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Transcriptome analysis of wheat seedling and spike tissues in the hybrid Jingmai 8 uncovered genes involved in heterosis
杂交种景迈 8 号小麦幼苗和穗组织转录组分析揭示了与优势有关的基因

Yong-jie Liu 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Shi-qing Gao 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Yi-miao Tang 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Jie Gong 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Xiao Zhang 3 ⋅ 3 ⋅ ^(3)*{ }^{3} \cdot Yong-bo Wang 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Li-ping Zhang 1 ⋅ 1 ⋅ ^(1)*^{1} \cdot Ren-wei Sun 1 , 2 1 , 2 ^(1,2){ }^{1,2}. Quan Zhang 4 ⋅ 4 ⋅ ^(4)*{ }^{4} \cdot Zhao-bo Chen 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Xiang Wang 5 ⋅ 5 ⋅ ^(5)*{ }^{5} \cdot Cai-juan Guo 6 ⋅ 6 ⋅ ^(6)*{ }^{6} \cdot Sheng-quan Zhang 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Feng-ting Zhang 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Jian-gang Gao 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Hui Sun 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Wei-bing Yang 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Wei-wei Wang 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot Chang-ping Zhao 1 1 ^(1){ }^{1}
永杰， 刘 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 世庆， 高 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 义淼， 唐 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 杰， 龚 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 晓， 张 3 ⋅ 3 ⋅ ^(3)*{ }^{3} \cdot 永波， 王 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 立平， 张 1 ⋅ 1 ⋅ ^(1)*^{1} \cdot 任伟， 孙 1 , 2 1 , 2 ^(1,2){ }^{1,2} 仁伟.全 张 4 ⋅ 4 ⋅ ^(4)*{ }^{4} \cdot 昭波 陈 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 翔 王 5 ⋅ 5 ⋅ ^(5)*{ }^{5} \cdot 彩娟 郭 6 ⋅ 6 ⋅ ^(6)*{ }^{6} \cdot 胜权 张 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 凤婷 张 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 建刚 高 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 辉 孙 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 伟兵 杨 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 伟伟 王 1 ⋅ 1 ⋅ ^(1)*{ }^{1} \cdot 长平 赵 1 1 ^(1){ }^{1}

Received: 29 September 2017 / Accepted: 12 January 2018 / Published online: 5 March 2018
收稿日期： 2017-09-29 / 录用日期： 2018-01-12 / 在线出版日期：2018-03-05
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
© 德国 Springer-Verlag GmbH，2018 年 Springer Nature 的一部分

Abstract  抽象

Main conclusion Transcriptome analysis was carried out for wheat seedlings and spikes from hybrid Jingmai 8 and both inbred lines to unravel mechanisms underlying heterosis.
主要结论 对杂交种景脉 8 号和两个自交系的小麦幼苗和穗状花序进行转录组分析，以揭示优势的机制。

Heterosis, known as one of the most successful strategies for increasing crop yield, has been widely exploited in plant breeding systems. Despite its great importance, the molecular mechanism underlying heterosis remains elusive. In the present study, RNA sequencing (RNA-seq) was performed on the seedling and spike tissues of the wheat (Triticum aestivum) hybrid Jingmai 8 (JM8) and its homozygous parents to unravel the underlying mechanisms of wheat heterosis. In total, 1686 and 2334 genes were identified as differentially expressed genes (DEGs) between the hybrid and the two inbred lines in seedling and spike tissues, respectively. Gene Ontology analysis revealed that DEGs from seedling tissues were significantly enriched in processes involved in photosynthesis and carbon fixation, and the majority of these DEGs expressed at a higher level in JM8 compared to both inbred lines. In addition, cell wall biogenesis and protein biosynthesis-related pathways were also significantly represented. These results confirmed that a combination of different pathways could contribute to heterosis. The DEGs between the hybrid and the two inbred progenitors from the spike tissues were significantly enriched in biological processes related to transcription, RNA biosynthesis and molecular function categories related to transcription factor activities. Furthermore, transcription factors such as NAC, ERF, and TIF-IIA were highly expressed in the hybrid JM8. These results may provide valuable insights into the molecular mechanisms underlying wheat heterosis.
优势被认为是提高作物产量的最成功策略之一，已在植物育种系统中得到广泛利用。尽管优势非常重要，但优势的分子机制仍然难以捉摸。本研究对小麦 （Triticum aestivum） 杂交种精麦 8 号 （JM8） 及其纯合亲本的幼苗和刺突组织进行 RNA 测序 （RNA-seq），以揭示小麦优势的潜在机制。总共有 1686 个和 2334 个基因被鉴定为杂交种和两个自交系之间的差异表达基因 （DEGs），分别在幼苗和刺突组织中。基因本体分析显示，来自幼苗组织的 DEGs 在光合作用和固碳过程中显著富集，与两个自交系相比，这些 DEGs 中的大多数在 JM8 中表达水平更高。此外，细胞壁生物发生和蛋白质生物合成相关途径也显著存在。这些结果证实了不同途径的组合可能导致优势。杂交种和来自刺突组织的两个自交祖细胞之间的 DEGs 在与转录、RNA 生物合成和转录因子活性相关的分子功能类别相关的生物过程中显著富集。此外，NAC、ERF 和 TIF-IIA 等转录因子在杂交 JM8 中高度表达。这些结果可能为小麦优势的分子机制提供有价值的见解。

