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18 pages, 2888 KiB

Open AccessArticle

Selection and Validation of Reference Genes for the qRT-PCR Assays of Populus ussuriensis Gene Expression under Abiotic Stresses and Related ABA Treatment

by Ming Wei, Yingxi Chen, Mengqiu Zhang, Jingli Yang, Han Lu, Xin Zhang and Chenghao Li

Forests 2020, 11(4), 476; https://doi.org/10.3390/f11040476 - 23 Apr 2020

Cited by 12 | Viewed by 2979

Abstract

Populus ussuriensis Kom. is one of the most important tree species for forest renewal in the eastern mountainous areas of Northeast China due to its fast growth, high yield, and significant commercial and ecological value. The selection of optimal reference genes for the [...] Read more.

Populus ussuriensis Kom. is one of the most important tree species for forest renewal in the eastern mountainous areas of Northeast China due to its fast growth, high yield, and significant commercial and ecological value. The selection of optimal reference genes for the normalization of qRT-PCR data is essential for the analysis of relative gene expression. In this study, fourteen genes were selected and assessed for their expression stability during abiotic stress (drought, high salinity, and cold stress) and after the treatment with the drought-related hormone ABA. Three algorithms were used, geNorm, NormFinder, and BestKeeper, and a comprehensive ranking of candidate reference genes was produced based on their output. The most appropriate reference genes were UBQ10 and RPL24 for drought and ABA treatment, UBQ10 and TUB3 for cold stress, and UBQ10 and 60S rRNA for high salinity. Overall, UBQ10 was the most stable reference gene for use as an internal control, whereas PP2A was the least stable. The expression of two target genes (P5CS2 and GI) was used to further verify that the selected reference genes were suitable for gene expression normalization. This work comprehensively assesses the stability of reference genes in Populus ussuriensis and identifies suitable reference genes for normalization during qRT-PCR analysis. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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14 pages, 3818 KiB

Open AccessArticle

Transcriptome Sequencing and Differential Expression Analysis Reveal Molecular Mechanisms for Starch Accumulation in Chestnut

by Shengxing Li, Haiying Liang, Liang Tao, Liquan Xiong, Wenhui Liang, Zhuogong Shi and Zhiheng Zhao

Forests 2020, 11(4), 388; https://doi.org/10.3390/f11040388 - 1 Apr 2020

Cited by 8 | Viewed by 2562

Abstract

Chestnuts are popular edible nuts that are rich in starch. In order to enhance the transcriptomic resources and further understand starch and sucrose metabolism in maturing chestnuts, a comparative transcriptomic study of Chinese chestnut kernels was conducted at three ripening stages (70, 82, [...] Read more.

Chestnuts are popular edible nuts that are rich in starch. In order to enhance the transcriptomic resources and further understand starch and sucrose metabolism in maturing chestnuts, a comparative transcriptomic study of Chinese chestnut kernels was conducted at three ripening stages (70, 82, and 94 DAF). At 82 and 94 days after flowering (DAF), starch continued to accumulate, and the amylopectin/amylose ratio increased. Transcriptomic profiling of kernels at 70 (stage I), 82 (stage II), and 94 DAF (stage III) indicated that soluble starch synthase and α-1,4-glucan branching enzyme genes are actively expressed at 82 and 94 DAF. The starch degradation enzymes amylase, phosphoglucan phosphatase DSP4, and maltose exporter did not show differential gene expression, while glycogen phosphorylase-encoding unigenes were significantly down-regulated at 94 DAF. In addition to starch and sucrose metabolism, RNA transport, RNA degradation, pyrimidine metabolism, purine metabolism, plant hormone signal transduction, plant–pathogen interactions, and glycerophospholipid metabolism were found to be significantly enriched in all comparisons included in the study. As Chinese chestnut matured, the unique enriched pathways switched from ribosomal biogenesis and RNA polymerase of eukaryotes to endocytosis and spliceosomes. These genomic resources and findings are valuable for further understanding starch and sucrose metabolism in the Chinese chestnut. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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16 pages, 741 KiB

Open AccessArticle

Identification and in Silico Characterization of Novel and Conserved MicroRNAs in Methyl Jasmonate-Stimulated Scots Pine (Pinus sylvestris L.) Needles

by Baiba Krivmane, Ilze Šņepste, Vilnis Šķipars, Igor Yakovlev, Carl Gunnar Fossdal, Adam Vivian-Smith and Dainis Ruņģis

Forests 2020, 11(4), 384; https://doi.org/10.3390/f11040384 - 28 Mar 2020

Cited by 4 | Viewed by 3550

Abstract

MicroRNAs (miRNAs) are non-protein coding RNAs of ~20–24 nucleotides in length that play an important role in many biological and metabolic processes, including the regulation of gene expression, plant growth and developmental processes, as well as responses to stress and pathogens. The aim [...] Read more.

