Zhiyuan Fu

Proteomic analysis of heterosis during maize seed germination

Heterosis is observed for most phenotypic traits and developmental stages in many plants. In this study, the embryos, from germinating seeds after 24 h of soaking, for five elite maize hybrids and their parents were selected to unravel the genetic basis of heterosis using 2‐D proteomic method. In total, 257 (80.06%), 363 (58.74%), 351 (79.95%), 242 (54.50%), and 244 (46.30%) nonadditively expressed proteins were identified in hybrids Zhengdan 958, Nongda 108, Yuyu 22, Xundan 20, and Xundan 18, respectively. The nonadditive proteins were divided into above high‐parent (++; 811, 55.66%), high‐parent (+; 121, 8.30%), partial dominance (+−; 249, 17.09%), low‐parent (−; 30, 2.06%), below low‐parent (− −; 62, 4.26%), and D (different; 184, 12.63%) expression patterns. The observed patterns indicate the important roles of dominance, partial dominance, and overdominance in regulating seed germination in maize. Additionally, 54 different proteins were identified by mass spectrometry and classified into nine functional groups: metabolism (9), cell detoxification (8), unknown functional proteins (8), chaperones (7), signal transduction (6), development process (5), other (5), transporter (3), and stress response (3). Of these, the most interesting are those involved with germination‐related hormone signal transduction and the abscisic acid and gibberellin regulation networks.

MicroRNA Transcriptomic Analysis of Heterosis during Maize Seed Germination

Heterosis has been utilized widely in the breeding of maize and other crops, and plays an important role in increasing yield, improving quality and enhancing stresses resistance, but the molecular mechanism responsible for heterosis is far from clear. To illustrate whether miRNA-dependent gene regulation is responsible for heterosis during maize germination, a deep-sequencing technique was applied to germinating embryos of a maize hybrid, Yuyu22, which is cultivated widely in China and its parental inbred lines, Yu87-1 and Zong3. The target genes of several miRNAs showing significant expression in the hybrid and parental lines were predicted and tested using real-time PCR. A total of 107 conserved maize miRNAs were co-detected in the hybrid and parental lines. Most of these miRNAs were expressed non-additively in the hybrid compared to its parental lines. These results indicated that miRNAs might participate in heterosis during maize germination and exert an influence via the decay of their target genes. Novel miRNAs were predicted follow a rigorous criterion and only the miRNAs detected in all three samples were treated as a novel maize miRNA. In total, 34 miRNAs belonged to 20 miRNA families were predicted in germinating maize seeds. Global repression of miRNAs in the hybrid, which might result in enhanced gene expression, might be one reason why the hybrid showed higher embryo germination vigor compared to its parental lines.

QTL analysis of Kernel-related traits in maize using an immortalized F2 population.

Kernel size and weight are important determinants of grain yield in maize. In this study, multivariate conditional and unconditional quantitative trait loci (QTL), and digenic epistatic analyses were utilized in order to elucidate the genetic basis for these kernel-related traits. Five kernel-related traits, including kernel weight (KW), volume (KV), length (KL), thickness (KT), and width (KWI), were collected from an immortalized F2 (IF2) maize population comprising of 243 crosses performed at two separate locations over a span of two years. A total of 54 unconditional main QTL for these five kernel-related traits were identified, many of which were clustered in chromosomal bins 6.04–6.06, 7.02–7.03, and 10.06–10.07. In addition, qKL3, qKWI6, qKV10a, qKV10b, qKW10a, and qKW7a were detected across multiple environments. Sixteen main QTL were identified for KW conditioned on the other four kernel traits (KL, KWI, KT, and KV). Thirteen main QTL were identified for KV conditioned on three kernel-shape traits. Conditional mapping analysis revealed that KWI and KV had the strongest influence on KW at the individual QTL level, followed by KT, and then KL; KV was mostly strongly influenced by KT, followed by KWI, and was least impacted by KL. Digenic epistatic analysis identified 18 digenic interactions involving 34 loci over the entire genome. However, only a small proportion of them were identical to the main QTL we detected. Additionally, conditional digenic epistatic analysis revealed that the digenic epistasis for KW and KV were entirely determined by their constituent traits. The main QTL identified in this study for determining kernel-related traits with high broad-sense heritability may play important roles during kernel development. Furthermore, digenic interactions were shown to exert relatively large effects on KL (the highest AA and DD effects were 4.6% and 6.7%, respectively) and KT (the highest AA effects were 4.3%).