Keywords Additivity • Breeding • Carbohydrate metabolism • Dominance gene expression • Photosynthesis • RNA biosynthesis ⋅ ⋅ *\cdot Triticum aestivum
关键词 加性 • 育种 • 碳水化合物代谢 • 显性基因表达 • 光合作用 • RNA 生物合成 ⋅ ⋅ *\cdot 小麦
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00425-018-2848-3) contains supplementary material, which is available to authorized users.
电子补充材料 本文 （https://doi.org/10.1007/s00425-018-2848-3） 的在线版本包含补充材料，可供授权用户使用。

Abbreviations  缩写

BAE Biallelic expression  双等位基因表达
DEGs  DEG Differentially expressed genes
差异表达基因
ELD Expression-level dominance
表达式级控制
GO Gene Ontology  基因本体论
JM8 Jingmai 8  景迈 8
BAE Biallelic expression DEGs Differentially expressed genes ELD Expression-level dominance GO Gene Ontology JM8 Jingmai 8| BAE | Biallelic expression | | :--- | :--- | | DEGs | Differentially expressed genes | | ELD | Expression-level dominance | | GO | Gene Ontology | | JM8 | Jingmai 8 |
MAE Monoallelic expression
MAE 单等位基因表达
MPV Mid-parental value  MPV 中父值
NAGs Non-additive genes  NAGs 非加性基因
PAE Preferallelic expression
PAE Prefer 等位基因表达
TGMS Thermosensitive genic male sterile
TGMS 热敏基因男性无菌
TY806 Taiyuan 806  TY806 太原 806

