Chloroplast genome data of five Amygdalus species: Clarifying genome structure and phylogenetic relationships.

Amygdalus species have considerable ecological and economic value, however, the phylogenetic relationships among Amygdalus remain controversy. In this study, we sequenced and assembled the chloroplast (cp) genomes of five Amygdalus species: Prunus communis, P. mongolica, P. pedunculata, P. triloba, and P. mira. We then conducted comparative genomic analyses and constructed their phylogenetic relationships. The genome length ranged from 157,870 to 158,451 bp, and 131 genes were annotated (86 protein-coding genes, 37 tRNAs, and 8 rRNAs). Additionally, 49-57 simple sequence repeats were detected, with most in the large single-copy region and with AT base preferences. Comparative genomic analyses revealed high similarities in structure, order, and gene content. However, we identified four highly divergent sequences: trnR-UCU-atpA, nbdhC-trnV-UAC, ycf4-cemA, and rpl32-trnL-UAG. The phylogenomic relationship analysis suggested that the Amygdalus species were grouped together, in which P. pedunculata, P. triloba, and Prunus tangutica were categorized into a branch, P. mongolica and Prunus davidiana were clustered a branch. This study provides an improved understanding of the genetic relationships among the Amygdalus and provides a basis for the development and utilization of Amygdalus resources.

Sequencing and Phylogenetic Analysis of the Chloroplast Genome of Three Apricot Species.

The production and quality of apricots in China is currently limited by the availability of germplasm resource characterizations, including identification at the species and cultivar level. To help address this issue, the complete chloroplast genomes of Prunus armeniaca L., P. sibirica L. and kernel consumption apricot were sequenced, characterized, and phylogenetically analyzed. The three chloroplast (cp) genomes ranged from 157,951 to 158,224 bp, and 131 genes were identified, including 86 protein-coding genes, 37 rRNAs, and 8 tRNAs. The GC content ranged from 36.70% to 36.75%. Of the 170 repetitive sequences detected, 42 were shared by all three species, and 53-57 simple sequence repeats were detected with AT base preferences. Comparative genomic analysis revealed high similarity in overall structure and gene content as well as seven variation hotspot regions, including psbA-trnK-UUU, rpoC1-rpoB, rpl32-trnL-UAG, trnK-rps16, ndhG-ndhI, ccsA-ndhD, and ndhF-trnL. Phylogenetic analysis showed that the three apricot species clustered into one group, and the genetic relationship between P. armeniaca and kernel consumption apricot was the closest. The results of this study provide a theoretical basis for further research on the genetic diversity of apricots and the development and utilization of molecular markers for the genetic engineering and breeding of apricots.

Genome-Wide Identification and Function of Aquaporin Genes During Dormancy and Sprouting Periods of Kernel-Using Apricot (Prunus armeniaca L.).

