Molecular Biology Reports

, Volume 39, Issue 4, pp 3773–3784

Gene expression profiles of two intraspecific Larix lines and their reciprocal hybrids

Authors

  • Ai Li
    • College of Life SciencesNankai University
  • Meng-Die Fang
    • College of Life SciencesNankai University
  • Wen-Qin Song
    • College of Life SciencesNankai University
  • Cheng-Bin Chen
    • College of Life SciencesNankai University
  • Li-Wang Qi
    • College of Life SciencesNankai University
    • College of Life SciencesNankai University
Article

DOI: 10.1007/s11033-011-1154-y

Cite this article as:
Li, A., Fang, M., Song, W. et al. Mol Biol Rep (2012) 39: 3773. doi:10.1007/s11033-011-1154-y

Abstract

Heterosis has been widely explored in Larix breeding for more than a century, but the molecular mechanisms underlying this phenomenon remain elusive. In the present study, the genome-wide transcript profiles from two Larix genotypes and their reciprocal hybrids were analyzed using Arabidopsis 70-mer oligonucleotide microarrays. Despite sharing the same two parental lines, one of the hybrids showed obvious heterosis, while the other did not. In total, 1,171 genes were differentially expressed between the heterotic hybrid and its parents, of which 133 genes were nonadditive expression. The number of differentially expressed genes between the non-heterotic hybrid and the parents was 939, but only 54 of these genes were nonadditive expression. Further, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses indicated that most of these differentially expressed genes in the heterotic hybrid were associated with several important biological functions such as physiological processes, responses to stimulus, and starch and sucrose metabolism. The reliability of the microarray data was further validated by the Real-time quantitative RT-PCR. A high Pearson linear correlation coefficient value was detected (r = 0.759, P < 0.01). In conclusion, the gene expression profile in the Larix heterotic hybrid was significantly different from that obtained from the non-heterotic hybrid, and more nonadditive differentially expressed genes were detected in the heterotic hybrid, implying that nonadditive effects may be closely associated with the formation of heterosis in the intraspecific Larix hybridization.

Keywords

Larix kaempferi (Lamb.)HeterosisNonadditive expressionTranscript profile

Introduction

As a driving force for speciation and evolution, hybridization events have played their part in the formation of many plant species [1]. Heterosis or hybrid vigor in plants refers to increases in grain yield, vegetative growth rate, resistance to pests and environmental stresses, accelerated maturity, and many other changes in desirable plant characteristics relative to their parents [2]. Hybrid breeding has been widely used in many crop and forest plants, and this has made significant contributions to the world and brought great economic and societal benefits [3]. However, the nature of heterosis remains ambiguous, and three main explanatory hypotheses have been proposed, namely the dominance, the over-dominance and the epistasis hypotheses [47]. Each of these hypotheses can be supported by results from quantitative trait locus (QTL) mapping studies [810]. However, no strong consensus has emerged concerning which of these hypotheses best explains heterosis, especially at the molecular level [11].

Recent investigations of the molecular mechanisms of heterosis have demonstrated that there are often considerable differences in gene expression between heterotic hybrids and their parents, and most of the differentially expressed genes have been associated with particular biological process, including metabolism and the syntheses of carbohydrates and starch [1214]. Further analyses indicate that these differentially expressed genes in hybrids are correlated with certain heterosis phenotypic traits, of which nonadditive gene expression in the F1 hybrids has been documented in several cases [15, 16]. Moreover, a number of reports that examined global gene expression or some specific classes of genes in inbred lines and their hybrids showed that nonadditive gene expression in hybrids is more closely associated with heterosis [1719]. However, most of these investigations have concerned model plants such as Arabidopsis or important crops, including rice, maize and Brassica campestris [1517, 20], which are all herbaceous plants. In contrast, little attention has been dedicated to woody perennial plants, especially gymnosperms, mainly due to their large genome sizes, complex genetic backgrounds and long growth cycles.