Introduction  介绍

Heterosis, or hybrid vigor, refers to the phenomenon in which progeny of two inbred varieties exhibits enhanced agronomic performance such as biomass production, growth rate, and fertility relative to both inbred parents (Shull 1908). Heterosis was first described by Darwin (1876) and later rediscovered independently by Shull (1908) and East (1908). It has been widely exploited in commercial breeding systems and has become one of the most important strategies for increasing productivity in maize (Moll et al. 1965), rice (Maurya and Singh 1978) and livestock (Gao et al. 2013). Despite its successful employment for yield improvement in crop breeding systems, the detailed mechanism behind heterosis remains elusive. Multiple models have been proposed to explain heterosis, and the three most classical and popular quantitative genetic explanations are dominance (Davenport 1908), over-dominance (East 1908; Shull 1908), and epistasis (Li et al. 2001; Thiemann et al. 2009). The “dominance” model posits that deleterious alleles at different loci in the two homozygous parental genomes are complemented in the heterozygous F 1 F 1 F_(1)\mathrm{F}_{1} hybrid, resulting in a superior phenotype. The “over-dominance” model suggests that improved performance of the F 1 F 1 F_(1)\mathrm{F}_{1} hybrid relative to its inbred parents is a consequence of favorable allelic interactions at heterozygous loci that outperform either homozygous state. Both the dominance and over-dominance hypotheses are based on single locus theory. However, the interaction of different loci may also contributes to heterosis, one example being the effect of epistasis (Birchler et al. 2003). “Epistasis” is classically defined as the interactions of superior alleles at different loci from two parents, and the effects may show additivity, dominance, or over-dominance (Li et al. 2001).
优势或杂交活力是指两个自交品种的后代相对于两个近交亲本表现出增强的农艺性能的现象，例如生物量生产、生长速率和生育能力（Shull 1908）。优势首先由 Darwin （1876） 描述，后来由 Shull （1908） 和 East （1908） 独立重新发现。它已在商业育种系统中得到广泛利用，并已成为提高玉米（Moll 等人，1965 年）、水稻（Maurya 和 Singh 1978 年）和畜牧业（Gao 等人，2013 年）生产力的最重要策略之一。尽管它在作物育种系统中成功地用于提高产量，但优势背后的详细机制仍然难以捉摸。已经提出了多种模型来解释优势，三种最经典和流行的定量遗传解释是显性 （Davenport 1908）、过度显性 （East 1908;Shull 1908） 和上位性 （Li et al. 2001;Thiemann 等人，2009 年）。“显性”模型假设两个纯合亲本基因组中不同位点的有害等位基因在杂合 F 1 F 1 F_(1)\mathrm{F}_{1} 杂交中互补，从而产生优越的表型。“过度显性”模型表明， F 1 F 1 F_(1)\mathrm{F}_{1} 杂交种相对于其近交亲本的性能改善是杂合位点上有利的等位基因相互作用的结果，其表现优于任一纯合状态。支配和过度支配假说都基于单位点理论。然而，不同基因座的相互作用也可能有助于优势，一个例子是上位性的影响（Birchler 等人，2003 年）。 “上位性”经典地定义为来自两个亲本的不同位点的优越等位基因的相互作用，其效应可能显示加性、显性或过度显性（Li et al. 2001）。
Numerous studies have been implemented to study the genetic and molecular determinants for heterosis. Many quantitative trait loci (QTL) have been shown to putatively underlie heterotic characteristics like yield (Guo et al. 2014; Li et al. 2016; Zhu et al. 2016) and plant height (Li et al. 2015, 2017; Shang et al. 2016), but few of them have been cloned for confirmation of their involvement. Recently, various pioneer studies have described differences in hybrid genome organization and gene expression from their parental inbred lines and correlated these differences with heterosis manifestation (Song et al. 2013; Zhai et al. 2013; Li et al. 2014, 2016; Gu et al. 2016). Throughout the entire
已经实施了大量研究来研究优势的遗传和分子决定因素。许多数量性状位点 （QTL） 已被证明是产量等特性的基础（Guo 等人，2014 年;Li 等人，2016 年;Zhu et al. 2016） 和株高 （Li et al. 2015， 2017;Shang et al. 2016），但其中很少有被克隆以确认他们的参与。最近，各种先驱研究描述了杂交基因组组织和基因表达与其亲本自交系的差异，并将这些差异与优势表现相关联（Song 等人，2013 年;Zhai 等人，2013 年;Li et al. 2014， 2016;Gu 等人，2016 年）。贯穿整个
life cycle of higher plants, all metabolic activities under plant growth and development are determined by complex, tightly, and collaboratively regulated global gene expression networks (Long et al. 2008). Hence, the superior vigor of highly heterozygous hybrid plants compared to their homozygous parental inbred lines is assumed to be a result of global differences in gene expression between inbred lines and hybrids. For example, correlations between the number of differentially expressed genes and the degree of heterosisassociated traits have been suggested (Li et al. 2009; Riddle et al. 2010). The differential expression in genes involved in CO 2 CO 2 CO_(2)\mathrm{CO}_{2} assimilation (Wang et al. 2002; Bao et al. 2005) and energy metabolism (Wei et al. 2009) could be related to the improved hybrid rice production. It has also been reported that allelic variation effects on gene expression may have an impact on hybrid vigor in maize (Guo et al. 2004; Springer and Stupar 2007) and rice (Song et al. 2007, 2013). In rice, the interplay between transcription factors and the polymorphic promoter cis-regulatory elements in hybrids has been suggested as a plausible molecular mechanism underlying heterotic gene expression (Zhang et al. 2008). As an important type of gene expression regulation, epigenetic modification and small RNA-directed gene regulation have also been shown to be related to heterosis (Ha et al. 2009; He et al. 2010; Li et al. 2014; Kawanabe et al. 2016).
高等植物的生命周期，植物生长和发育下的所有代谢活动都由复杂、紧密和协同调节的全球基因表达网络决定（Long 等人，2008 年）。因此，与其纯合亲本自交系相比，高度杂合杂交植物的活力优越，被认为是自交系和杂交种之间基因表达全球差异的结果。例如，已经提出了差异表达基因的数量与异种相关性状程度之间的相关性（Li 等人，2009 年;Riddle 等人，2010 年）。参与 CO 2 CO 2 CO_(2)\mathrm{CO}_{2} 同化的基因的差异表达（Wang 等人，2002 年;Bao et al. 2005） 和能量代谢 （Wei et al. 2009） 可能与杂交水稻产量的提高有关。据报道，等位基因变异对基因表达的影响可能对玉米的杂交活力产生影响（Guo 等人，2004 年;Springer 和 Stupar 2007 年）和水稻（Song 等人，2007 年，2013 年）。在水稻中，杂交种中转录因子和多态性启动子顺式调节元件之间的相互作用已被证明是异种基因表达的合理分子机制（Zhang 等人，2008 年）。作为一种重要的基因表达调控类型，表观遗传修饰和小 RNA 导向的基因调控也被证明与优势有关（Ha 等人，2009 年;He et al. 2010;Li 等人，2014 年;Kawanabe 等人，2016 年）。
In this study, RNA sequencing (RNA-seq) was performed to investigate the global transcriptomes of wheat spike and seedling tissues at the five-leaf stage from the hybrid Jingmai 8 (JM8) and its two parental inbred lines, BS366 (maternal, a thermosensitive genic male sterile line, TGMS line) (Tang et al. 2011) and Taiyuan 806 (TY806, paternal). Differentially expressed genes and expression patterns in the hybrid and both parents were analyzed. This genome-wide transcriptome comparison offers a foundation for analyzing the causative role of the altered gene expression in the hybrid’s superior performance and the molecular mechanism underlying heterosis for wheat spike and seedling.
在本研究中，进行了 RNA 测序 （RNA-seq） 以研究杂交精麦 8 （JM8） 及其两个亲本自交系 BS366 （母系，热敏基因雄性不育系，TGMS 系） （Tang et al. 2011） 和太原 806 （TY806，父系）的五叶期小麦穗和幼苗组织的全局转录组。分析杂交种和亲本中的差异表达基因和表达模式。这种全基因组转录组比较为分析改变的基因表达在杂交种卓越性能中的致病作用以及小麦穗和幼苗优势的分子机制提供了基础。