MicroRNAs (miRNAs) are non-protein coding RNAs of ~20–24 nucleotides in length that play an important role in many biological and metabolic processes, including the regulation of gene expression, plant growth and developmental processes, as well as responses to stress and pathogens. The aim of this study was to identify and characterize novel and conserved microRNAs expressed in methyl jasmonate-treated Scots pine needles. In addition, potential precursor sequences and target genes of the identified miRNAs were determined by alignment to the Pinus unigene set. Potential precursor sequences were identified using the miRAtool, conserved miRNA precursors were also tested for the ability to form the required stem-loop structure, and the minimal folding free energy indexes were calculated. By comparison with miRBase, 4975 annotated sequences were identified and assigned to 173 miRNA groups, belonging to a total of 60 conserved miRNA families. A total of 1029 potential novel miRNAs, grouped into 34 families were found, and 46 predicted precursor sequences were identified. A total of 136 potential target genes targeted by 28 families were identified. The majority of previously reported highly conserved plant miRNAs were identified in this study, as well as some conserved miRNAs previously reported to be monocot specific. No conserved dicot-specific miRNAs were identified. A number of potential gymnosperm or conifer specific miRNAs were found, shared among a range of conifer species. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

18 pages, 1707 KiB

Open AccessArticle

Update of Genetic Linkage Map and QTL Analysis for Growth Traits in Eucommia ulmoides Oliver

by Cangfu Jin, Zhouqi Li, Yu Li, Shuhui Wang, Long Li and Minhao Liu

Forests 2020, 11(3), 311; https://doi.org/10.3390/f11030311 - 12 Mar 2020

Cited by 7 | Viewed by 2703

Abstract

Eucommia ulmoides (Tu-chung) is an economically and ecologically important tree species which has attracted worldwide attention due to its application in pharmacology, landscaping, wind sheltering and sand fixation. Molecular marker technologies can elucidate the genetic mechanism and substantially improve the breeding efficiency of [...] Read more.

Eucommia ulmoides (Tu-chung) is an economically and ecologically important tree species which has attracted worldwide attention due to its application in pharmacology, landscaping, wind sheltering and sand fixation. Molecular marker technologies can elucidate the genetic mechanism and substantially improve the breeding efficiency of E. ulmoides. The current research updated the original linkage map, and quantitative trait loci (QTL) analysis was performed on tree growth traits measured over 10 consecutive years in an E. ulmoides F1 population (“Xiaoye” × “Qinzhong No.1”). In total, 452 polymorphic markers were scored from 365 simple sequence repeat (SSR) primers, with an average of 1.24 polymorphic markers per primer combination. The integrated map was 1913.29 cM (centimorgan) long, covering 94.10% of the estimated genome and with an average marker density of 2.20 cM. A total of 869 markers were mapped into 19 major independent linkage groups. Growth-related traits measured over 10 consecutive years showed a significant correlation, and 89 hypothetical QTLs were forecasted and divided into 27 distinct loci. Three traits for tree height, ground diameter and crown diameter detected 25 QTLs (13 loci), 32 QTLs (17 loci) and 15 QTLs (10 loci), respectively. Based on BLASTX search results in the NCBI database, six candidate genes were obtained. It is important to explore the growth-related genetic mechanism and lay the foundation for the genetic improvement of E. ulmoides at the molecular level. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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15 pages, 3450 KiB

Open AccessArticle

The Chloroplast Genome of Carya illinoinensis: Genome Structure, Adaptive Evolution, and Phylogenetic Analysis

by Zhenghai Mo, Wenrui Lou, Yaqi Chen, Xiaodong Jia, Min Zhai, Zhongren Guo and Jiping Xuan

Forests 2020, 11(2), 207; https://doi.org/10.3390/f11020207 - 12 Feb 2020

Cited by 45 | Viewed by 3874

Abstract

Research Highlights: For the first time, the complete chloroplast (cp) genome of Carya illinoinensis cv. ‘Pawnee’ was de novo assembled. Comprehensive analysis the cp genome of C. illinoinensis revealed potential cpDNA markers for intraspecies identification, genes involved in adaptation, and its phylogenetic position. [...] Read more.

Research Highlights: For the first time, the complete chloroplast (cp) genome of Carya illinoinensis cv. ‘Pawnee’ was de novo assembled. Comprehensive analysis the cp genome of C. illinoinensis revealed potential cpDNA markers for intraspecies identification, genes involved in adaptation, and its phylogenetic position. Background and Objectives: C. illinoinensis is an economically important nut tree in the family Juglandaceae. Cp-derived markers are helpful for genetic research, but they still need to be developed in C. illinoinensis. Additionally, the adaptation and phylogenetic relationships of C. illinoinensis have not been revealed based on the complete cp genome. Materials and Methods: Chloroplast genomic DNA of C. illinoinensis cv. ‘Pawnee’ was extracted and subjected to Illumina sequencing. Results: The cp genome is 160,819 bp in size, exhibiting a typical quadripartite structure with a large single copy (LSC) of 90,022 bp, a small single copy (SSC) of 18,791 bp, and a pair of inverted repeats (IRA and IRB) regions of 26,003 bp each. The genome was predicted to encode 112 unique genes, including 79 protein-coding genes, 29 tRNAs, and four rRNAs, with 19 duplicates in the IR regions. In total, 213 SSRs and 44 long repeats were identified in the cp genome. A comparison of two different C. illinoinensis genotypes, ‘Pawnee’ and 87MX3-2.11, obtained 143 SNPs and 74 indels. The highly variable regions such as atpF, clpP, and ndhA genes, and matK-rps16, trnS-trnG, and trnT-psbD intergenic spacers might be helpful for future intraspecific identification. Positive selection was acting on the ccsA and rps12 cp genes based on the Ka/Ks ratios. Phylogenetic analysis indicated that C. illinoinensis forms a sister clade to Asian Carya species, represented by C. kweichowensis and Annamocarya sinensis. Conclusions: The genome information in our study will have significance for further research on the intraspecies identification and genetic improvement of C. illinoinensis. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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17 pages, 2284 KiB

Open AccessArticle

Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

by Dandan Li, Sen Yu, Minzhen Zeng, Xiao Liu, Jia Yang and Chenghao Li

Forests 2020, 11(2), 193; https://doi.org/10.3390/f11020193 - 10 Feb 2020

Cited by 8 | Viewed by 2352

Abstract

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time [...] Read more.