In Larix breeding, heterosis has been widely exploited for more than a century. Some investigations have shown that the Larix F1 can be superior to both parents in terms of wood quality [21], vole resistance [22, 23] and growth rate [24, 25]. Unfortunately, there is still a very limited understanding of the underlying genetic and molecular mechanisms for these observations. In a previous study, the characteristics of genomic DNA methylation between a set of Larix reciprocal hybrids and their corresponding parents have been analyzed. The results indicated that the heterotic hybrid showed lower DNA methylation levels, and demethylation events were predominant compared with their corresponding parents (data not shown). These findings indicated that alterations of the genomic DNA methylation profile may be associated with heterosis in intraspecific Larix. In the present study, to further elucidate the molecular mechanisms of heterosis in intraspecific Larix, the genome transcriptome profiles of reciprocal hybrids and their parents were analyzed using the Arabidopsis 70-mer oligonucleotide microarray. The objectives of this study were to gain a global view of gene expression changes in the reciprocal hybrids compared to their parental lines, and the different characteristics of gene expression profiles between the two reciprocal hybrids. This genome-wide transcriptome comparison should allow a deeper understanding of the causative mechanisms of altered gene expression in the hybrid and, ultimately, the molecular mechanisms underlying heterosis in Larix.

Materials and methods

Plant materials

Two intraspecific Larix genotypes Larix13 and Larix82 and their reciprocal hybrids Larix82 × 13-6 and Larix13 × 82-21 that have been grown for more than 20 years were used in this study. The hybrid Larix82 × 13-6 (generated by crossing Larix13 with Larix82 as the female recipient of pollen) exhibited obvious heterosis, while the hybrid Larix13 × 82-21 (generated by crossing Larix82 with Larix13 as the female recipient of pollen) showed no signs of heterosis.

RNA extraction

Total RNA was isolated from young fresh leaves using the CTAB method with some modifications [26], and was purified using the NucleoSpin® RNA clean-up kit according to manufacturer protocols (MACHEREY–NAGEL, Germany). Each quantified RNA sample was confirmed by measuring its A260/A280 ratio using NanoDrop®ND-1000 (Nanodrop Technologies, USA) and by 1% agarose electrophoresis.

Preparation of microarray slides

A total of 29,110 unique Arabidopsis 70-mer oligonucleotides representing 26,173 Arabidopsis genes and 28,964 transcripts were spotted onto 75 × 25 mm microarray slides using the SmartArrayTM (CapitalBio Corp., Beijing, China). The details of the 70-mer oligos can be found at http://www.Operon.com.

RNA labeling and microarray hybridization

Dye-Swap labeled microarray hybridizations were performed according to a reference design over the reciprocal hybrids and their parents. Each sample was labeled with Cy5-dCTP or Cy3-dCTP using Crystal Core® cRNA amplified RNA labeling kit. In brief, Larix13, Larix82, Larix82 × 13-6 and Larix13 × 82-21 were labeled with Cy5-dCTP or Cy3-dCTP, respectively. Then four probes were made for pairwise comparisons between the hybrids and their parental lines by mixing in equal proportions the differently-labeled RNA samples, which were Larix82 × 13-6 (Cy5-dCTP)/Larix13 (Cy3-dCTP), Larix82 × 13-6 (Cy5-dCTP)/Larix82 (Cy3-dCTP), Larix13 × 82-21 (Cy5-dCTP)/Larix13 (Cy3-dCTP) and Larix13 × 82-21 (Cy5-dCTP)/Larix82 (Cy3-dCTP). Each probe was hybridized with the Arabidopsis 70-mer oligonucleotide microarray. Simultaneously, to ensure the reliability of the microarray hybridization, another four identical probes with a reversed fluorescent labeling combination were also prepared to hybridize with the microarray.