Materials and methods  材料和方法

Plant materials and phenotype evaluation of hybrid and parental lines
杂交系和亲本系的植物材料及表型评价

The wheat hybrid Jingmai 8 (JM8), maternal parent BS366 (a thermosensitive genic male sterile, TGMS line), and paternal parent Taiyuan806 (TY806) were used in this study. All wheat seeds were provided by Beijing Engineering Research Center for Hybrid Wheat and sown in the experimental farm at the Beijing Academy of Agriculture and Forestry Sciences in early October with regular watering and fertilizer application. For each genotype, three biological replicates were included for phenotype evaluation.
本研究采用小麦杂交种景迈 8 号 （JM8）、母系亲本 BS366 （一种热敏基因雄性不育，TGMS 系） 和父系太原 806 （TY806）。所有小麦种子均由北京市杂交小麦工程研究中心提供，并于 10 月初在北京农林科学院的试验场播种，并定期浇水和施肥。对于每种基因型，包括 3 个生物学重复用于表型评估。
For each replicate, five rows ( 1.5 − m 1.5 − m 1.5-m1.5-\mathrm{m}-long rows and spaced 0.2 m apart) with 30 variable seeds per line were planted. To access the heterotic performance of the hybrid wheat, phenotype evaluation was carried out at the five-leaf stage seedling. We manually collected and measured plant height and tiller numbers of 15 seedlings for each genotype with three biological replicates.
对于每个重复，种植 5 行 （ 1.5 − m 1.5 − m 1.5-m1.5-\mathrm{m} -长行，间隔 0.2 m），每行 30 颗可变种子。为了获得杂交小麦的优势性能，在五叶期幼苗进行了表型评估。我们手动收集并测量了每种基因型 15 株幼苗的株高和分蘖数，并进行了 3 次生物学重复。