Aquaporins (AQPs) are essential channel proteins that play a major role in plant growth and development, regulate plant water homeostasis, and transport uncharged solutes across biological membranes. In this study, 33 AQP genes were systematically identified from the kernel-using apricot (Prunus armeniaca L.) genome and divided into five subfamilies based on phylogenetic analyses. A total of 14 collinear blocks containing AQP genes between P. armeniaca and Arabidopsis thaliana were identified by synteny analysis, and 30 collinear blocks were identified between P. armeniaca and P. persica. Gene structure and conserved functional motif analyses indicated that the PaAQPs exhibit a conserved exon-intron pattern and that conserved motifs are present within members of each subfamily. Physiological mechanism prediction based on the aromatic/arginine selectivity filter, Froger's positions, and three-dimensional (3D) protein model construction revealed marked differences in substrate specificity between the members of the five subfamilies of PaAQPs. Promoter analysis of the PaAQP genes for conserved regulatory elements suggested a greater abundance of cis-elements involved in light, hormone, and stress responses, which may reflect the differences in expression patterns of PaAQPs and their various functions associated with plant development and abiotic stress responses. Gene expression patterns of PaAQPs showed that PaPIP1-3, PaPIP2-1, and PaTIP1-1 were highly expressed in flower buds during the dormancy and sprouting stages of P. armeniaca. A PaAQP coexpression network showed that PaAQPs were coexpressed with 14 cold resistance genes and with 16 cold stress-associated genes. The expression pattern of 70% of the PaAQPs coexpressed with cold stress resistance genes was consistent with the four periods [Physiological dormancy (PD), ecological dormancy (ED), sprouting period (SP), and germination stage (GS)] of flower buds of P. armeniaca. Detection of the transient expression of GFP-tagged PaPIP1-1, PaPIP2-3, PaSIP1-3, PaXIP1-2, PaNIP6-1, and PaTIP1-1 revealed that the fusion proteins localized to the plasma membrane. Predictions of an A. thaliana ortholog-based protein-protein interaction network indicated that PaAQP proteins had complex relationships with the cold tolerance pathway, PaNIP6-1 could interact with WRKY6, PaTIP1-1 could interact with TSPO, and PaPIP2-1 could interact with ATHATPLC1G. Interestingly, overexpression of PaPIP1-3 and PaTIP1-1 increased the cold tolerance of and protein accumulation in yeast. Compared with wild-type plants, PaPIP1-3- and PaTIP1-1-overexpressing (OE) Arabidopsis plants exhibited greater tolerance to cold stress, as evidenced by better growth and greater antioxidative enzyme activities. Overall, our study provides insights into the interaction networks, expression patterns, and functional analysis of PaAQP genes in P. armeniaca L. and contributes to the further functional characterization of PaAQPs.

AprGPD: the apricot genomic and phenotypic database.

BACKGROUND: Apricot is cultivated worldwide because of its high nutritive content and strong adaptability. Its flesh is delicious and has a unique and pleasant aroma. Apricot kernel is also consumed as nuts. The genome of apricot has been sequenced, and the transcriptome, resequencing, and phenotype data have been increasely generated. However, with the emergence of new information, the data are expected to integrate, and disseminate. RESULTS: To better manage the continuous addition of new data and increase convenience, we constructed the apricot genomic and phenotypic database (AprGPD, http://apricotgpd.com ). At present, AprGPD contains three reference genomes, 1692 germplasms, 306 genome resequencing data, 90 RNA sequencing data. A set of user-friendly query, analysis, and visualization tools have been implemented in AprGPD. We have also performed a detailed analysis of 59 transcription factor families for the three genomes of apricot. CONCLUSION: Six modules are displayed in AprGPD, including species, germplasm, genome, variation, product, tools. The data integrated by AprGPD will be helpful for the molecular breeding of apricot.

Dynamic transcriptome analysis identifies genes related to fatty acid biosynthesis in the seeds of Prunus pedunculata Pall.

BACKGROUND: Prunus pedunculata Pall, the deciduous shrub of Amygdalus subgenus in Rosaceae, is a new kind of desert oil-bearing tree. It has a long story of being planted in the West and North of China for sand fixation and desert control. In addition, the seeds of P. pedunculata are rich of oil, especially the monounsaturated fatty acid and polyunsaturated fatty acid. However, little is known about the molecular mechanisms of oil accumulation during the seed development of P. pedunculata. RESULTS: The seeds of P. pedunculata from three independent plants at 10, 18, 24, 31, 39, 45, 59 and 73 days after flowering (DAF) were obtained and the oil compositions were evaluated. It showed that oleic acid was the dominant type of oil content in the mature seeds (from 32.724% at 10DAF to 72.06% at 73DAF). Next, transcriptome sequencing for the developing seeds produced 988.795 million high quality reads and TRINITY assembled 326,271 genes for the first transcriptome for P. pedunculata. After the assembled transcriptome was evaluated by BUSCO with 85.9% completeness, we identified 195,342, 109,850 and 121,897 P. pedunculata genes aligned to NR, GO and KEGG pathway databases, respectively. Then, we predicted 23,229 likely proteins from the assembled transcriptome and identified 1917 signal peptides and 5512 transmembrane related proteins. In the developing seeds we detected 91,362 genes (average FPKM > 5) and correlation analysis indicated three possible development stages - early (10 ~ 24DAF), middle (31 ~ 45DAF) and late (59 ~ 73DAF). We next analyzed the differentially expressed genes (DEGs) in the developing seeds. Interestingly, compared to 10DAF the number of DEGs was increased from 4406 in 18DAF to 27,623 in 73DAF. Based on the gene annotation, we identified 753, 33, 8 and 645 DEGs related to the fatty acid biosynthesis, lipid biosynthesis, oil body and transcription factors. Notably, GPAT, DGD1, LACS2, UBC and RINO were highly expressed at the early development stage, ω6-FAD, SAD, ACP, ACCA and AHG1 were highly expressed at the middle development stage, and LACS6, DGD1, ACAT1, AGPAT, WSD1, EGY2 and oleosin genes were highly expressed at the late development stage. CONCLUSIONS: This is the first time to study the developing seed transcriptome of P. pedunculata and our findings will provide a valuable resource for future studies. More importantly, it will improve our understanding of molecular mechanisms of oil accumulation in P. pedunculata.

