaCl. In term of mineral accumulation, sodium content increased with an increase in NaCl levels. It indicates that A. breslerianus calli could acclimatize to salt stress by high osmotic adjustment. These suggested that, under in vitro salt-induced osmotic stress, A. breslerianus is reflected as salt resistant which tolerate NaCl up to 109.4 mM.

Detecting the Candidate Gender Determinants by Bioinformatic Prediction of miRNAs and Their Targets from Transcriptome Sequences of the Male and Female Flowers in Salix suchowensis.

MicroRNAs (miRNAs) belong to a class of small, noncoding, and endogenous single-stranded RNAs that negatively regulate gene expression at the posttranscriptional level. Potential miRNAs can be identified based on sequence homology since miRNAs are highly conserved in plants. In this study, we aligned the expressed sequence tags derived from flower buds of male and female S. suchowensis to miRNAs in the miRBase, which enable us to identify 34 potential miRNAs from flower buds of the alternate sexes. Among them, 11 were from the female and 23 were from the male. Analyzing sequence complementarity led to identification of 124 and 55 miRNA targets in the male and female flower buds, respectively. By mapping the target genes of the predicted miRNAs to the sequence assemblies of S. suchowensis, a miR156 mediated gene was detected at the gender locus of willow, which was a transcription factor involved in flower development. It is noteworthy that this target is not expressed in male flower, while it is expressed fairly highly in female flower based on the transcriptome data derived from the alternate sexes of willows. This study provides new bioinformatic clue for further exploring the genetic mechanism underlying gender determination in willows.