Sample preparation, RNA isolation, and real-time qRT-PCR
样品制备、RNA 分离和实时 qRT-PCR

The spikes and seedlings of hybrid JM8 and both parental inbred lines at the five-leaf stage when the flag leaf had just emerged from the collar penultimate were included in the transcriptome analysis. The main stem spikes of BS366, TY806, and JM8 were harvested and used as spike samples. Every sample consisted of 15 plants pooled together for spike RNA extraction. Five entire seedling plants of BS366, TY806, and JM8 were harvested separately as the seedling samples. All samples were immediately frozen in liquid nitrogen and stored at − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} for RNA extraction. Total RNA from roots, leaves, stems, and spikes from the seedlings samples were extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The concentration and quality of total RNA were determined with a Nanodrop spectrophotometer and 1% agarose gel electrophoresis. The RNAs from different tissues for a specific seedling sample were mixed together equally for transcriptional analysis. For real-time qRT-PCR, cDNA was synthesized using the PrimeScript TM TM ^(TM){ }^{\mathrm{TM}} RT reagent Kit with gDNA Eraser (Takara). Differentially expressed genes were validated with a CFX96 Touch TM TM  ^("TM "){ }^{\text {TM }} Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green II (Takara). Expression levels of genes in samples were normalized using endogenous wheat 18 S gene with primer sequences 5 ′ 5 ′ 5^(')5^{\prime}-TGC TGGAATCGGAATAGTTGAG- 3 ′ 3 ′ 3^(')3^{\prime} and 5 ′ 5 ′ 5^(')5^{\prime}-ACTACGCAG GCTCATCAAACAG-3’; the relative expression levels were calculated using the 2 − Δ Δ C t 2 − Δ Δ C t 2^(-Delta DeltaC_(t))2^{-\Delta \Delta C_{\mathrm{t}}} method. Primer sequences were designed using Primer3 input version 4.0.0 (http://prime r3.ut.ee/) and listed in Supplementary Table S1.
将杂交 JM8 的穗状花序和幼苗以及旗叶刚从领倒数第二片中出来的五叶期亲本自交系纳入转录组分析。收获 BS366 、 TY806 和 JM8 的主茎穗，用作穗状样品。每个样品由 15 株植物混合在一起进行刺突 RNA 提取。分别收获 BS366 、 TY806 和 JM8 的 5 株整株幼苗作为幼苗样品。所有样品立即在液氮中冷冻并储存 − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} 用于 RNA 提取。使用 TRIzol 试剂（Invitrogen，Carlsbad，CA，USA）提取幼苗样品的根、叶、茎和刺突的总 RNA。使用 Nanodrop 分光光度计和 1% 琼脂糖凝胶电泳测定总 RNA 的浓度和质量。将特定幼苗样品中来自不同组织的 RNA 均匀混合在一起进行转录分析。对于实时 qRT-PCR，使用含 gDNA Eraser 的 PrimeScript TM TM ^(TM){ }^{\mathrm{TM}} RT 试剂盒 （Takara） 合成 cDNA。使用 SYBR Green II （Takara） 的 CFX96 Touch TM TM  ^("TM "){ }^{\text {TM }} 实时 PCR 检测系统 （Bio-Rad Laboratories， Hercules， CA， USA） 验证差异表达基因。使用内源小麦 18 S 基因和引物序列 5 ′ 5 ′ 5^(')5^{\prime} -TGC、TGGAATCGGAATAGTTGAG- 3 ′ 3 ′ 3^(')3^{\prime} 和 5 ′ 5 ′ 5^(')5^{\prime} -ACTACGCAG、GCTCATCAAACAG-3' 对样品中基因的表达水平进行归一化;使用该方法 2 − Δ Δ C t 2 − Δ Δ C t 2^(-Delta DeltaC_(t))2^{-\Delta \Delta C_{\mathrm{t}}} 计算相对表达水平。使用 Primer3 input 版本 4.0.0 （http://primer3.ut.ee/） 设计引物序列，并列于补充表 S1 中。