Data analysis and biological function classification of differentially expressed genes

Microarrays were scanned with a confocal LuxScanTM scanner (CapitalBio Corp., Beijing, China) and the data from these images was extracted using the SpotData software (CapitalBio Corp., Beijing, China). A space and intensity-dependent normalization method based on the LOWESS program was employed [27]. Genes with a signal intensity (Cy3 or Cy5) > 800 were regarded as expressed. Those genes whose alteration tendencies remained consistent in both arrays and had mean expression ratios above twofold were selected as differentially expressed genes. The formula used for ratio calculation was ratio = (ratio1 × ratio2)0.5. Clustering analysis was processed by Cluster and Treeview software to identify gene expression patterns, and then Molecule Annotation System V4.0 (CapitalBio Corp., Beijing, China) was used to categorize the differentially expressed genes according to biological function.

Expression validation via real-time quantitative RT-PCR (qRT-PCR)

qRT-PCR was conducted to validate the gene expression patterns detected by the microarray analyses. For this, 16 genes were selected that had differential expression levels of at least twofold outside the range of the parents. Total RNA samples were prepared as those for the microarrays, and the first strand cDNA synthesis was performed using M-MLV reverse transcriptase (promega, USA) according to the manufacturer’s instructions. qRT-PCR was carried out using the icycler Thermal Cycler (Bio-RAD, USA). To ensure the reliability of the quantitative analyses, the actin gene was selected as the control in each experiment, and three biological and technical replicates were carried out for each sample. Reactions were set up by combining 12.5 μl of 2 × Reaction Mix (Roche, Germany) with 1 μl cDNA templates (100 ng), 1 μl of forward and 1 μl of reverse primer (10 μM each), and 9.5 μl of ddH20, under the following conditions: 95°C for 15 s; 40 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s with melting curve analysis in the thermocycler. Data on the threshold cycle at which the fluorescent intensity of each sample first increased above background levels were collected and normalized to the levels associated with actin. Then the relative expression levels of the selected genes were calculated using the comparative 2−ΔΔCT method according to the manufacturer’s recommendations, respectively. Fold change = 2−ΔΔCt. ΔΔCt = [(Ctgene of interest − Ctinternal control) sample A − (Ctgene of interest − Ctinternal control) sample B].

Results

Transcriptome profiles of the reciprocal hybrids and their parental lines

In this present study, the microarray hybridizations were designed mainly to detect four pairwise comparisons between the two reciprocal hybrids (Larix82 × 13-6 and Larix13 × 82-21) and their corresponding parental lines (Larix13 and Larix82). To further calibrate the hybridization, eleven housekeeping genes from Arabidopsis were used as positive controls (Table 1), with Hex as a spotting positive control, 50% DMSO as a negative control, and eight yeast genes as exogenous controls. For all the slide hybridizations, fluorescent intensity was scanned for each spot. Then the intensity ratio (hybrid/one of parental lines) was calculated. A global representation of the changes in the expression of all genes was depicted in Fig. 1. As it was designed, each of these points denoted one gene that corresponded to an Arabidopsis 70-mer oligo-ID. In total, 2,792 and 3,827 genes were detected by the corresponding probes Larix82 × 13-6/Larix13 and Larix82 × 13-6/Larix82, respectively. While the hybridization using the Larix13 × 82-21/Larix13 and Larix13 × 82-21/Larix82 as probes indicated that 3,041 and 4,506 genes showed distinct hybridization signals, respectively (Table 2). Further cluster analyses revealed that each pairwise comparison of the dye-swap labeled microarray hybridizations formed the primary groups. This suggests that the microarray hybridization data was reliable. In broad terms, the transcriptome profiles of Larix82 × 13-6/Larix13 were similar to Larix13 × 82-21/Larix13, while Larix82 × 13-6/Larix82 was similar to Larix13 × 82-21/Larix82 (Fig. 2a). The data also showed that more up-regulated genes were identified in the reciprocal hybrids compared to the Larix13 than in comparison with Larix82 (Figs. 1, 2a).
Table 1

Microarray positive control of Arabidopsis thaliana housekeeping genes

Name

Description

At1g64740

Tubulin alpha-1 chain (TUA1) nearly identical to SP|P11139 Tubulin alpha-1 chain

At1g17745

D-3-phosphoglycerate dehydrogenase/3-PGDH identical to SP|O04130

At1g74970

Ribosomal protein S9 (RPS9) identical to ribosomal protein S9 [Arabidopsis thaliana] GI:5456946

