Exploring the molecular basis of heterosis for plant breeding

Since approximate a century ago, many hybrid crops have been continually developed by crossing two inbred varieties. Owing to heterosis(hybrid vigor) in plants, these hybrids often have superior agricultural performances in yield or disease resistance succeeding their inbred parental lines. Several classical hypotheses have been proposed to explain the genetic causes of heterosis. During recent years, many new genetics and genomics strategies have been developed and used for the identifications of heterotic genes in plants. Heterotic effects of the heterotic loci and molecular functions of the heterotic genes are being investigated in many plants such as rice, maize, sorghum, Arabidopsis and tomato.More and more data and knowledge coming from the molecular studies of heterotic loci and genes will serve as a valuable resource for hybrid breeding by molecular design in future. This review aims to address recent advances in our understanding of the genetic and molecular mechanisms of heterosis in plants. The remaining scientific questions on the molecular basis of heterosis and the potential applications in breeding are also proposed and discussed.     

Comparative analysis of inbred and hybrid maize at the diploid and tetraploid levels

Heterosis often occurs in offspring derived from a cross between inbred or divergent parents and can be observed as the superior performance of these hybrids for a wide variety of characters. Heterosis was compared in maize lines at two ploidy levels, diploid and tetraploid, to gain a better understanding of the interaction of heterosis and ploidy level. Employing genetically identical diploid and tetraploid maize derived from four different inbred lines, we investigated heterosis for 11 morphological traits, including several plant height measures, as well as flowering time for both silks and anthers. We find that the heterotic response of a certain hybrid differs between diploid and tetraploid lines, and that the response at one ploidy cannot serve as a predictor for the other. Also, progressive heterosis was found for several of the characters in the tetraploid double-cross hybrid, which can have four different alleles at one locus, compared to the double-cross diploid hybrids, which can only possess two alleles per locus. Overall, the results indicate that the heterotic response of tetraploid maize lines differs significantly from that of the diploid. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Effect of Low Growth Temperature on Hill Reaction and Photosystem 1 Activities and Pigment Contents in Maize Inbred Lines and Their F1 Hybrids

Young plants of maize inbred lines CE777, CE704, and CE810 and their F₁ hybrids displaying a positive heterotic effect in various photosynthetic characteristics were exposed to low temperature during their early growth developmental stage. The photochemical activity of isolated mesophyll chloroplasts and the contents of photosynthetic pigments in leaves of stressed and non-stressed plants were compared with the aim to find out the possible changes in the relationship between parents and hybrids, and to determine the genetic basis of heterosis in F₁ generation. Strong decrease in the content of chlorophylls was observed for all genotypes examined when plants were subjected to low growth temperature. Similar change was recorded for Hill reaction activity (HRA) of inbred lines but not of their F₁ hybrids, and no significant response at all was found for photosystem 1 (PS1) activity or the total carotenoids content. The intraspecific variation due to differences between genotypes was found for most of photosynthetic characteristics examined. This variation was caused by the additive and dominance genetic effects. Positive dominance was the main cause of positive heterosis in HRA and in the contents of photosynthetic pigments and was much more pronounced in the stressed plants compared to the non-stressed ones. The maternal additive effects participated in the inheritance of contents of photosynthetic pigments in plants exposed to low temperature, too. This revised version was published online in June 2006 with corrections to the Cover Date.     