Illumina sequencing and data analysis
Illumina 测序和数据分析

All samples were sequenced using the Illumina HiSeq 2000 platform. Transcriptome sequence data for all samples can be found in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under accession number PRJNA398700. Raw reads were filtered to obtain high-quality reads by removing low-quality reads containing more than 30 % 30 % 30%30 \% bases with Q < 20 Q < 20 Q < 20Q<20. After trimming low-quality bases ( Q < 20 Q < 20 Q < 20Q<20 ) from the 5 ′ 5 ′ 5^(')5^{\prime} and 3 ′ 3 ′ 3^(')3^{\prime} ends of the remaining reads, the resulting high-quality clean reads in each sample were mapped to the wheat reference genome
所有样本均使用 Illumina HiSeq 2000 平台进行测序。所有样品的转录组序列数据都可以在国家生物技术信息中心 （NCBI） 序列读取档案 （SRA） 的 PRJNA398700 号下找到。通过删除包含多个 30 % 30 % 30%30 \% 碱基的低质量读数，对原始读数进行过滤，以获得高质量的读数 Q < 20 Q < 20 Q < 20Q<20 。从 5 ′ 5 ′ 5^(')5^{\prime} 剩余读数的 3 ′ 3 ′ 3^(')3^{\prime} 末端修剪低质量碱基 （ Q < 20 Q < 20 Q < 20Q<20 ） 后，将每个样品中产生的高质量干净读数映射到小麦参考基因组
(Triticum_aestivum.TGACv1) using HISAT (v2.0.6) (Kim et al. 2015). Only reads that can be mapped to only one location in the reference genome (unique hits) were kept for further analysis. Fragments per kilobase of exon model per million mapped reads (FPKM) were used to estimate transcript expression levels in all samples and genes with expression higher than 1 FPKM in at least one sample of seedling or spike tissues were used for further analysis. Differentially expressed genes (DEGs) in each comparison were identified by EdgeR (Robinson et al. 2009), using a threshold P P PP value < 0.05 < 0.05 < 0.05<0.05 and a fold change ≥ 2 ≥ 2 >= 2\geq 2. The identified DEGs were subjected to Gene Ontology (GO) enrichment analyses as previously described (Ashburner et al. 2000).
（Triticum_aestivum。TGACv1） 使用 HISAT （v2.0.6） （Kim et al. 2015）。仅保留只能映射到参考基因组中一个位置的 reads（唯一命中）以供进一步分析。使用每千碱基外显子模型每百万个映射读数 （FPKM） 的片段来估计所有样品中的转录表达水平，并使用至少一个幼苗或刺突组织样品中表达高于 1 FPKM 的基因进行进一步分析。EdgeR （Robinson et al. 2009） 使用阈值 P P PP < 0.05 < 0.05 < 0.05<0.05 和倍数变化 ≥ 2 ≥ 2 >= 2\geq 2 来识别每次比较中的差异表达基因 （DEG）。如前所述，对鉴定出的 DEGs 进行基因本体论 （GO） 富集分析（Ashburner et al. 2000）。

Cluster analysis  聚类分析

Cluster analysis was carried out for all transcripts in hybrid JM8 and both inbred lines from spike and seedling. The normalized log ⁡ 2 log ⁡ 2 log 2\log 2 transformed read counts in each sample were used for hierarchical clustering using the Cluster 3.0 software and visualized using Treeview (Eisen et al. 1998).
对杂交 JM8 中的所有转录本以及来自穗状和幼苗的自交系进行了聚类分析。每个样本中的标准化 log ⁡ 2 log ⁡ 2 log 2\log 2 转换读取计数用于使用 Cluster 3.0 软件进行分层聚类，并使用 Treeview 进行可视化（Eisen 等人，1998 年）。