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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14 pages, 2639 KiB

Open AccessArticle

SLAF-seq Uncovers the Genetic Diversity and Adaptation of Chinese Elm (Ulmus parvifolia) in Eastern China

by Yun-zhou Lyu, Xiao-yun Dong, Li-bin Huang, Ji-wei Zheng, Xu-dong He, Hai-nan Sun and Ze-ping Jiang

Forests 2020, 11(1), 80; https://doi.org/10.3390/f11010080 - 9 Jan 2020

Cited by 15 | Viewed by 3181

Abstract

The Chinese elm is an important tree ecologically; however, little is known about its genetic diversity and adaptation mechanisms. In this study, a total of 107 individuals collected from seven natural populations in eastern China were investigated by specific locus amplified fragment sequencing [...] Read more.

The Chinese elm is an important tree ecologically; however, little is known about its genetic diversity and adaptation mechanisms. In this study, a total of 107 individuals collected from seven natural populations in eastern China were investigated by specific locus amplified fragment sequencing (SLAF-seq). Based on the single nucleotide polymorphisms (SNPs) detected by SLAF-seq, genetic diversity and markers associated with climate variables were identified. All seven populations showed medium genetic diversity, with PIC values ranging from 0.2632 to 0.2761. AMOVA and Fst indicated that a low genetic differentiation existed among populations. Environmental association analyses with three climate variables (annual rainfall, annual average temperature, and altitude) resulted in, altogether, 43 and 30 putative adaptive loci by Bayenv2 and LFMM, respectively. Five adaptive genes were annotated, which were related to the functions of glycosylation, peroxisome synthesis, nucleic acid metabolism, energy metabolism, and signaling. This study was the first on the genetic diversity and local adaptation in Chinese elms, and the results will be helpful in future work on molecular breeding. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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25 pages, 14549 KiB

Open AccessArticle

Effects of Metaxenia on Stone Cell Formation in Pear (Pyrus bretschneideri) Based on Transcriptomic Analysis and Functional Characterization of the Lignin-Related Gene PbC4H2

by Xi Cheng, Jinyun Zhang, Han Wang, Tianzhe Chen, Guohui Li, Chongchong Yan, Qing Jin, Yi Lin and Yongping Cai

Forests 2020, 11(1), 53; https://doi.org/10.3390/f11010053 - 1 Jan 2020

Cited by 11 | Viewed by 4013

Abstract

The deposition of lignin in flesh parenchyma cells for pear stone cells, and excessive stone cells reduce the taste and quality of the fruit. The effect of metaxenia on the quality of fruit has been heavily studied, but the effect of metaxenia on [...] Read more.

The deposition of lignin in flesh parenchyma cells for pear stone cells, and excessive stone cells reduce the taste and quality of the fruit. The effect of metaxenia on the quality of fruit has been heavily studied, but the effect of metaxenia on stone cell formation has not been fully elucidated to date. This study used P. bretschneideri (Chinese white pear) cv. ‘Yali’ (high-stone cell content) and P. pyrifolia (Sand pear) cv. ‘Cuiguan’ (low-stone cell content) as pollination trees to pollinate P. bretschneideri cv. ‘Lianglizaosu’ separately to fill this gap in the literature. The results of quantitative determination, histochemical staining and electron microscopy indicated that the content of stone cells and lignin in YL fruit (‘Yali’ (pollen parent) × ‘Lianglizaosu’ (seed parent)) was significantly higher than that in CL fruit (‘Cuiguan’ (pollen parent) × ‘Lianglizaosu’ (seed parent)). The transcriptome sequencing results that were obtained from the three developmental stages of the two types of hybrid fruits indicated that a large number of differentially expressed genes (DEGs) related to auxin signal transduction (AUX/IAAs and ARFs), lignin biosynthesis, and lignin metabolism regulation (MYBs, LIMs, and KNOXs) between the CL and YL fruits at the early stage of fruit development. Therefore, metaxenia might change the signal transduction process of auxin in pear fruit, thereby regulating the expression of transcription factors (TFs) related to lignin metabolism, and ultimately affecting lignin deposition and stone cell development. In addition, we performed functional verification of a differentially expressed gene, PbC4H2 (cinnamate 4-hydroxylase). Heterologous expression of PbC4H2 in the c4h mutant not only restored its collapsed cell wall, but also significantly increased the lignin content in the inflorescence stem. The results of our research help to elucidate the metaxenia-mediated regulation of pear stone cell development and clarify the function of PbC4H2 in cell wall development and lignin synthesis, which establishes a foundation for subsequent molecular breeding. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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10 pages, 973 KiB

Open AccessArticle

Pinus sylvestris Breeding for Resistance against Natural Infection of the Fungus Heterobasidion annosum

by Raitis Rieksts-Riekstiņš, Pauls Zeltiņš, Virgilijus Baliuckas, Lauma Brūna, Astra Zaļuma and Rolands Kāpostiņš

Forests 2020, 11(1), 23; https://doi.org/10.3390/f11010023 - 22 Dec 2019

Cited by 11 | Viewed by 3054

Abstract

Increasing resistance against biotic and abiotic factors is an important goal of forest tree breeding. The aim of the present study was to develop a root rot resistance index for Scots pine breeding and evaluate its effectiveness. The productivity, branch diameter, branchiness, stem [...] Read more.