The use of SSRs for predicting the hybrid yield and yield heterosis in 15 key inbred lines of Chinese maize

A challenge to maize breeders is to predict and identify inbred lines that can produce highly heterotic hybrids precisely. In the present study we surveyed the genetic diversity among 15 elite inbred lines of maize in China with SSR markers and assessed the relationship between SSR marker and hybrid yield/yield heterosis in a diallel set of 105 crosses. Forty-three SSR primers selected from all sixty-three primers gave stable profiles amplified in the sample of 15 inbred lines, which could clearly resolve on 4% metaphor agarose gel. The average number of alleles per SSR locus was 4.44 with a range from 2 to 9. The polymorphism information content (PIC) for the SSR loci varied from 0.28 to 0.81 with a mean of 0.6281. Genetic similarity (GS) among 15 lines was estimated with 191 alleles identified as raw data, the Nei's coefficient of GS ranged from 0.492 for 478 vs HZ4 up to 0.745 for E28 to ZH64 with a mean of 0.619. The cluster diagram based upon the SSR data grouped the 15 lines into families consistent with the yield heterotic response of these. Genetic distance (GD) based on SSR data was significantly correlated with hybrid yield/yield heterosis, the correlation coefficient (r) being 0.5432 and 0.4271 in 1999 and 0.4305 and 0.3614 in 1998 field test, respectively, whereas the determination coefficient (r2) was lower. The correlation between GD based on SSR data and hybrid yield/yield heterosis changed alone with the difference of number and pedigree relationship among parents that were used in this study. SSR makers showed high polymorphism and could be used to assess the relationship between inbred lines of maize, but it was difficult to predict the yield heterosis of maize.     

Manifestation of heterosis during early maize (Zea mays L.) root development

Heterosis is typically detected in adult hybrid plants as increased yield or vigor compared to their parental inbred lines. Only little is known about the manifestation of heterosis during early postembryonic development. Objective of this study was to identify heterotic traits during early maize root development. Four German inbred lines of the flint (UH002 and UH005) and dent (UH250 and UH301) pool and the 12 reciprocal hybrids generated from these inbred lines were subjected to a morphological and histological analysis during early root development. Primary root length and width were measured daily in a time course between 3 and 7 days after germination (DAG) and displayed average midparent heterosis (MPH) of 17–25% and 1–7%, respectively. Longitudinal size of cortical cells in primary roots was determined 5 DAG and displayed on average 24% MPH thus demonstrating that enlarged primary roots of hybrids can mainly be attributed to elongated cortical cells. The number of seminal roots determined 14 DAG showed on average 18% MPH. Lateral root density of all tested hybrids was determined 5 DAG. This root trait showed the highest degree of heterosis with an average MPH value of 51%. This study demonstrated that heterosis is already manifesting during the very early stages of root development a few days after germination. The young root system is therefore a suitable model for subsequent molecular studies of the early stages of heterosis manifestation during seedling development.     

Genome‐wide identification and analysis of heterotic loci in three maize hybrids

Heterosis, or hybrid vigour, is a predominant phenomenon in plant genetics, serving as the basis of crop hybrid breeding, but the causative loci and genes underlying heterosis remain unclear in many crops. Here, we present a large‐scale genetic analysis using 5360 offsprings from three elite maize hybrids, which identifies 628 loci underlying 19 yield‐related traits with relatively high mapping resolutions. Heterotic pattern investigations of the 628 loci show that numerous loci, mostly with complete–incomplete dominance (the major one) or overdominance effects (the secondary one) for heterozygous genotypes and nearly equal proportion of advantageous alleles from both parental lines, are the major causes of strong heterosis in these hybrids. Follow‐up studies for 17 heterotic loci in an independent experiment using 2225 F₂ individuals suggest most heterotic effects are roughly stable between environments with a small variation. Candidate gene analysis for one major heterotic locus (ub3) in maize implies that there may exist some common genes contributing to crop heterosis. These results provide a community resource for genetics studies in maize and new implications for heterosis in plants.     