Definition of expression patterns of DEGs
DEGs 表达模式的定义

The expression pattern of DEGs was determined by comparing their expression levels in hybrid JM8 with the average value of that in two inbred lines in the respective tissues. We defined gene expression in JM8 as E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} and gene expression of the two inbred line as E B S 366 E B S 366 E_(BS 366)\mathrm{E}_{B S 366} and E T Y 806 E T Y 806 E_(TY 806)\mathrm{E}_{T Y 806}. In addition, we defined the average value of both inbred lines as MPV (midparental value). Thus
通过比较 DEGs 在杂交 JM8 中的表达水平与各自组织中两个自交系中的表达水平的平均值来确定 DEGs 的表达模式。我们将 JM8 中的基因表达定义为 E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} ，两个自交系的基因表达定义为 E B S 366 E B S 366 E_(BS 366)\mathrm{E}_{B S 366} 和 E T Y 806 E T Y 806 E_(TY 806)\mathrm{E}_{T Y 806} 。此外，我们将两个自交系的平均值定义为 MPV （中亲值）。因此
MPV = 1 2 ( E B S 366 + E T Y 806 ) MPV = 1 2 E B S 366 + E T Y 806 MPV=(1)/(2)(E_(BS 366)+E_(TY 806))\mathrm{MPV}=\frac{1}{2}\left(\mathrm{E}_{B S 366}+\mathrm{E}_{T Y 806}\right).
The Student’s t t tt test method was conducted to test the difference between E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} and MPV. If the E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} was significantly ( P P PP value < 0.05 < 0.05 < 0.05<0.05 and fold change > 2 > 2 > 2>2 ) different from MPV, we then defined these genes as non-additive genes (NAGs); if there was no significant difference between E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} and MPV, these genes were defined as additive genes.
学生的 t t tt 测试方法是为了测试 MPV 之间的差异 E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} 。 E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} 如果 （ P P PP 值 < 0.05 < 0.05 < 0.05<0.05 和倍数变化 > 2 > 2 > 2>2 ） 与 MPV 显著不同，我们将这些基因定义为非加性基因 （NAG）;如果 和 MPV 之间 E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} 没有显着差异，则这些基因被定义为加性基因。
DEGs between hybrid JM8 and both inbred lines were defined as DGhp. Classification of DGhp was carried out according to E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} relative to E B S 366 E B S 366 E_(BS 366)\mathrm{E}_{B S 366} and E T Y 806 E T Y 806 E_(TY 806)\mathrm{E}_{T Y 806}. "="means statistically similar and " > > >> " and " < < << " means statistically higher or lower. If E J M 8 > E B S 366 = E T Y 806 E J M 8 > E B S 366 = E T Y 806 E_(JM8) > E_(BS 366)=E_(TY 806)\mathrm{E}_{J M 8}>\mathrm{E}_{B S 366}=\mathrm{E}_{T Y 806}, or E J M 8 > E B S 366 > E T Y 806 E J M 8 > E B S 366 > E T Y 806 E_(JM8) > E_(BS 366) > E_(TY 806)\mathrm{E}_{J M 8}>\mathrm{E}_{B S 366}>\mathrm{E}_{T Y 806}, or E J M 8 > E T Y 806 > E B S 366 E J M 8 > E T Y 806 > E B S 366 E_(JM8) > E_(TY 806) > E_(BS 366)\mathrm{E}_{J M 8}>\mathrm{E}_{T Y 806}>\mathrm{E}_{B S 366}, these genes were defined as higher than both parents (H2P); if E J M 8 = E B S 366 > E T Y 806 E J M 8 = E B S 366 > E T Y 806 E_(JM8)=E_(BS 366) > E_(TY 806)\mathrm{E}_{J M 8}=\mathrm{E}_{B S 366}>\mathrm{E}_{T Y 806}, or E J M 8 = E T Y 806 > E B S 366 E J M 8 = E T Y 806 > E B S 366 E_(JM8)=E_(TY 806) > E_(BS 366)\mathrm{E}_{J M 8}=\mathrm{E}_{T Y 806}>\mathrm{E}_{B S 366}, these genes were defined as close to higher parent (CHP); if E B S 366 > E J M 8 > E T Y 806 E B S 366 > E J M 8 > E T Y 806 E_(BS 366) > E_(JM8) > E_(TY 806)\mathrm{E}_{B S 366}>\mathrm{E}_{J M 8}>\mathrm{E}_{T Y 806}, or E T Y 806 > E