Insights Into the Molecular Mechanisms of Late Flowering in Prunus sibirica by Whole-Genome and Transcriptome Analyses.

Freezing during the flowering of Prunus sibirica is detrimental to fruit production. The late flowering (LF) type, which is delayed by 7-15 days compared with the normal flowering (NF) type, avoids damages at low temperature, but the molecular mechanism of LF remains unclear. Therefore, this study was conducted to comprehensively characterize floral bud differentiation. A histological analysis showed that initial floral bud differentiation was delayed in the LF type compared to the NF type. Genome-wide associated studies (GWAS) showed that a candidate gene (PaF106G0600023738.01) was significantly associated with LF type. It was identified as trehalose-6-phosphate phosphatase (PsTPPF), which is involved in trehalose-6-phosphate (Tre6P) signaling pathway and acts on floral transition. A whole-transcriptome RNA sequencing analysis was conducted, and a total of 6,110 differential expression (DE) mRNAs, 1,351 DE lncRNAs, and 148 DE miRNAs were identified. In addition, 24 DE mRNAs related with floral transition were predicted, and these involved the following: three interactions between DE lncRNAs and DE mRNAs of photoperiod pathway with two mRNAs (COP1, PaF106G0400018289.01 and CO3, MXLOC_025744) and three lncRNAs (CCLR, LTCONS_00031803, COCLR1, LTCONS_00046726, and COCLR2, LTCONS_00046731); one interaction between DE miRNAs and DE mRNAs with one mRNA, encoding trehalose-6-phosphate synthase (PsTPS1, PaF106G0100001132.01), and one miRNA (miRNA167h). Combined with the expression profiles and Tre6P levels, functions of PsTPPF and PsTPS1 in Tre6P regulation were considered to be associated with flowering time. A new network of ceRNAs correlated with LF was constructed, and it consisted of one mRNA (PsTPS1), one lncRNA (TCLR, LTCONS_00034157), and one miRNA (miR167h). This study provided insight into the molecular regulatory mechanism of LF in Prunus sibirica.

Proline-rich protein gene PdPRP regulates secondary wall formation in poplar.

Proline-rich protein (PRP) is a plant cell wall associated protein. Its distinct patterns of regulation and localization studied in a number of plants indicate that it may play important roles in growth and development. However, the mechanism of how these genes control secondary cell wall development in tree species is largely unknown. Here, we report that a Populus deltoides (Marsh.) proline-rich protein gene PdPRP was preferentially expressed in immature/mature phloem and immature xylem in P. deltoides. PdPRP overexpression increased poplar plant height and diameter as well as the radial width of the phloem and xylem regions, facilitated secondary wall deposition, and induced expression of genes related to microfibril angle (MFA) and secondary wall biosynthesis. Downregulation of PdPRP retarded poplar growth, decreased the radial width of the secondary phloem and secondary xylem regions, reduced secondary wall thickening in fibers and vessels, and decreased the expression of genes related to MFA and secondary wall biosynthesis. These results suggest that PdPRP might positively regulate secondary cell wall formation by promoting secondary wall thickening and expansion in poplar. PdPRP-overexpressing poplar had a lower MFA, indicating that PdPRP may be useful for improving wood stiffness and properties in plants. Together, our results demonstrate that PdPRP is a proline-rich protein associated with cell wall development, playing a critical role in regulating secondary cell wall formation in poplar.