Increasing resistance against biotic and abiotic factors is an important goal of forest tree breeding. The aim of the present study was to develop a root rot resistance index for Scots pine breeding and evaluate its effectiveness. The productivity, branch diameter, branchiness, stem straightness, spike knots, and damage from natural infection of root rot in 154 Scots pine open-pollinated families from Latvia were evaluated through a progeny field trial at the age of 38 years. Trees with decline symptoms were sampled for fungal isolations. Based on this information and kriging estimates of root rot, 35 affected areas (average size: 108 m²; total 28% from the 1.5 ha trial) were delineated. Resistance index of a single tree was formed based on family adjusted proportion of live to infected trees and distance to the center of affected area. Heritability for resistance to root rot based on the value of this index, was high (0.37) and comparable to indices of growth traits. Correlations of family breeding estimates between resistance to root rot and the other traits were not significant, except for a weak, yet significant, positive correlation with diameter at breast height and branch diameter. Selection index including only growth traits (height and stem volume) had a negligible effect on damage by root rot. We detected a maximum genetic gain in resistance index of 33.7% when incorporating it into the selection index with positive gains for growth traits (6.5–11.0%). Two-stage selection with prior selection of the most resistant families was not superior to the use of selection index with only rot resistance included. Overall; rot resistance index appeared to be an effective tool in tree breeding for the selection of more resistant families, using the existing trials with natural (uncontrolled) infection Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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13 pages, 2273 KiB

Open AccessArticle

Sub-Genome Polyploidization Effects on Metabolomic Signatures in Triploid Hybrids of Populus

by Shiping Cheng, Yuxia Zong and Xuewen Wang

Forests 2019, 10(12), 1091; https://doi.org/10.3390/f10121091 - 1 Dec 2019

Cited by 6 | Viewed by 2594

Abstract

Allopolyploids are known to have superior advantages such as high growth speed. Triploids have even greater heterozygosity, explaining more phenotypic variance than 2n hybrid F1 and have therefore become new resources in breeding. To date, the metabolomic basis underlying polyploidization vigor remains unclear. [...] Read more.

Allopolyploids are known to have superior advantages such as high growth speed. Triploids have even greater heterozygosity, explaining more phenotypic variance than 2n hybrid F1 and have therefore become new resources in breeding. To date, the metabolomic basis underlying polyploidization vigor remains unclear. Here, we identified and compared 235 metabolites in the shoot apical buds between multiple allo-triploid populations and parental 2n hybrid F1 in Populus via metabolome profiling using liquid chromatography–mass spectrometry (LC–MS) assays. Associations with growth vigor in three types of allo-triploid populations, namely first division restitution (FDR), second division restitution (SDR) and postmeiotic restitution (PMR) generated from doubled 2n female gametes and male gametes of 2n hybrid, were also investigated. Each allo-triploid population has different sub-genome duplicated. Major metabolomes were amino acids, secondary metabolism associated, and carbohydrates. We mapped 181 metabolites into known metabolism pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Ten compounds, i.e., fructose 1,6-diphosphate and xylulose, were more abundant in all allo-triploids than the 2n hybrid. Principal component analysis revealed the abundance of metabolites fell into distinct clusters corresponding to ploidy composition. Heterozygosity in triploids mainly effected the contents of carbohydrates and secondary metabolites rather than lipids. Comparisons between subgroups with different growth rates revealed some carbohydrates and secondary metabolites of flavonoids were positively associated with gene expression and the high growth vigor. The results provided an informative metabolomic basis for factors conferring growth vigor in polyploid Populus. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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19 pages, 2372 KiB

Open AccessArticle

Combining Quantitative Data on Growth, Wood Density and Other Traits with SSR Markers to Evaluate Genetic Diversity and Structure in a Planted Population of Eucalyptus camaldulensis Dehn.

by Xiuhua Shang, Roger J. Arnold, Zhihua Wu, Peijian Zhang, Guo Liu, Jianzhong Luo and Ni Zhan

Forests 2019, 10(12), 1090; https://doi.org/10.3390/f10121090 - 1 Dec 2019

Cited by 5 | Viewed by 2639

Abstract

Eucalyptus camaldulensis Dehn. is one of the most morphologically and genetically variable Eucalyptus species. Growth, Leptocybe invasa Fisher & La Salle susceptibility, pilodyn penetration and other traits up to age 36 months were assessed in a seed source/family trial in China comprising 112 [...] Read more.