Proteomic identification of genes associated with maize grain-filling rate.

Grain filling during the linear phase contributes most of the dry matter accumulated in the maize kernel, which in turn determines the final grain yield. Endosperms and embryos of three elite maize hybrids (Zhengdan 958, Nongda 108, and Pioneer 335) were sampled 17, 22, 25, and 28 days after pollination, during the linear phase of grain filling, for proteomic analysis to explore the regulatory factors critical for grain filling rate. In total, 39 and 43 protein spots that showed more than 2-fold changes in abundance at P<0.01 between any two sampling stages in the endosperm and embryo were analyzed by protein mass spectrometry. The changing patterns in expression index of these proteins in the endosperm were evenly distributed, whereas up-regulation patterns predominated (74%) in the embryo. Functional analysis revealed that metabolism was the largest category, represented by nine proteins in the endosperm and 12 proteins in the embryo, of the proteins that significantly changed in abundance. Glycolysis, a critical process both for glucose conversion into pyruvate and for release of free energy and reducing power, and proteins related to redox homeostasis were emphasized in the endosperm. Additionally, lipid, nitrogen, and inositol metabolism related to fatty acid biosynthesis and late embryogenesis abundant proteins were emphasized in the embryo. One protein related to cellular redox equilibrium, which showed a more than 50-fold change in abundance and was co-localized with a quantitative trait locus for grain yield on chromosome 1, was further investigated by transcriptional profile implying consistent expression pattern with protein accumulation. The present results provide a first step towards elucidation of the gene network responsible for regulation of grain filling in maize.

Quantitative trait loci for mercury accumulation in maize (Zea mays L.) identified using a RIL population.

To investigate the genetic mechanism of mercury accumulation in maize (Zea mays L.), a population of 194 recombinant inbred lines derived from an elite hybrid Yuyu 22, was used to identify quantitative trait loci (QTLs) for mercury accumulation at two locations. The results showed that the average Hg concentration in the different tissues of maize followed the order: leaves > bracts > stems > axis > kernels. Twenty-three QTLs for mercury accumulation in five tissues were detected on chromosomes 1, 4, 7, 8, 9 and 10, which explained 6.44% to 26.60% of the phenotype variance. The QTLs included five QTLs for Hg concentration in kernels, three QTLs for Hg concentration in the axis, six QTLs for Hg concentration in stems, four QTLs for Hg concentration in bracts and five QTLs for Hg concentration in leaves. Interestingly, three QTLs, qKHC9a, qKHC9b, and qBHC9 were in linkage with two QTLs for drought tolerance. In addition, qLHC1 was in linkage with two QTLs for arsenic accumulation. The study demonstrated the concentration of Hg in Hg-contaminated paddy soil could be reduced, and maize production maintained simultaneously by selecting and breeding maize Hg pollution-safe cultivars (PSCs).

Identification and Characterization of microRNAs during Maize Grain Filling

The grain filling rate is closely associated with final grain yield of maize during the period of maize grain filling. To identify the key microRNAs (miRNAs) and miRNA-dependent gene regulation networks of grain filling in maize, a deep-sequencing technique was used to research the dynamic expression patternsof miRNAs at four distinct developmental grain filling stages in Zhengdan 958, which is an elite hybrid and cultivated widely in China. The sequencing result showed that the expression amount of almost all miRNAs was changing with the development of the grain filling and formed in seven groups. After normalization, 77 conserved miRNAs and 74 novel miRNAs were co-detected in these four samples. Eighty-one out of 162 targets of the conserved miRNAs belonged to transcriptional regulation (81, 50%), followed by oxidoreductase activity (18, 11%), signal transduction (16, 10%) and development (15, 9%). The result showed that miRNA 156, 393, 396 and 397, with their respective targets, might play key roles in the grain filling rate by regulating maize growth, development and environment stress response. The result also offered novel insights into the dynamic change of miRNAs during the developing process of maize kernels and assistedin the understanding of how miRNAs are functioning about the grain filling rate.