At3g51840

Short-chain acyl-CoA oxidase identical to Short-chain acyl CoA oxidase

At5g49910

Heat shock protein 70/HSP70 (HSC70-7) identical to heat shock protein 70

At5g42300

Ubiquitin family protein contains INTERPRO:IPR000626 ubiquitin domain

At5g46430

60S ribosomal protein L32 (RPL32B)

At2g17190

Ubiquitin family protein contains INTERPRO:IPR000626 ubiquitin domain

At3g18780

Actin 2 (ACT2) identical to SP|Q96292 Actin 2; nearly identical to SP|Q96293 Actin 8

At5g62690

Tubulin beta-2/beta-3 chain (TUB2) nearly identical to SP|P29512 Tubulin beta-2/beta-3 chain

At1g75780

Spot and immobilization positive control

https://static-content.springer.com/image/art%3A10.1007%2Fs11033-011-1154-y/MediaObjects/11033_2011_1154_Fig1_HTML.gif
Fig. 1

Scatter plots for hybridization signals by 70-mer oligonucleotide microarray analyses

Table 2

Number and classification of differentially expressed genes

Sample

Parents

Checked genes

Differentially expressed genes

DG

DGU

DGD

NAG

Larix82 × 13-6

Larix 82(m)

3,827

540

288

252

133

Larix 13(p)

2,792

775

563

212

Larix13 × 82-21

Larix 13(m)

3,041

559

396

163

54

Larix 82(p)

4,506

448

193

255

p male parent, m female parent, DG differentially expressed genes, DGU up-regulated differentially expressed genes, DGD down-regulated differentially expressed genes, NAG nonadditive expression genes

https://static-content.springer.com/image/art%3A10.1007%2Fs11033-011-1154-y/MediaObjects/11033_2011_1154_Fig2_HTML.gif
Fig. 2

The models and functional categories of differential expression genes in each pairwise comparison. a Clustering analyses of all gene models based on expression data. Each horizontal line refers to a gene. The color represents the logarithmic intensity of the expressed genes. b Biological functions of genes demonstrating differential expression in all four pairwise comparisons. 13, 82, 6, 21 represent Larix13, Larix82, Larix82 × 13-6 and Larix13 × 82-21, respectively. U and D indicate the up-regulated and down-regulated genes, respectively

Differentially expressed genes detected in the reciprocal hybrids compared to the parental lines

Among all these detected genes, those that showed differential expression in each pairwise comparison were considered to be of greatest interest. The expression levels of 775 genes (27.76% of the identified transcripts) were altered by greater than twofold in the heterotic hybrid Larix82 × 13-6 compared to Larix13, of which 563 of these genes were up-regulated while 212 were down-regulated. In the comparison of the heterotic Larix82 × 13-6 with another parent Larix82, 540 genes (14.11% of the identified transcripts) showed greater than twofold changes in expression, of which 288 genes were up-regulated and 252 genes were down-regulated (Table 2). However, compared to these same parental lines (Larix13 and Larix82), only 559 (18.38% of the identified transcripts) and 448 (9.94% of the identified transcripts) genes were differentially expressed in the non-heterotic hybrid Larix13 × 82-21, respectively. The characteristics of the differentially expressed genes could be found in Table 2. To further understand the differentially expressed genes, all these genes were classified according to function and relatedness. The results suggested that the differentially expressed genes were mostly enriched in physiological processes, metabolism, catalytic activity and response to external stimuli (Fig. 2b). This implied that these functional categories were associated with the phenotypic differences between the heterotic hybrid Larix82 × 13-6 and the non-heterotic hybrid Larix13 × 82-21, even though they shared the same parent lines.