J M 8 > E B S 366 E T Y 806 > E J M 8 > E B S 366 E_(TY 806) > E_(JM8) > E_(BS 366)\mathrm{E}_{T Y 806}>\mathrm{E}_{J M 8}>\mathrm{E}_{B S 366}, these genes were defined as between two parents (B2P); if E J M 8 = E B S 366 < E T Y 806 E J M 8 = E B S 366 < E T Y 806 E_(JM8)=E_(BS 366) < E_(TY 806)\mathrm{E}_{J M 8}=\mathrm{E}_{B S 366}<\mathrm{E}_{T Y 806}, or E J M 8 = E T Y 806 < E B S 366 E J M 8 = E T Y 806 < E B S 366 E_(JM8)=E_(TY 806) < E_(BS 366)\mathrm{E}_{J M 8}=\mathrm{E}_{T Y 806}<\mathrm{E}_{B S 366}, these genes were defined as close to lower parent (CLP);
杂交 JM8 和两个自交系之间的 DEGs 被定义为 DGhp。DGhp 的分类是根据 E J M 8 E J M 8 E_(JM8)\mathrm{E}_{J M 8} 相对于 E B S 366 E B S 366 E_(BS 366)\mathrm{E}_{B S 366} 和 E T Y 806 E T Y 806 E_(TY 806)\mathrm{E}_{T Y 806} 进行的。“=”表示统计上相似，“ > > >> ” 和 “ < < << ” 表示统计上较高或较低。如果 E J M 8 > E B S 366 = E T Y 806 E J M 8 > E B S 366 = E T Y 806 E_(JM8) > E_(BS 366)=E_(TY 806)\mathrm{E}_{J M 8}>\mathrm{E}_{B S 366}=\mathrm{E}_{T Y 806} 、 E J M 8 > E B S 366 > E T Y 806 E J M 8 > E B S 366 > E T Y 806 E_(JM8) > E_(BS 366) > E_(TY 806)\mathrm{E}_{J M 8}>\mathrm{E}_{B S 366}>\mathrm{E}_{T Y 806} 或 E J M 8 > E T Y 806 > E B S 366 E J M 8 > E T Y 806 > E B S 366 E_(JM8) > E_(TY 806) > E_(BS 366)\mathrm{E}_{J M 8}>\mathrm{E}_{T Y 806}>\mathrm{E}_{B S 366} ，这些基因被定义为高于父母双方 （H2P）;如果 E J M 8 = E B S 366 > E T Y 806 E J M 8 = E B S 366 > E T Y 806 E_(JM8)=E_(BS 366) > E_(TY 806)\mathrm{E}_{J M 8}=\mathrm{E}_{B S 366}>\mathrm{E}_{T Y 806} ， 或 E J M 8 = E T Y 806 > E B S 366 E J M 8 = E T Y 806 > E B S 366 E_(JM8)=E_(TY 806) > E_(BS 366)\mathrm{E}_{J M 8}=\mathrm{E}_{T Y 806}>\mathrm{E}_{B S 366} ，这些基因被定义为接近高亲本 （CHP）;如果 E B S 366 > E J M 8 > E T Y 806 E B S 366 > E J M 8 > E T Y 806 E_(BS 366) > E_(JM8) > E_(TY 806)\mathrm{E}_{B S 366}>\mathrm{E}_{J M 8}>\mathrm{E}_{T Y 806} ， 或 E T Y 806 > E J M 8 > E B S 366 E T Y 806 > E J M 8 > E B S 366 E_(TY 806) > E_(JM8) > E_(BS 366)\mathrm{E}_{T Y 806}>\mathrm{E}_{J M 8}>\mathrm{E}_{B S 366} ，这些基因被定义为两个亲本之间 （B2P）;如果 E J M 8 = E B S 366 < E T Y 806 E J M 8 = E B S 366 < E T Y 806 E_(JM8)=E_(BS 366) < E_(TY 806)\mathrm{E}_{J M 8}=\mathrm{E}_{B S 366}<\mathrm{E}_{T Y 806} ， 或 E J M 8 = E T Y 806 < E B S 366 E J M 8 = E T Y 806 < E B S 366 E_(JM8)=E_(TY 806) < E_(BS 366)\mathrm{E}_{J M 8}=\mathrm{E}_{T Y 806}<\mathrm{E}_{B S 366} ，这些基因被定义为接近低亲本 （CLP）;
  1. Yong-jie Liu, Shi-qing Gao, and Yi-miao Tang have contributed equally to this work
    Yong-jie Liu、Shi-qing Gao 和 Yi-miao Tang 对这项工作做出了同样的贡献
  2. Shi-qing Gao  高世青
    gaoshiqing@baafs.net.cn; gshiq@126.com
    gaoshiqing@baafs.net.cn;gshiq@126.com
    ⊠ ⊠ ⊠\boxtimes Chang-ping Zhao
    ⊠ ⊠ ⊠\boxtimes 赵昌平
    zhaochangping@baafs.net.cn; cp_zhao@vip.sohu.com
    zhaochangping@baafs.net.cn;cp_zhao@vip.sohu.com
    1 Beijing Engineering Research Center for Hybrid Wheat, The Municipal Key Laboratory of the Molecular Genetics of Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
    1 北京市农林科学院杂交小麦分子遗传学市级重点实验室，北京市杂交小麦工程研究中心，北京100097
    2 Beijing University of Agriculture, Beijing 100036, China
    2 北京农业大学，中国北京100036
  3. 3 Hebei Normal University, Shijiazhuang 050024, China
    3 河北师范大学，石家庄050024
    4 Shandong Normal University, Jinan 250014, China
    4 山东师范大学，济南250014
    5 Huazhong Agricultural University, Wuhan 430070, China
    5 华中农业大学， 湖北 武汉 430070
    6 Yangtze University, Jingzhou 434023, China
    6 长江大学，中国荆州434023