Investigating the molecular genetic basis of heterosis for internode expansion in maize by microRNA transcriptomic deep sequencing

Heterosis has been used widely in the breeding of maize and other crops and plays an important role in increasing yield, improving quality, and enhancing stress resistance, but its molecular mechanism is far from clear. To determine whether microRNA (miRNA)-dependent gene regulation is responsible for heterosis of elongating internodes below the ear and ear height in maize, a deep-sequencing strategy was applied to the elite hybrid Xundan20, which is currently cultivated widely in China, and its two parents. RNA was extracted from the eighth internode because it shows clear internode length heterosis. A total of 99 conserved maize miRNAs were detected in both the hybrid and parental lines. Most of these miRNAs were expressed nonadditively in the hybrid compared with its parental lines. These results indicated that miRNAs might participate in heterosis during internode expansion in maize and exert an influence on ear and plant height via the repression of their target genes. In total, eight novel miRNAs belonging to four miRNA families were predicted in the expanding internode. Global repression of miRNAs in the hybrid, which might result in enhanced gene expression, might be one reason why the hybrid shows longer internodes and taller seedlings compared with its parental lines.

Proteomic analysis reveals that auxin homeostasis influences the eighth internode length heterosis in maize ( Zea mays )

Ear height is an important maize morphological trait that influences plant lodging resistance in the field, and is based on the number and length of internodes under the ear. To explore the effect of internodes on ear height, the internodes under the ear were analysed in four commercial hybrids (Jinsai6850, Zhengdan958, Xundan20, and Yuyu22) from different heterotic groups in China. The eighth internode, which is the third aboveground extended internode, exhibited high-parent or over high-parent heterosis and contributed considerably to ear height. Thus, the proteome of the eighth internode was examined. Sixty-six protein spots with >1.5-fold differences in accumulation (P < 0.05) among the four hybrids were identified by mass spectrometry and data analyses. Most of the differentially accumulated proteins exhibited additive accumulation patterns, but with epistatic effects on heterosis performance. Proteins involved in phenylpropanoid and benzoxazinoid metabolic pathways were observed to influence indole-3-acetic acid biosynthesis and polar auxin transport during internode development. Moreover, indole-3-acetic acid content was positively correlated with the eighth internode length, but negatively correlated with the extent of the heterosis of the eighth internode length.

Identification of heterotic loci associated with grain yield and its components using two CSSL test populations in maize

Heterosis has widely been used to increase grain yield and quality. In this study, the genetic basis of heterosis on grain yield and its main components in maize were examined over 2 years in two locations in two test populations constructed from a set of 184 chromosome segment substitution lines (CSSLs) and two inbred lines (Zheng58 and Xun9058). Of the 169 heterotic loci (HL) associated with grain yield and its five components identified in CSSL × Zheng58 and CSSL × Xun9058 test populations, only 25 HL were detected in both populations. The comparison of quantitative trait loci (QTLs) detected in the CSSL population with HL detected in the two test populations revealed that only 15.46% and 17.35% of the HL in the given populations respectively, shared the same chromosomal regions as that of the corresponding QTLs and showed dominant effects as well as pleiotropism with additive and dominant effects. In addition, most of the HL (74.23% and 74.49%) had overdominant effects. These results suggest that overdominance is the main contributor to the effects of heterosis on grain yield and its components in maize, and different HL are associated with heterosis for different traits in different hybrids.

Transcriptomic analysis of maize kernel row number-associated miRNAs between a single segment substitution line and its receptor parent

To detect microRNAs (miRNAs) involved in determining kernel row number in maize, next generation deep sequencing was performed on an elite inbred line Zong3 (row number 14–16) of maize in China and a single segment substitution line (SSSL) SSL-10 (row number 8–10) derived from the same genetic background. In SSL-10, the single segment is inserted in chromosome 1 between molecular marker bnlg1953 and bnlg1811. Twenty-eight miRNAs belonging to 11 conserved miRNA families in maize showed expression differences >2-fold in the two lines, among which 14 members from four miRNA families were up-regulated and 14 members from 7 miRNA families were repressed in SSL-10. A genome wide degradome was sequenced to validate the miRNA target genes in solid experiment. In addition, novel miRNAs associated with ear development were predicted using a series of strict criteria, and 29 miRNAs representing eight families were predicted as novel miRNAs. Among the novel miRNAs, only one showed an expression difference >2-fold. The conserved and novel miRNAs with >2-fold expression differences were treated as candidate miRNAs involved in maize kernel row number determination. MiRNA-dependent gene expression regulation and physiological and morphological effects on ear development may explain why the SSSL changed kernel row number compared with its recurrent parent. Based on the interaction of miRNAs and their target genes, a possible miRNA-dependent pathway leading to the given DNA fragment inducing a change in kernel row number was proposed.