The heterotic hybrid showed more differentially expressed genes compared to the parents

The results of the pairwise comparisons indicated that more genes showed changes in their expression levels in the heterotic hybrid Larix82 × 13-6 compared to the parental lines Larix82 or Larix13. However, this was not the case in the non-heterotic hybrid Larix13 × 82-21 (Table 2). The initial data suggested that the differential expression of genes may play an important role in the formation of heterosis in the heterotic hybrid Larix82 × 13-6. Accordingly, it may be expected that the heterosis-related genes would be more enriched in the subset of genes showing differential expression compared with both parents. As a result the common differentially expressed genes in the reciprocal hybrids were selected and analyzed further. Briefly, 144 common differentially expressed genes were identified in the heterotic hybrid Larix82 × 13-6, while in the non-heterotic Larix13 × 82-21 the corresponding genes were only 68 (Fig. 3a). These differentially expressed genes could be further grouped into different expression classes. Expression level that was significantly higher in the hybrid than in both parental lines was designed as “above both parents, ABP”. Similarly, if the expression level in the hybrid was significantly lower than that in both parents, it was classified as “below both parents, BBP”. Furthermore, if the expression level in the hybrid was higher than male parent but lower than female parent, this pattern was designated in the category “above paternal, below maternal, APBM”. Finally, genes that displayed an expression pattern that was higher than female parent but lower than male parent were classified as “above maternal, below paternal, AMBP”. For the heterotic hybrid Larix82 × 13-6, most of the common differentially expressed genes fell into classes “ABP” (101 genes) and “BBP” (32 genes), while the same classifications in the non-heterotic Larix 13 × 82-21 contained 48 genes and six genes, respectively. Only four genes were classified as “AMBP” and seven genes were classified as “APBM” in the heterotic hybrid Larix82 × 13-6. In the non-heterotic Larix13 × 82-21, 14 genes were classified as “AMBP”, while no genes were designated “APBM” (Fig. 3b).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-011-1154-y/MediaObjects/11033_2011_1154_Fig3_HTML.gif
Fig. 3

The analyses of differentially expressed genes and nonadditive expression genes in the heterotic and non-heterotic hybrids. a The common differentially expressed genes between the heterotic or non-heterotic hybrids and their parental lines. b Gene expression patterns in the heterotic or non-heterotic hybrids compared to both of the parents. ABP indicates above both parents, BBP indicates below both parents, APBM denotes above male parent, below female parent, AMBP denotes above female parent, below male parent, respectively. 0.5 and 2.0 represent the ratios of the hybrids in comparison with each of their parents. c Biological functions of genes demonstrating nonadditive expression in the heterotic and non-heterotic hybrids. 13, 82, 6, 21 represent Larix13, Larix82, Larix82 × 13-6 and Larix13 × 82-21, respectively. U and D indicate the up-regulated and down-regulated genes, respectively

Nonadditive expression genes were more prevalent in the heterotic rather than the non-heterotic hybrid

Additive gene expression in hybrids exhibits a cumulative mode, contributed by each allele from the respective parents, which is attributable mostly to cis-regulation, while nonadditive gene expression deviates from the mid-parent level, which leads to activation (>2), repression (<2), dominance or over-dominance [28]. In nonadditive genes, other regulators probably contribute to the altered expression of the corresponding alleles in the hybrid, which is attributable mostly to trans-regulation. The dramatic changes in nonadditive gene regulation can be induced by interspecific or intraspecific hybridization [14, 20]. Here, we were mostly interested in the nonadditive genes with expression levels that fell outside of the parental values (>2 or <2). The microarray hybridization data indicated that these genes numbered 133 in the heterotic Larix82 × 13-6, while in the non-heterotic Larix13 × 82-21 there were only 54 (Table 2). To further understand these genes, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were performed at Molecular Annotation System (http://bioinfo.capitalbio.com/mas). The results of the GO analysis indicated that the nonadditive genes in the heterotic hybrid Larix82 × 13-6 were mostly enriched in physiological processes, cellular processes, catalytic activity, metabolism and responses to stimuli (Fig. 3c). KEGG analyses showed that the nonadditive genes participated in several important pathways, including phenylpropanoid biosynthesis, fructose and mannose metabolism, and starch and sucrose metabolism (Table 3).
Table 3