The Hardy Rubber Tree Genome Provides Insights into the Evolution of Polyisoprene Biosynthesis.

Eucommia ulmoides, also called hardy rubber tree, is an economically important tree; however, the lack of its genome sequence restricts the fundamental biological research and applied studies of this plant species. Here, we present a high-quality assembly of its ∼1.2-Gb genome (scaffold N50 = 1.88 Mb) with at least 26 723 predicted genes for E. ulmoides, the first sequenced genome of the order Garryales, which was obtained using an integrated strategy combining Illumina sequencing, PacBio sequencing, and BioNano mapping. As a sister taxon to lamiids and campanulids, E. ulmoides underwent an ancient genome triplication shared by core eudicots but no further whole-genome duplication in the last ∼125 million years. E. ulmoides exhibits high expression levels and/or gene number expansion for multiple genes involved in stress responses and the biosynthesis of secondary metabolites, which may account for its considerable environmental adaptability. In contrast to the rubber tree (Hevea brasiliensis), which produces cis-polyisoprene, E. ulmoides has evolved to synthesize long-chain trans-polyisoprene via farnesyl diphosphate synthases (FPSs). Moreover, FPS and rubber elongation factor/small rubber particle protein gene families were expanded independently from the H. brasiliensis lineage. These results provide new insights into the biology of E. ulmoides and the origin of polyisoprene biosynthesis.

Genome-Wide Identification of Mitogen-activated Protein Kinase Cascade Genes and Transcriptional Profiling Analysis during Organ Development in Eucommia ulmoides.

The mitogen-activated protein kinase (MAPK) cascades, which play crucial roles in plant development processes, are universal modules of signal transduction in eukaryotes and consist of a core module of three sequentially phosphorylated kinases: MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). This is the first report on the identification and analysis of MAPK cascades in Eucommia ulmoides. We conducted a genome-wide screening and identified 13 EuMAPKs, five EuMAPKKs, and 57 EuMAPKKKs. The construction of phylogenetic trees revealed that EuMAPKs and EuMAPKKs were divided into four groups (A, B, C, and D), and EuMAPKKKs were divided into three subfamilies (MEKK, RAF, and ZIK). These subfamilies were further confirmed by conserved domain/motif analysis and gene structure analysis. Based on the expression profiles of all identified EuMAPK cascades in various organs at different developmental stages, three genes (EuRAF22-2, EuRAF34-1, and EuRAF33-2) with stable expression patterns at all stages of fruit or leaf development, three genes (EuRAF2-3, EuMPK11, and EuMEKK21) with differential expression patterns, and two highly expressed genes (EuZIK1 and EuMKK2) were screened and validated by qRT-PCR. Overall, our results could be used for further research on the precise role of MAPK cascades during organ development in E. ulmoides.

Genome-Wide Identification of MicroRNAs and Their Targets in the Leaves and Fruits of Eucommia ulmoides Using High-Throughput Sequencing.

MicroRNAs (miRNAs), a group of endogenous small non-coding RNAs, play important roles in plant growth, development, and stress response processes. Eucommia ulmoides Oliver (hardy rubber tree) is one of the few woody plants capable of producing trans-1, 4-polyisoprene (TPI), also known as Eu-rubber, which has been utilized as an industrial raw material and is extensively cultivated in China. However, the mechanism of TPI biosynthesis has not been identified in E. ulmoides. To characterize small RNAs and their targets with potential biological roles involved in the TPI biosynthesis in E. ulmoides, in the present study, eight small RNA libraries were constructed and sequenced from young and mature leaves and fruits of E. ulmoides. Further analysis identified 34 conserved miRNAs belonging to 20 families (two unclassified families), and 115 novel miRNAs seemed to be specific to E. ulmoides. Among these miRNAs, fourteen conserved miRNAs and 49 novel miRNAs were significantly differentially expressed and identified as Eu-rubber accumulation related miRNAs. Based on the E. ulmoides genomic data, 202 and 306 potential target genes were predicted for 33 conserved and 92 novel miRNAs, respectively; the predicted targets are mostly transcription factors and functional genes, which were enriched in metabolic pathways and biosynthesis of secondary metabolites. Noticeably, based on the expression patterns of miRNAs and their target genes in combination with the Eu-rubber accumulation, the negative correlation of expression of six miRNAs (Eu-miR14, Eu-miR91, miR162a, miR166a, miR172c, and miR396a) and their predicted targets serving as potential regulators in Eu-rubber accumulation. This study is the first to detect conserved and novel miRNAs and their potential targets in E. ulmoides and identify several candidate genes potentially controlling rubber accumulation, and thus provide molecular evidence for understanding the roles of miRNAs in regulating the TPI biosynthesis in E. ulmoides.