Eucalyptus camaldulensis Dehn. is one of the most morphologically and genetically variable Eucalyptus species. Growth, Leptocybe invasa Fisher & La Salle susceptibility, pilodyn penetration and other traits up to age 36 months were assessed in a seed source/family trial in China comprising 112 seedlots representing five natural stand and six exotic seed sources. Genetic diversity and population structure of this trial population were also analyzed using 48 simple sequence repeat (SSR) markers. The key objective was to examine whether the genomic data could provide value over information obtained from just quantitative trait data. Significant genetic variation was found among seed sources and among families within seed sources for most quantitative traits. The ratio of variance among seed sources to variance among families within seed sources, based on variances estimated from quantitative trait data, varied from 0.1% (height at 9 months) up to 75.2% (bark thickness). Equivalent ratios estimated from the AMOVA on SSR loci data were similar for height (ages 24 and 36 months) and also pilodyn penetration at 36 months, but not for 9-month height or 36-month bark thickness. From 48 SSR loci examined, the genetic differentiation coefficient (among seed sources) was 0.086, indicating low genetic differentiation among seed sources. While overall genetic diversity in the trial population examined was high, the levels within the different seed sources varied markedly. Prior to this study, genetic distances among families from the three exotic seed sources (from domesticated Indian populations) in the trial, along with their genetic distances from, and relatedness to, families from five natural stand seed sources (Australian) in the trial were unknown. The SSR loci data removed uncertainties and revealed that the exotic sources increased the breadth of genetic origins represented in the trial population—information that could not have been obtained from just the quantitative trait data. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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18 pages, 5329 KiB

Open AccessArticle

Identification and Expression of NAC Transcription Factors of Vaccinium corymbosum L. in Response to Drought Stress

by Kehao Liang, Aibin Wang, Yongjiang Sun, Mingxin Yu and Lingyun Zhang

Forests 2019, 10(12), 1088; https://doi.org/10.3390/f10121088 - 1 Dec 2019

Cited by 9 | Viewed by 2925

Abstract

Research Highlights: Phenotypic changes and expression profiles, phylogeny, conserved motifs, and expression correlations of NAC (NAM, ATAF1, ATAF2 and CUC2) transcription factors (TFs) in blueberry genome were detected under drought stress, and the expression patterns and functions of 12 NACs were analyzed. Background [...] Read more.

Research Highlights: Phenotypic changes and expression profiles, phylogeny, conserved motifs, and expression correlations of NAC (NAM, ATAF1, ATAF2 and CUC2) transcription factors (TFs) in blueberry genome were detected under drought stress, and the expression patterns and functions of 12 NACs were analyzed. Background and Objectives: Blueberry is an important shrub species with a high level of flavonoids in fruit, which are implicated in a broad range of health benefits. However, the molecular mechanism of this shrub species in response to drought stress still remains elusive. NAC TFs widely participate in stress tolerance in many plant species. The characterization and expression profiles of NAC TFs were analyzed on the basis of genome data in blueberry when subjected to drought stress. Materials and Methods: Combined with the analysis of chlorophyll a fluorescence and endogenous phytohormones, the phenotypic changes of blueberry under drought stress were observed. The phylogenetic tree, conserved motifs, differently expressed genes, and expression correlation were determined by means of multiple bioinformatics analysis. The expression profiles of NACs in different organs were examined and compared through RNA-seq and qRT-PCR assay. Results: The chlorophyll a fluorescence parameters φ_Po, φ_Eo, φ_Ro, and PIabs of leaves were significantly inhibited under drought stress. ABA (abscisic acid) content noticeably increased over the duration of drought, whereas GA₃ (gibberellic acid) and IAA (indole acetic acid) content decreased continuously. A total of 158 NACs were identified in blueberry genome and 62 NACs were differently expressed in leaf and root of blueberry under drought stress. Among them, 14 NACs were significantly correlated with the expression of other NAC genes. Conclusions: Our results revealed the phenotypic changes of this shrub under drought stress and linked them with NAC TFs, which are potentially involved in the process of response to drought stress. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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13 pages, 2380 KiB

Open AccessArticle

Low Divergence Among Natural Populations of Cornus kousa subsp. chinensis Revealed by ISSR Markers

by Jia-Qiu Yuan, Qin Fang, Guo-Hua Liu and Xiang-Xiang Fu

Forests 2019, 10(12), 1082; https://doi.org/10.3390/f10121082 - 28 Nov 2019

Cited by 2 | Viewed by 2480

Abstract

Research Highlights: Taking Chinese dogwood (Cornus kousa subsp. chinensis) as an example, the genetic characteristics of natural populations collected from main a distribution area were evaluated using intersimple sequence repeat (ISSR) markers to reveal the genetic basis for further selection and [...] Read more.

Research Highlights: Taking Chinese dogwood (Cornus kousa subsp. chinensis) as an example, the genetic characteristics of natural populations collected from main a distribution area were evaluated using intersimple sequence repeat (ISSR) markers to reveal the genetic basis for further selection and breeding. Background and Objectives: Chinese dogwood is a small understory tree that is widely distributed in China. Chinese dogwood has attracted interest for its potential horticultural and ornamental values, and its natural resource potential urgently needs to be estimated. Materials and Methods: In this study, the genetic diversity of 12 natural populations collected from six provinces containing 223 individuals was evaluated based on ISSR markers. Results: Relatively high levels of genetic diversity were found at both the population and individual levels. The Shannon’s diversity index (I) among individuals (0.504) was higher than that among populations (0.338). Analysis of molecular variance (AMOVA) revealed that genetic variation mainly existed within populations (61.55%) rather than among populations (38.45%). According to the STRUCTURE analysis, 12 populations were assigned to two groups, i.e., the northern and southern ecological regions, which are separated by the Yangtze River. A Mantel test analysis showed that there was no significant correlation between genetic distance and geographic distance. Conclusions: Considering the breeding system of dogwoods, we speculated that the genetic characteristics of the natural populations of this species would be affected by the dispersal mode of its pollens and seeds; additionally, genetic drift could play an important role in its genetic differentiation. In conclusion, in situ conservation is recommended for Chinese dogwood based on our results. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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20 pages, 5993 KiB

