Gene expression profiles of two intraspecific Larix lines and their reciprocal hybrids
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- Li, A., Fang, M., Song, W. et al. Mol Biol Rep (2012) 39: 3773. doi:10.1007/s11033-011-1154-y
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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.
KeywordsLarix kaempferi (Lamb.)HeterosisNonadditive expressionTranscript profile
As a driving force for speciation and evolution, hybridization events have played their part in the formation of many plant species . 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 . 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 . 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 [4–7]. Each of these hypotheses can be supported by results from quantitative trait locus (QTL) mapping studies [8–10]. However, no strong consensus has emerged concerning which of these hypotheses best explains heterosis, especially at the molecular level .
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 [12–14]. 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 [17–19]. However, most of these investigations have concerned model plants such as Arabidopsis or important crops, including rice, maize and Brassica campestris [15–17, 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 , 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
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.
Total RNA was isolated from young fresh leaves using the CTAB method with some modifications , 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 . 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].
Transcriptome profiles of the reciprocal hybrids and their parental lines
Microarray positive control of Arabidopsis thaliana housekeeping genes
Tubulin alpha-1 chain (TUA1) nearly identical to SP|P11139 Tubulin alpha-1 chain
D-3-phosphoglycerate dehydrogenase/3-PGDH identical to SP|O04130
Ribosomal protein S9 (RPS9) identical to ribosomal protein S9 [Arabidopsis thaliana] GI:5456946
Short-chain acyl-CoA oxidase identical to Short-chain acyl CoA oxidase
Heat shock protein 70/HSP70 (HSC70-7) identical to heat shock protein 70
Ubiquitin family protein contains INTERPRO:IPR000626 ubiquitin domain
60S ribosomal protein L32 (RPL32B)
Ubiquitin family protein contains INTERPRO:IPR000626 ubiquitin domain
Actin 2 (ACT2) identical to SP|Q96292 Actin 2; nearly identical to SP|Q96293 Actin 8
Tubulin beta-2/beta-3 chain (TUB2) nearly identical to SP|P29512 Tubulin beta-2/beta-3 chain
Spot and immobilization positive control
Number and classification of differentially expressed genes
Differentially expressed genes
Larix82 × 13-6
Larix13 × 82-21
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
Nonadditive expression genes were more prevalent in the heterotic rather than the non-heterotic hybrid
Some important pathways of the nonadditive expression genes
Calcium signaling pathway
Fructose and mannose metabolism
Starch and sucrose metabolism
Bile acid biosynthesis
One carbon pool by folate
Differential expression of genes were validated by qRT-PCR
Summary of genes analyzed by qRT-PCR
Larix13 × 82-21
4CL3 (4-coumarate: CoA ligase 3)
UBC35; ubiquitin-protein ligase
AP2 domain-containing transcription factor
Larix82 × 13-6
TUA6 (tubulin alpha-6 chiain)
Transcription elongation factor-related
CSD2; copper, zinc superoxide dismutase
Caffeoyl-CoA 3-O-methyltransferase, putative
60S ribosomal protein L10A (RPL10aC)
Thaumatin-like protein/pathogenesis-related protein
AP2 domain-containing transcription factor
SHM6; glycine hydroxymethyltransferase
60S ribosomal protein L37a (RPL37aB)
The precise molecular basis of heterosis remains enigmatic despite its wide exploitation in agriculture and forestry . 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 [8–10, 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 [12–15]. 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 . 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 . 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 [34–36]. 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 [38–40]. 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 . 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.
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).