Comparison of maize (Zea mays L.) F1-hybrid and parental inbred line primary root transcriptomes suggests organ-specific patterns of nonadditive gene expression and conserved expression trends

The phenomenon of heterosis describes the increased agronomic performance of heterozygous F(1) plants compared to their homozygous parental inbred plants. Heterosis is manifested during the early stages of root development in maize. The goal of this study was to identify nonadditive gene expression in primary roots of maize hybrids compared to the average expression levels of their parental inbred lines. To achieve this goal a two-step strategy was used. First, a microarray preselection of nonadditively expressed candidate genes was performed. Subsequently, gene expression levels in a subset of genes were determined via high-throughput quantitative real-time (qRT)-PCR experiments. Initial microarray experiments identified 1941 distinct microarray features that displayed nonadditive gene expression in at least 1 of the 12 analyzed hybrids compared to the midparent value of their parental inbred lines. Most nonadditively expressed genes were expressed between the parental values (>89%). Comparison of these 1941 genes with nonadditively expressed genes identified in maize shoot apical meristems via the same experimental procedure in the same genotypes revealed significantly less overlap than expected by pure chance. This finding suggests organ-specific patterns of nonadditively expressed genes. qRT-PCR analyses of 64 of the 1941 genes in four different hybrids revealed conserved patterns of nonadditively expressed genes in different hybrids. Subsequently, 22 of the 64 genes that displayed nonadditive expression in all four hybrids were analyzed in 12 hybrids that were generated from four inbred lines. Among those genes a superoxide dismutase 2 was expressed significantly above the midparent value in all 12 hybrids and might thus play a protective role in heterosis-related antioxidative defense in the primary root of maize hybrids. The findings of this study are consistent with the hypothesis that both global expression trends and the consistent differential expression of specific genes contribute to the organ-specific manifestation of heterosis.     

Isozyme Studies in Predicting the Potential Yield of Heterosis in Maize

Electrophoretic isozyme zymogram patterns of peroxidase (POD), cytochrome oxidase (COD), esterase (Est), α-amylase (α-Amy), catalase (Cat), Glutamate dehydrogenase (GDH), Malate dehydrogenase (MDH), superoxide dismutase (SOD), acid phosphatase (Acp), etc. obtained from 108 maize inbred lines and their 199 hybrids were analyzed. The soluble protein patterns from these materials were tested as well. The authors had probed into the relationship between the index of zymogram difference and potential yield of heterosis in maize. The results indicated that hybrids from parents which showed high zymogram difference index could produce high heterosis and those from parents which had low zymogram difference index could also produce high heterosis as well. But hybrids from the sister lines which had lower zymogram difference index could only produce lower heterosis. There was no significant statistical difference between isozyme zymogram difference index and potential yield of heteroSis on a genetic background with complex combinaton.     

Specific changes in total and mitochondrial proteomes are associated with higher levels of heterosis in maize hybrids

The phenomenon of hybrid vigor (heterosis) has long been harnessed by plant breeders to improve world food production. However, the changes that are essential for heterotic responses and the mechanisms responsible for heterosis remain undefined. Large increases in biomass and yield in high-heterosis hybrids suggest that alterations in bioenergetic processes may contribute to heterosis. Progeny from crosses between various inbred lines vary in the extent of vigor observed. Field-grown maize F(1) hybrids that consistently exhibited either low or high heterosis across a variety of environments were examined for changes in proteins that may be correlated with increased plant vigor and yield. Unpollinated ears at the time of flowering (ear shoots) were selected for the studies because they are metabolically active, rich in mitochondria, and the sizes of the ears are diagnostic of yield heterosis. Total protein and mitochondrial proteomes were compared among low- and higher-heterosis hybrids. Two-dimensional difference gel electrophoresis was used to identify allelic and/or isoform differences linked to heterosis. Identification of differentially regulated spots by mass spectrometry revealed proteins involved in stress responses as well as primary carbon and protein metabolism. Many of these proteins were identified in multiple spots, but analysis of their abundances by label-free mass spectrometry suggested that most of the expression differences were due to isoform variation rather than overall protein amount. Thus, our proteomics studies suggest that expression of specific alleles and/or post-translational modification of specific proteins correlate with higher levels of heterosis.     