Some important pathways of the nonadditive expression genes

Pathway name

Number

P-value

Q-value

Pyruvate metabolism

1

0.011668

0.014701

Phenylalanine metabolism

1

0.014701

0.014701

Phenylpropanoid biosynthesis

5

0.009266

0.004308

Calcium signaling pathway

1

0.031279

0.040205

Fructose and mannose metabolism

1

0.072394

0.040205

Starch and sucrose metabolism

1

0.107933

0.043173

Flavonoid biosynthesis

1

0.010771

0.004308

Retinol metabolism

1

0.023801

0.002209

Methane metabolism

4

0.024853

0.002209

Bile acid biosynthesis

1

0.041283

0.003176

One carbon pool by folate

1

0.049907

0.003697

Circadian rhythm

1

0.011664

0.083316

Differential expression of genes were validated by qRT-PCR

Microarray analyses provided a large set of candidate genes that were differentially expressed in the reciprocal hybrids compared to their parental lines. However, the fold changes obtained via microarray hybridizations may not always correspond to actual changes in gene expression. Therefore, qRT-PCR was conducted to validate the differential gene expression profiles identified by the microarray data. In total, 16 genes, that were up- or down-regulated nonadditive expression in the heterotic hybrid Larix82 × 13-6 or the non-heterotic hybrid Larix13 × 82-21, were subjected to qRT-PCR analyses (Table 4). The annotated information suggested that most of these genes played important roles in growth and development. The qRT-PCR results demonstrated a good correlation between the magnitudes of the gene expression measured by microarray and qRT-PCR (Pearson linear correlation coefficient value r = 0.759, P < 0.01) (Fig. 4a, b), although some inconsistent events were apparent. In brief, a total of 64 pairwise comparisons of the 16 selected genes in the four plant materials used in the microarrays were performed, of which only nine pairwise comparison data detected by qRT-PCR was not in agreement with the corresponding microarray data. Thus, 85.94% of the pairwise comparison results were consistent in both methods. This further suggested that the data obtained by the microarrays was reliable, and can be used accurately for generating differential gene expression profiles between the reciprocal hybrids (Larix82 × 13-6 and Larix13 × 82-21) and their parents (Larix82 and Larix13).
Table 4

Summary of genes analyzed by qRT-PCR

Oligo ID

Pattern

qRT-PCR primers

Length(bp)

Biological function

Larix13 × 82-21

At1g65060

BBP

F-GCCGCTCTTCCACATCTATTC

200

4CL3 (4-coumarate: CoA ligase 3)

R-TTCGGACGGACGAGACATC

At1g78870

ABP

F-TCAAGACTAAGGTTTACCATCCAA

135

UBC35; ubiquitin-protein ligase

R-TAGGGTCAGTCAGAAGAGAGCAA

At2g47520

ABP

F-CAGCATCGTGGTGTTACATTCTA

204

AP2 domain-containing transcription factor

R-CATCTGTTTCACATCCTCCTCATA

Larix82 × 13-6

At2g29570

ABP

F-TGTCGCCTCCATCGTCG

157

PCNA2

R-GCCCTCGTTTCCCTGTTG

At4g14960

ABP

F-GCCAATGTTCAGAGAGCCGT

204

TUA6 (tubulin alpha-6 chiain)

R-GCCAACCTCCTCATAATCCTT

At4g18720

ABP

F-CGTGAGATTCTTTATGAGGCTTT

150

Transcription elongation factor-related

R-TTGGGCTCCATTAGACCGC

At4g34350

ABP

F-AATCCCACCGTGAATGAGAGA

208

CLB6

R-TGTTCCAAACCTTAGACACCCA

At2g28190

ABP

F-TGCGGGTTCTGATGGAGTT

206

CSD2; copper, zinc superoxide dismutase

R-AGCCGTAAATGGGAGTGAGTC

At4g34050

BBP

F-GCCGACAAAGACAACTATCTAAAC

166

Caffeoyl-CoA 3-O-methyltransferase, putative

R-GCTCCATCACGAAGTCTCTGTA

At5g22440

ABP

F-GAGTGATGTGTTGAGGGAGGC

166

60S ribosomal protein L10A (RPL10aC)