Open AccessArticle

Effect of Phosphinothricin on Transgenic Downy Birch (Betula pubescens Ehrh.) Containing bar or GS1 Genes

by Vadim G. Lebedev, Konstantin V. Krutovsky and Konstantin A. Shestibratov

Forests 2019, 10(12), 1067; https://doi.org/10.3390/f10121067 - 24 Nov 2019

Cited by 4 | Viewed by 2900

Abstract

Weeds are a big problem in agriculture and forestry, and herbicides are the main tools to control them. Phosphinotricin (ammonium glufosinate, PPT) is one of the most effective non-selective herbicides, to which weeds hardly gain resistance, but the reasons for its effect and [...] Read more.

Weeds are a big problem in agriculture and forestry, and herbicides are the main tools to control them. Phosphinotricin (ammonium glufosinate, PPT) is one of the most effective non-selective herbicides, to which weeds hardly gain resistance, but the reasons for its effect and toxicity to plants are still unclear, and especially, it is little studied in trees, including transgenic ones. We studied the physiological responses of downy birch (Betula pubescens Ehrh.) containing the herbicide resistance bar gene or the cytosol glutamine synthetase GS1 gene (the target enzyme of the herbicide) to PPT-based Basta herbicide treatment in various doses under open-air conditions during two years. Birch saplings with the bar gene were resistant to a double field dose (10 L/ha), but the expression of the GS1 gene only slightly increased resistance compared to the control. Herbicide treatment increased the ammonium level in leaf tissue by 3–8 times, but this, apparently, was not the main cause of plant death. Among leaf pigments, chlorophyll B was the most resistant to PPT, and carotenoids were the most sensitive. Responses of birch trees with the GS1 gene (accumulation of ammonium, pigment content, and dehydration) during treatment with a low dose of herbicide were less pronounced than in control plants. One-year-old control and transgenic plants with the GS gene died after 2.5 L/ha treatment, and two-year-old plants lost foliage after such treatment but remained alive and developed buds four weeks after treatment. Herbicide treatment of plants with the bar gene did not cause significant deviations in height (first year) or the accumulation of aboveground biomass (second year). The obtained results improve our understanding of the effect of PPT on woody plants and can be used both to clarify mechanisms of herbicide action and in plantation forestry. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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17 pages, 3003 KiB

Open AccessArticle

Genetic Diversity and Population Genetic Structure of Cinnamomum camphora in South China Revealed by EST-SSR Markers

by Yongda Zhong, Aihong Yang, Zhiting Li, Hui Zhang, Lipan Liu, Zhaoxiang Wu, Yanqiang Li, Tengyun Liu, Meng Xu and Faxin Yu

Forests 2019, 10(11), 1019; https://doi.org/10.3390/f10111019 - 13 Nov 2019

Cited by 22 | Viewed by 3718

Abstract

Cinnamomum camphora is a valuable broad-leaf tree indigenous to South China and East Asia and has been widely cultivated and utilized by humans since ancient times. However, owing to its overutilization for essential oil extraction, the Transplanting Big Trees into Cities Program, and [...] Read more.

Cinnamomum camphora is a valuable broad-leaf tree indigenous to South China and East Asia and has been widely cultivated and utilized by humans since ancient times. However, owing to its overutilization for essential oil extraction, the Transplanting Big Trees into Cities Program, and over deforestation to make furniture, its wild populations have been detrimentally affected and are declining rapidly. In the present study, the genetic diversity and population structure of 180 trees sampled from 41 populations in South China were investigated with 22 expressed sequence tag-simple sequence repeat (EST-SSR) markers. In total, 61 alleles were harbored across 180 individuals, and medium genetic diversity level was inferred from the observed heterozygosity (Ho), expected heterozygosity (He), and Nei’ gene diversity (GD), which were 0.45, 0.44, and 0.44, respectively. Among the 41 wild populations, C. camphora had an average of 44 alleles, 2.02 effective alleles, and He ranging from 0.30 (SC) to 0.61 (HK). Analysis of molecular variance (AMOVA) showed that 17% of the variation among populations and the average pairwise genetic differentiation coefficient (F_ST) between populations was 0.162, indicating relatively low genetic population differentiations. Structure analysis suggested two groups for the 180 individuals, which was consistent with the principal coordinate analysis (PCoA) and unweighted pair-group method with arithmetic means (UPGMA). Populations grouped to cluster I were nearly all distributed in Jiangxi Province (except population XS in Zhejiang Province), and cluster II mainly comprised populations from other regions, indicating a significant geographical distribution. Moreover, the Mantel test showed that this geographical distance was significantly correlated with genetic distance. The findings of this research will assist in future C. camphora conservation management and breeding programs. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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17 pages, 5189 KiB

Open AccessArticle

Differential Expression of Genes Related to the Formation of Giant Leaves in Triploid Poplar

by Kang Du, Qiang Han, Ying Zhang and Xiangyang Kang

Forests 2019, 10(10), 920; https://doi.org/10.3390/f10100920 - 19 Oct 2019

Cited by 5 | Viewed by 2977

Abstract

Plant polyploids tend to have large leaves, but their formation mechanism has not yet been well explained. Therefore, daily transcriptomic differences between triploids and diploids from a synthetic Populus sect. Tacamahaca three times a day (i.e., 04:00, 09:00, and 21:00) were investigated using [...] Read more.