Heterotic patterns of sugar and amino acid components in developing maize kernels

Heterosis is the superior performance of hybrids over their inbred parents. Despite its importance, little is known about the genetic and molecular basis of this phenomenon. Heterosis has been extensively exploited in plant breeding, particularly in maize (Zea mays, L.), and is well documented in the B73 and Mo17 maize inbred lines and their F1 hybrids. In this study, we determined the dry matter, the levels of starch and protein components and a total of 24 low-molecular weight metabolites including sugars, sugar-phosphates, and free amino acids, in developing maize kernels between 8 and 30 days post-pollination (DPP) of the hybrid B73 × Mo17 and its parental lines. The tissue specificity of amino acid and protein content was investigated between 16 and 30 DPP. Key observations include: (1) most of the significant differences in the investigated tissue types occurred between Mo17 and the other two genotypes; (2) heterosis of dry matter and metabolite content was detectable from the early phase of kernel development onwards; (3) the majority of metabolites exhibited an additive pattern. Nearly 10% of the metabolites exhibited nonadditive effects such as overdominance, underdominance, and high-parent and low-parent dominance; (4) The metabolite composition was remarkably dependent on kernel age, and this large developmental effect could possibly mask genotypic differences; (5) the metabolite profiles and the heterotic patterns are specific for endosperm and embryo. Our findings illustrate the power of metabolomics to characterize heterotic maize lines and suggest that the metabolite composition is a potential marker in the context of heterosis research.     

Heterotic trait locus (HTL) mapping identifies intra-locus interactions that underlie reproductive hybrid vigor in Sorghum bicolor

Identifying intra-locus interactions underlying heterotic variation among whole-genome hybrids is a key to understanding mechanisms of heterosis and exploiting it for crop and livestock improvement. In this study, we present the development and first use of the heterotic trait locus (HTL) mapping approach to associate specific intra-locus interactions with an overdominant heterotic mode of inheritance in a diallel population using Sorghum bicolor as the model. This method combines the advantages of ample genetic diversity and the possibility of studying non-additive inheritance. Furthermore, this design enables dissecting the latter to identify specific intra-locus interactions. We identified three HTLs (3.5% of loci tested) with synergistic intra-locus effects on overdominant grain yield heterosis in 2 years of field trials. These loci account for 19.0% of the heterotic variation, including a significant interaction found between two of them. Moreover, analysis of one of these loci (hDPW4.1) in a consecutive F2 population confirmed a significant 21% increase in grain yield of heterozygous vs. homozygous plants in this locus. Notably, two of the three HTLs for grain yield are in synteny with previously reported overdominant quantitative trait loci for grain yield in maize. A mechanism for the reproductive heterosis found in this study is suggested, in which grain yield increase is achieved by releasing the compensatory tradeoffs between biomass and reproductive output, and between seed number and weight. These results highlight the power of analyzing a diverse set of inbreds and their hybrids for unraveling hitherto unknown allelic interactions mediating heterosis.     

玉米种质资源抗矮花叶病鉴定

采用摩擦接种方法,鉴定了3995份玉米种质对玉米矮花叶病毒(SCMV-MDB)的抗病性。经重复鉴定,筛选出抗病优良自交系73份,抗病丰产杂交种80份。玉米不同类群种质,对SCMV-MDB的抗病性有明显差异,改良Reid、Lancaster、旅大红骨类群种质大部分表现感病;塘四平头、外杂先锋选系类群种质大多数表现抗病。在不同类型种质中,硬粒型、马齿型和半马齿型表现抗病的较多,糯质型较少,甜质型和爆裂型中尚未发现抗源。     