R-GGGGCGAGGAATGTGAGG

At1g73620

BBP

F-GAAGAATGAGCAAGGCTGTGT

227

Thaumatin-like protein/pathogenesis-related protein

R-GAGGGCAAAAGGTGATGAGATA

At2g47520

ABP

F-CAGCATCGTGGTGTTACATTCTA

204

AP2 domain-containing transcription factor

R-CATCTGTTTCACATCCTCCTCATA

At1g22020

ABP

F-TGGTGCCGTGCTCTTATGTG

133

SHM6; glycine hydroxymethyltransferase

R-CCCTGCTCTTGGACCTCTCA

At3g10950

ABP

F-CTGTGAATGCTGTGGCAAGTT

166

60S ribosomal protein L37a (RPL37aB)

R-TTATCCATCTGTCTGCTCCCTC

At5g60790

ABP

F-CAGCACAGATGGGAACAAGAG

188

ATGCN1

R-CCAACACCAGTGAAACGGAAC

At2g40890

BBP

F-ACTTGTTAGCCATCCCCGA

173

Cytochrome P450

R-CTTTATGTGGAAGCATCAGAGG

ABP above both parents, BBP below both parents

https://static-content.springer.com/image/art%3A10.1007%2Fs11033-011-1154-y/MediaObjects/11033_2011_1154_Fig4_HTML.gif
Fig. 4

Gene expression value comparison of qRT-PCR results with those from microarray analyses. a qRT-PCR confirmation of genes displaying up- and down-regulated nonadditive expression patterns in the heterotic and non-heterotic hybrids. b Correlation analyses of expression ratios measured by qRT-PCR and microarray in the selected 16 genes, with 64 different pairwise comparisons

Discussion

The precise molecular basis of heterosis remains enigmatic despite its wide exploitation in agriculture and forestry [11]. The dominance and over-dominance hypotheses were proposed to explain heterosis before the molecular concepts of genetics were formulated. Following that QTL mapping methods were conducted widely to explore the nature of heterosis in different plant species [810, 29]. However, undoubtedly, more investigations from not only the view of genetics but also genome, transcriptome, molecular biology or epigenetics were still required to explore this phenomenon. Recently, investigations concentrated on gene expression profiles between the hybrids and their parental lines in some plants have offered new insights into the molecular understanding of heterosis [1215]. Most of these reports revealed that differential gene expression, especially nonadditive gene expression, was prevalent in the hybrids and these genes might be associated with the heterosis phenotype [14, 20].

In this present study, we attempted to identify the molecular mechanisms of heterosis in Larix from genome-wide quantitative assessments of gene expression. The results indicated that the gene expression profile in the heterotic hybrid Larix82 × 13-6 was significantly different from that in the non-heterotic hybrid Larix13 × 82-21, despite these hybrids sharing the same parental lines. More differentially expressed genes were detected in the heterotic hybrid Larix82 × 13-6 (Table 2; Fig. 3a). Moreover, the data indicated that among these differentially expressed genes, the genes whose expression patterns belonged to “above both parents” and “below both parents” patterns (which both are regarded as nonadditive gene expression) accounted for 20.9% and 10.9% of differentially expressed genes in the heterotic and non-heterotic hybrid, respectively. A similar result was observed in a study of maize heterosis where nonadditive genes were found to be 2.2% of the total genes and 22% of differentially expressed genes [17]. Most investigations revealed that nonadditive gene expression was considered as mid-parent heterosis or heterotic expression [30, 31]. This implied that these differentially expressed genes, especially nonadditive genes, may also play a crucial role in driving the formation of heterosis in the intraspecific Larix hybridization. Moreover, it must be noticed that in the present microarray analysis the expressions of parental alleles in the F1 hybrid could not be accurately distinguished, and only two extreme types of nonadditive expressed genes such as “above both parents” and “below both parents” were counted and emphasized. Consequently, the nonadditive expressed genes in the hybrids may be underestimated.