Plant polyploids tend to have large leaves, but their formation mechanism has not yet been well explained. Therefore, daily transcriptomic differences between triploids and diploids from a synthetic Populus sect. Tacamahaca three times a day (i.e., 04:00, 09:00, and 21:00) were investigated using high-throughput RNA-seq analysis. In this study, we identified several transcription factors associated with giant leaves. The combined effects included the high expression of several transcription factors (WRKY, MYB, etc.) and hormone-related genes (e.g., activates auxin, cytokine, and brassinosteroid synthesis-related genes) that accelerate the synthesis and accumulation of endogenous hormones. High levels of growth hormones were maintained by reducing the genes’ expression of hormone metabolism and degradation. The coordination of hormones accumulated sufficient materials and energy for leaf growth and development. Thereby, cell division and growth were accelerated which enhanced the photosynthesis of leaves, and the increased accumulation of photosynthetic products led to giant triploid leaves. This study lays the foundation for revealing the molecular mechanisms in the formation of giant leaves in polyploids. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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13 pages, 6599 KiB

Open AccessArticle

Identification of Reference Genes for Quantitative Gene Expression Studies in Pinus massoniana and Its Introgression Hybrid

by Jiaxing Mo, Jin Xu, Wenjing Jin, Liwei Yang, Tongming Yin and Jisen Shi

Forests 2019, 10(9), 787; https://doi.org/10.3390/f10090787 - 11 Sep 2019

Cited by 6 | Viewed by 2621

Abstract

qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and [...] Read more.

qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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14 pages, 2477 KiB

Open AccessArticle

Chinese Fir Breeding in the High-Throughput Sequencing Era: Insights from SNPs

by Huiquan Zheng, Dehuo Hu, Ruping Wei, Shu Yan and Runhui Wang

Forests 2019, 10(8), 681; https://doi.org/10.3390/f10080681 - 12 Aug 2019

Cited by 12 | Viewed by 3723

Abstract

Knowledge on population diversity and structure is of fundamental importance for conifer breeding programs. In this study, we concentrated on the development and application of high-density single nucleotide polymorphism (SNP) markers through a high-throughput sequencing technique termed as specific-locus amplified fragment sequencing (SLAF-seq) [...] Read more.

Knowledge on population diversity and structure is of fundamental importance for conifer breeding programs. In this study, we concentrated on the development and application of high-density single nucleotide polymorphism (SNP) markers through a high-throughput sequencing technique termed as specific-locus amplified fragment sequencing (SLAF-seq) for the economically important conifer tree species, Chinese fir (Cunninghamia lanceolata). Based on the SLAF-seq, we successfully established a high-density SNP panel consisting of 108,753 genomic SNPs from Chinese fir. This SNP panel facilitated us in gaining insight into the genetic base of the Chinese fir advance breeding population with 221 genotypes for its genetic variation, relationship and diversity, and population structure status. Overall, the present population appears to have considerable genetic variability. Most (94.15%) of the variability was attributed to the genetic differentiation of genotypes, very limited (5.85%) variation occurred on the population (sub-origin set) level. Correspondingly, low F_ST (0.0285–0.0990) values were seen for the sub-origin sets. When viewing the genetic structure of the population regardless of its sub-origin set feature, the present SNP data opened a new population picture where the advanced Chinese fir breeding population could be divided into four genetic sets, as evidenced by phylogenetic tree and population structure analysis results, albeit some difference in membership of the corresponding set (cluster vs. group). It also suggested that all the genetic sets were admixed clades revealing a complex relationship of the genotypes of this population. With a step wise pruning procedure, we captured a core collection (core 0.650) harboring 143 genotypes that maintains all the allele, diversity, and specific genetic structure of the whole population. This generalist core is valuable for the Chinese fir advanced breeding program and further genetic/genomic studies. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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11 pages, 4226 KiB

Open AccessArticle

A Weighted Mean Value Analysis to Identify Biological Pathway Activity Changes during Poplar Seed Germination

by Chunpu Qu, Yuqing Zhang, Jinyuan Chen, Shuang Zhang, Jiajie Yu, Chengjun Yang, Xiuli Zhang, Zhi-Ru Xu and Guan-Jun Liu

Forests 2019, 10(8), 664; https://doi.org/10.3390/f10080664 - 7 Aug 2019

Cited by 3 | Viewed by 2846

Abstract

Poplar (Populus × xiaohei T. S. Hwang et Liang) is an excellent model plant, with a known genome sequence, for studying woody plant developmental processes, such as seed germination. Here, we report the transcriptional profiling of poplar seeds at five germination stages [...] Read more.