一个新矮生玉米种质资源的发现与遗传鉴定

玉米矮生种质资源在育种工作中具有重要的利用价值。2002年在玉米种质资源扩繁与鉴定过程中,从玉米自交系K36中发现一株矮生突变体。随后通过连续自交,获得了纯合一致、稳定的矮生自交系,命名为矮2003。该矮秆材料在北京表现株高62．1cm,植株清秀,茎秆坚硬,结实正常。于不同时期用不同浓度赤霉素处理该材料显示其对赤霉素反应不敏感。矮2003与正常玉米自交系测交F1呈现高秆,F2与BC1高、矮秆分离比例分别符合3：1与1：1,遗传分析表明其矮生性状受一对主效单基因控制,表现为隐性遗传。所携带的矮生基因不同于已报道的玉米Dwarf8等。     

Heterosis for carotenoid concentration and profile in maize hybrids

Production of high-lutein maize grain is of particular interest as a value-added feed source to produce high-lutein eggs. In this paper, it is demonstrated that heterosis for total carotenoid concentration and for the ratio of lutein to zeaxanthin (L:Z ratio), or profile type, exists infrequently in yellow dent crosses. However, yellow dent inbred maize lines A619 and CG102, both possessing high-lutein profiles, produce F1 seed with a classic overdominant expression of lutein levels (i.e., 49 μg/g dry weight (DW) above the high-parent value). Reciprocal crosses of A619 and CG102 with one another and with two high-zeaxanthin (i.e., low lutein), high-carotenoid lines both suggest that the A619 and CG102 high-lutein phenotypes are achieved by different and complementary genotypes. The contribution of CG102 to the heterotic response was examined using a QTL-based approach that involved phenotyping the mapping population in a testcross to A619. Significant QTL were found at loci known to be involved in the carotenoid pathway but also at loci proximate to, but separate from, known carotenoid pathway steps. Exploiting an overdominant heterotic response for lutein and total carotenoids should be given strong consideration as a viable method of producing high-carotenoid hybrid maize lines.     

Gene Expression Analysis in Sorghum Hybrids and Their Parental Lines at Critical Developmental Stages in Relation to Grain Yield Heterosis by Exploiting Heterosis-Related Genes from Major Cereals

Relative expression levels of selected genes from the Heterosis-Related Gene Database exhibiting more than 90% homology with sorghum were studied in hybrids and their respective parental lines for a better understanding on the molecular basis of heterosis. A high (27A × RS 673) and a low heterotic hybrid (7A × CB 26) of sorghum along with their parental lines were used for this purpose. Twenty (15 maize and 5 rice) genes exhibiting more than 90% homology with that of sorghum were identified. The maize genes ZmHG13, ZmHG16, and ZmhG19 exhibited more than fourfold increase over the male parent (RS 673) of high heterotic hybrid during booting stage, which started decreasing during flowering stage. Similarly, the rice genes OsHG1 and OsHG12 recorded >?2.5-fold increase. However, these genes recorded less than twofold increase during the same stage of the plant in the low heterotic hybrid. Notably, among the genes that exhibited higher expression in the highly heterotic hybrid were those coding for proteins, which were known to play crucial roles in the manifestation of heterosis in plants.     

Temporal Regulation of the Metabolome and Proteome in Photosynthetic and Photorespiratory Pathways Contributes to Maize Heterosis

Heterosis or hybrid vigor is widespread in plants and animals. Although the molecular basis for heterosis has been extensively studied, metabolic and proteomic contributions to heterosis remain elusive. Here we report an integrative analysis of time-series metabolome and proteome data in maize (Zea mays) hybrids and their inbred parents. Many maize metabolites and proteins are diurnally regulated, and many of these show nonadditive abundance in the hybrids, including key enzymes and metabolites involved in carbon assimilation. Compared with robust trait heterosis, metabolic heterosis is relatively mild. Interestingly, most amino acids display negative mid-parent heterosis (MPH), i.e., having lower values than the average of the parents, while sugars, alcohols, and nucleoside metabolites show positive MPH. From the network perspective, metabolites in the photosynthetic pathway show positive MPH, whereas metabolites in the photorespiratory pathway show negative MPH, which corresponds to nonadditive protein abundance and enzyme activities of key enzymes in the respective pathways in the hybrids. Moreover, diurnally expressed proteins that are upregulated in the hybrids are enriched in photosynthesis-related gene-ontology terms. Hybrids may more effectively remove toxic metabolites generated during photorespiration, and thus maintain higher photosynthetic efficiency. These metabolic and proteomic resources provide unique insight into heterosis and its utilization for high yielding maize and other crop plants.     