It is well known that heterosis simply represents the manifestation of a phenotype in hybrids that is different from the expectation of the parental average value and resulting from the complex actions of many components of which the regulation of gene expression (including the regulation of the timing, magnitude and location of gene expression) is one of the most important factors [32]. Various investigations have identified that regulatory sequence/element and transcription factors are one of the causes of gene expression changes, and the effect of these regulatory genes have been proposed to affect the phenotypes of the hybrids [14, 33]. Based on the microarray analyses, many transcription factors exhibited differential expression in the heterotic hybrid Larix82 × 13-6, including the AP2/EREBP, zinc finger and WRKY families, which play critical roles in diverse developmental and physiological processes [3436]. It can be speculated that these transcription factors participate in the regulation of key genes that cause heterosis in the intraspecific Larix. In addition to these transcription factors, some epigenetic regulation-related genes were also identified in the differentially expressed genes in the heterotic hybrid Larix82 × 13-6. Among these genes the differential expression of several DNA methyltransferase genes, such as DRM1, MET2 and MET1, were of great interest, since recent reports have indicated that the modification of DNA methylation may be another important regulatory factor in the formation of heterosis [14, 37, 38]. Investigations from rice, sorghum and maize revealed that the levels and patterns of DNA methylation in hybrids were significantly different compared with their parental lines [3840]. Our previous study also indicated that the heterotic hybrid Larix82 × 13-6 had lower DNA methylation levels compared to the mid-parent value and demethylation events were predominant, while this was not the case in the non-heterotic hybrid Larix13 × 82-21 (data not shown). This suggested that, similar to the heterosis of rice, sorghum and maize, modifications in DNA methylation may also be important for the formation of heterosis in the intraspecific Larix, which could provide a new insight into understanding heterosis in gymnosperms.

Microarray-based expression data allowed for the exploration of differential gene expression profiles between the reciprocal hybrids and their parents. Further, GO and KEGG analyses indicated that the differentially expressed genes, especially nonadditive genes, were often involved in important pathways associated with growth and development, metabolism and cell physiology (Figs. 2b, 3c; Table 3). For example, more genes involved in carbohydrate metabolism, starch synthesis and calcium-mediated signaling pathway were identified up-regulated expression in the heterotic hybrid than in the parental lines, while in the non-heterotic hybrid these genes accounted for a smaller fraction of the differentially expressed genes. The data also showed that the genes participating in circadian rhythm pathway were up-regulated in the heterotic hybrid. An important discovery was that hybrids of Arabidopsis can gain advantage from the control of circadian-mediated physiological and metabolic pathways, leading to growth vigor and increased biomass [41]. Thus, it implied that the circadian rhythm pathway in Larix may play a similar role in the formation of heterosis as in Arabidopsis hybrids.

In summary, to understand the molecular mechanisms underlying the hybrid vigor of heterosis in Larix, the present study elucidated the differential gene expression profiles of reciprocal hybrids and their parents, and a suite of effect genes and regulatory genes were identified that exhibited differential expression. Several important metabolic pathways that may play crucial roles in the formation of heterosis were also identified. Therefore, although the molecular basis of heterosis in Larix may be more complex than some herbaceous plants which have been widely studied, due to their large genome, complex genetic backgrounds and long growth cycles, this present study could provide a series of significant new insights to further explore and understand the formation of heterosis in the Larix at the genetic and epigenetic levels.

Acknowledgments

Firstly, we thank the anonymous reviewers for critical reading of the manuscript. We are also grateful to Dr. Liwang Qi, Chinese Academy of Forestry, Beijing, China, for kindly providing the materials of Larix kaempferi (Lamb.). This work was carried out with the financial support from The National Key Basic Research Program (N0. 2009CB119105) and The National Natural Science Foundation of China and Tianjin (N0. 10JCZDJC17900 and N0. 07JCYBJC11700).

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© Springer Science+Business Media B.V. 2011