Poplar (Populus × xiaohei T. S. Hwang et Liang) is an excellent model plant, with a known genome sequence, for studying woody plant developmental processes, such as seed germination. Here, we report the transcriptional profiling of poplar seeds at five germination stages using RNA-Seq technology. We focused on identifying biological pathway activity changes during seed germination and transcription factors that play important roles in different stages. Among the 16 significantly changing clusters obtained using the STEM method, transcription was significantly enriched in five different clusters, 8, 21, 25, 27, and 35. The oxidative phosphorylation-related genes were only enriched in cluster 9, and expression patterns decreased in 6 and 24 HAI, while ubiquitin-dependent protein catabolic processes were only enriched in cluster 16, and expression patterns increased in 6 HAI. A weighted mean method analysis determined that most primary metabolism-associated categories, such as major carbohydrate metabolism, glycolysis, oxidative pentose phosphate, tricarboxylic acid cycle, lipid metabolism, nucleotide metabolism, amino acid metabolism, and protein metabolism, were elevated between 6 and 48 h after imbibition (HAI). ATP synthesis and C1 metabolism had highly active expression patterns between 0.75 and 48 HAI. The photosynthesis category-associated genes that were identified appeared highly active at 144 HAI. The homogenization of transcription factors in each cluster revealed that the HAP2, C3H zinc finger family, and C2C2(Zn) GATA transcription factors were present in relatively high numbers in cluster 8, while HAP5, Zn-finger (CCHC), FHA, and E2F/DP transcription factor families, as well as SNF7, were present in high numbers in cluster 25. Thus, we identified a series of biological pathway activity changes that occur, and transcription factors that are active, during poplar seed germination. Moreover, this study provides an integrated view of transcriptional regulation that can reveal the molecular events occurring during seed germination. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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12 pages, 3173 KiB

Open AccessArticle

Non-Targeted Metabolomics Reveals Patterns of Metabolic Changes during Poplar Seed Germination

by Chunpu Qu, Jinyuan Chen, Lina Cao, Xiangjin Teng, Jinbo Li, Chengjun Yang, Xiuli Zhang, Yuhong Zhang, Guanjun Liu and Zhiru Xu

Forests 2019, 10(8), 659; https://doi.org/10.3390/f10080659 - 6 Aug 2019

Cited by 3 | Viewed by 2981

Abstract

Research Highlights: This study was the first to use metabolomics techniques to investigate seed germination in poplar, a model woody plant. Our results lay a foundation for uncovering changes in metabolite levels during woody plant seed germination and for understanding the underlying mechanism. [...] Read more.

Research Highlights: This study was the first to use metabolomics techniques to investigate seed germination in poplar, a model woody plant. Our results lay a foundation for uncovering changes in metabolite levels during woody plant seed germination and for understanding the underlying mechanism. Background and Objectives: Poplar is a model woody plant. Because poplar can be easily propagated asexually, the molecular mechanism of poplar seed germination has not been well studied. However, long-term asexual reproduction of poplar leads to seedlings with weak resistance, high vulnerability to degradation, and reduced growth potential. Materials and Methods: The non-targeted metabolomics technique was used to analyze changing trends in metabolite contents during the poplar seed germination process. Results: We found that the number of differential metabolites increased as seed germination progressed. Metabolic pathway analysis of differential metabolites revealed that galactose metabolism and alanine, aspartate, and glutamate metabolism were significantly enriched during all germination periods. MapMan-based visual analysis of metabolic pathways of differential metabolites indicated that glutamine, glutamic acid, phenylalanine, arginine, and asparagine contents increased with germination time, with most metabolites related to glucose metabolism following similar trends. Contents of most metabolites related to the tricarboxylic acid cycle exhibited a fluctuating pattern. Conclusions: This study has revealed the major changes taking place in primary metabolite contents during poplar seed germination and has laid the foundation for elucidation of the molecular mechanism of poplar seed germination. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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18 pages, 3807 KiB

Open AccessArticle

Selection of Suitable Reference Genes in Pinus massoniana Lamb. Under Different Abiotic Stresses for qPCR Normalization

by Peihuang Zhu, Yinyan Ma, Lingzhi Zhu, Yu Chen, Rong Li and Kongshu Ji

Forests 2019, 10(8), 632; https://doi.org/10.3390/f10080632 - 27 Jul 2019

Cited by 36 | Viewed by 3599

Abstract

The normalization of data by choosing suitable reference genes is fundamental for obtaining accurate and reliable results in quantitative real-time polymerase chain reaction (qPCR) analyses. In this study, the expression stability of 12 candidate reference genes of Pinus massoniana under different abiotic stresses [...] Read more.

The normalization of data by choosing suitable reference genes is fundamental for obtaining accurate and reliable results in quantitative real-time polymerase chain reaction (qPCR) analyses. In this study, the expression stability of 12 candidate reference genes of Pinus massoniana under different abiotic stresses was evaluated using four statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. The results indicate that the following genes could be used as reference genes under different treatments: Actin 2 (ACT2) and F-box family gene (F-box) for salinity treatment, cyclophilin (CYP) and alpha-tubulin (TUA) for ABA treatment, actin 7 (ACT7) and CYP for drought treatment, actin 1 (ACT1) and ACT7 for cold treatment, ACT1 and CYP for heat treatment, and TUA and ACT2 for the “Total” group. To validate the suitability of the selected reference genes in this study, the Short-Root protein (SHR), Alpha-pinene synthase (APS), and Pyrabactin resistance-like protein (PYL) gene expression patterns were analyzed. The expression patterns had significant biases when the most unstable reference genes were used for normalization, compared with when the optimum reference gene or gene combinations were used for normalization. These results will be beneficial for further studies on gene transcription in early-stage, unlignified seedlings of P. massoniana. Full article

(This article belongs to the Special Issue Forest Genetics and Tree Improvement)

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