The genetic basis of heterosis: multiparental quantitative trait loci mapping reveals contrasted levels of apparent overdominance among traits of agronomical interest in maize (Zea mays L.)

Understanding the genetic bases underlying heterosis is a major issue in maize (Zea mays L.). We extended the North Carolina design III (NCIII) by using three populations of recombinant inbred lines derived from three parental lines belonging to different heterotic pools, crossed with each parental line to obtain nine families of hybrids. A total of 1253 hybrids were evaluated for grain moisture, silking date, plant height, and grain yield. Quantitative trait loci (QTL) mapping was carried out on the six families obtained from crosses to parental lines following the "classical" NCIII method and with a multiparental connected model on the global design, adding the three families obtained from crosses to the nonparental line. Results of the QTL detection highlighted that most of the QTL detected for grain yield displayed apparent overdominance effects and limited differences between heterozygous genotypes, whereas for grain moisture predominance of additive effects was observed. For plant height and silking date results were intermediate. Except for grain yield, most of the QTL identified showed significant additive-by-additive epistatic interactions. High correlation observed between heterosis and the heterozygosity of hybrids at markers confirms the complex genetic basis and the role of dominance in heterosis. An important proportion of QTL detected were located close to the centromeres. We hypothesized that the lower recombination in these regions favors the detection of (i) linked QTL in repulsion phase, leading to apparent overdominance for heterotic traits and (ii) linked QTL in coupling phase, reinforcing apparent additive effects of linked QTL for the other traits.     

Absence of heterosis in hybrid corn. Description of the effect

Three unrelated homozygous maize lines, TS11, P22, and ST156, that produced hybrids in which heterosis was either absent or insignificant were identified. These hybrids were phenotypically similar to self-pollinated homozygous lines. Reciprocal crosses showed that the absence of heterosis is controlled by nuclear genes and is not associated with the cytoplasm of inbred lines. Analysis of F2 plants demonstrated that lines TS11, P22, and ST156 contained allelic genes determining the absence of heterosis in hybrid plants. Crosses of lines TS11, P22, and ST156 with a common selection line 092 generated hybrids with normal heterosis. It was concluded that heterozygosity or homozygosity of particular genes in lines TS11, P22, and ST156 play a pivotal role in the manifestation or the absence of hybrid vigor in hybrids.     

Progressive heterosis in genetically defined tetraploid maize

Progressive heterosis, i.e., the additional hybrid vigor in double-cross tetraploid hybrids not found in their single-cross tetraploid parents, has been documented in a number of species including alfalfa,potato, and maize. In this study, four artificially induced maize tetraploids, directly derived from standard inbred lines, were crossed in pairs to create two single-cross hybrids. These hybrids were then crossed to create double-cross hybrids containing genetic material from all four original lines. Replicated fieldbased phenotyping of the materials over four years indicated a strong progressive heterosis phenotype in tetraploids but not in their diploid counterparts. In particular, the above ground dry weight phenotype of double-cross tetraploid hybrids was on average 34% and 56% heavier than that of the single-cross tetraploid hybrids and the double-cross diploid counterparts, respectively. Additionally,whole-genome resequencing of the original inbred lines and further analysis of these data did not show the expected spectrum of alleles to explain tetraploid progressive heterosis under the complementation of complete recessive model. These results underscore the reality of the progressive heterosis phenotype,its potential utility for increasing crop biomass production, and the need for exploring alternative hypothesis to explain it at a molecular level.