Cloning, characterization and promoter analysis of S-RNase gene promoter from Chinese pear (Pyrus pyrifolia).

The 5'-flanking region of the S(12)-, S(13)-, S(21)-RNase with a length of 854 bp, 1448 bp and 1137 bp were successfully isolated by TAIL-PCR from genomic DNA from 'Jinhua', 'Maogong' (Pyrus pyrifolia) and 'Yali' (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S(13)-, S(12)-, S(21)-RNase gene promoter sequences with S(2)-, S(3)-, S(4)-, S(5)-RNase 5'-flanking sequences indicated that a homology region of about 240 bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5'-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S(12)-pro-(1 to 5)-GUS-pBll01.2 transgenic pistils and progressively decreased from S(12)-pro-1-GUS-pBI l01.2 to S(12)-pro-5-GUS-pBll01.2. No GUS activity was detected in S(12)-pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S(12)-RNase promoter is pistil specific expression promoter.

[Effects of high concentration ozone on soybean growth and grain yield].

By using open top chambers (OTCs), soybean plants were grown in pots, and exposed to charcoal-filtered air ([O3] < 10 microg x kg(-1)) and elevated O3 (80 microg x kg(-1)) after anthesis, aimed to investigate the responses of soybean' s agronomic characters, leaf area, chlorophyll content, antioxidant system, and grain yield to elevated O3. Under elevated O3, the leaf area and chlorophyll content decreased significantly (P < 0.05), and the leaf catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities had a significant increase (P < 0.01) first but a gradual decrease then, compared with those under charcoal-filtered air. Elevated O3 decreased the leaf soluble protein and ascorbate content (AsA) contents while increased the leaf malonaldehyde (MDA) content (P < 0.05), suggesting that the leaf membrane lipid peroxidation was accelerated. The dry mass per plant, effective pod number, grain number, 100-grains weight, and grain yield under elevated O3 had somewhat decreased, among which, grain yield decreased significantly, with the decrement reached to 47% (P < 0.01).

Molecular identification of two new self-incompatible alleles (S-alleles) in Chinese pear (Pyrus bretschneideri).

Self-incompatibility (SI) is an important intraspecific reproductive barrier in flowering plants. To identify the S-alleles of Chinese pear species (Pyrus pyrifolia, P. bretschneideri, P. ussuriensis and P. sinkiangenis etc.), S-RNase-specific PCR amplification, sequence analyses and field pollination tests were performed using two cultivars 'Jingxiang' and 'Esu' of P. bretschneideri as materials. Two new S-RNase genes were identified from the two cultivars. They were 1,122 bp and 1,058 bp in length, and designated as S37-RNase (GenBank accession no. DQ839238) and S38-RNase (GenBank accession no. DQ839239). By comparison of their deduced amino acid sequences with those of S1-to S36-alleles of Oriental pear, it can be seen that both the two new S-alleles had their conserved regions C1 and C2, but their hypervariable regions (HV) were quite different from those of the others. S37 showed a higher similarity (96%) to S38 in the amino acid sequences deduced from them, whereas both of them displayed the highest similarity (98%) to S15 and the lowest (63%) to S32. The two S-alleles had introns of 786 bp and 723 bp, respectively, similar in size to that of S15 (777 bp). Finally, the S-genotypes of 'Jinxiang' and 'Esu' were unambiguously determined as S34S37 and S15S38, respectively.