Identification of ZmBK2 Gene Variation Involved in Regulating Maize Brittleness.

Maize stalk strength is a crucial agronomic trait that affects lodging resistance. We used map-based cloning and allelic tests to identify a maize mutant associated with decreased stalk strength and confirmed that the mutated gene, ZmBK2, is a homolog of Arabidopsis AtCOBL4, which encodes a COBRA-like glycosylphosphatidylinositol (GPI)-anchored protein. The bk2 mutant exhibited lower cellulose content and whole-plant brittleness. Microscopic observations showed that sclerenchymatous cells were reduced in number and had thinner cell walls, suggesting that ZmBK2 affects the development of cell walls. Transcriptome sequencing of differentially expressed genes in the leaves and stalks revealed substantial changes in the genes associated with cell wall development. We constructed a cell wall regulatory network using these differentially expressed genes, which revealed that abnormal cellulose synthesis may be a reason for brittleness. These results reinforce our understanding of cell wall development and provide a foundation for studying the mechanisms underlying maize lodging resistance.

A Maize Necrotic Leaf Mutant Caused by Defect of Coproporphyrinogen III Oxidase in the Porphyrin Pathway.

Lesion mimic mutants provide ideal genetic materials for elucidating the molecular mechanism of cell death and disease resistance. The maize necrotic leaf mutant (nec-t) is a recessive mutant with necrotic spots and yellow-green leaves. In this study, we found that nec-t was a light and temperature-dependent mutant. Map-based cloning and the allelic test revealed that nec-t was a novel allelic mutant of the Necrotic4 gene. Necrotic4 encodes the coproporphyrinogen III oxidase (CPX1), a key enzyme in the tetrapyrrole pathway, catalyzing coproporphyrinogen III oxidate to protoporphyrinogen IX. Subcellular localization showed that the necrotic4 protein was localized in the chloroplast. Furthermore, RNA-seq analysis showed that the Necrotic4 mutation caused the enhanced chlorophyll degradation and reactive oxygen species (ROS) response. The mechanism of plant lesion formation induced by light and temperature is not clear. Our research provides a basis for understanding the molecular mechanism of necrosis initiation in maize.

PPR647 Protein Is Required for Chloroplast RNA Editing, Splicing and Chloroplast Development in Maize.

Chloroplasts play an essential role in plant growth and development. Any factors affecting chloroplast development will lead to abnormal plant growth. Here, we characterized a new maize mutant, albino seedling mutant 81647 (as-81647), which exhibits an entirely albino phenotype in leaves and eventually died before the three-leaf stage. Transmission electron microscopy (TEM) demonstrated that the chloroplast thylakoid membrane was impaired and the granum lamellae significantly decreased in as-81647. Map-based cloning and transgenic analysis confirmed that PPR647 encodes a new chloroplast protein consisting of 11 pentratricopeptide repeat domains. Quantitative real-time PCR (qRT-PCR) assays and transcriptome analysis (RNA-seq) showed that the PPR647 mutation significantly disrupted the expression of PEP-dependent plastid genes. In addition, RNA splicing and RNA editing of multiple chloroplast genes showed severe defects in as-81647. These results indicated that PPR647 is crucial for RNA editing, RNA splicing of chloroplast genes, and plays an essential role in chloroplast development.

qMrdd2, a novel quantitative resistance locus for maize rough dwarf disease.

BACKGROUND: Maize rough dwarf disease (MRDD), a widespread disease caused by four pathogenic viruses, severely reduces maize yield and grain quality. Resistance against MRDD is a complex trait that controlled by many quantitative trait loci (QTL) and easily influenced by environmental conditions. So far, many studies have reported numbers of resistant QTL, however, only one QTL have been cloned, so it is especially important to map and clone more genes that confer resistance to MRDD. RESULTS: In the study, a major quantitative trait locus (QTL) qMrdd2, which confers resistance to MRDD, was identified and fine mapped. qMrdd2, located on chromosome 2, was consistently identified in a 15-Mb interval between the simple sequence repeat (SSR) markers D184 and D1600 by using a recombinant inbred line (RIL) population derived from a cross between resistant ("80007") and susceptible ("80044") inbred lines. Using a recombinant-derived progeny test strategy, qMrdd2 was delineated to an interval of 577 kb flanked by markers N31 and N42. We further demonstrated that qMrdd2 is an incompletely dominant resistance locus for MRDD that reduced the disease severity index by 20.4%. CONCLUSIONS: A major resistance QTL (qMrdd2) have been identified and successfully refined into 577 kb region. This locus will be valuable for improving maize variety resistance to MRDD via marker-assisted selection (MAS).

Maize Plant Architecture Is Regulated by the Ethylene Biosynthetic Gene ZmACS7.

Plant height and leaf angle are two crucial determinants of plant architecture in maize (Zea mays) and are closely related to lodging resistance and canopy photosynthesis at high planting density. These two traits are primarily regulated by several phytohormones. However, the mechanism of ethylene in regulating plant architecture in maize, especially plant height and leaf angle, is unclear. Here, we characterized a maize mutant, Semidwarf3 (Sdw3), which exhibits shorter stature and larger leaf angle than the wild type. Histological analysis showed that inhibition of longitudinal cell elongation in the internode and promotion in the auricle were mainly responsible for reduced plant height and enlarged leaf angle in the Sdw3 mutant. Through positional cloning, we identified a transposon insertion in the candidate gene ZmACS7, encoding 1-aminocyclopropane-1-carboxylic acid (ACC) Synthase 7 in ethylene biosynthesis of maize. The transposon alters the C terminus of ZmACS7. Transgenic analysis confirmed that the mutant ZmACS7 gene confers the phenotypes of the Sdw3 mutant. Enzyme activity and protein degradation assays indicated that the altered C terminus of ZmACS7 in the Sdw3 mutant increases this protein's stability but does not affect its catalytic activity. The ACC and ethylene contents are dramatically elevated in the Sdw3 mutant, leading to reduced plant height and increased leaf angle. In addition, we demonstrated that ZmACS7 plays crucial roles in root development, flowering time, and leaf number, indicating that ZmACS7 is an important gene with pleiotropic effects during maize growth and development.

Genetic dissection of grain water content and dehydration rate related to mechanical harvest in maize.

BACKGROUND: The low grain water content (GWC) at harvest is a prerequisite to mechanical harvesting in maize, or otherwise would cause massive broken kernels and increase drying costs. The GWC at harvest in turn depends on GWC at the physiological maturity (PM) stage and grain dehydration rate (GDR). Both GWC and GDR are very complex traits, governed by multiple quantitative trait loci (QTL) and easily influenced by environmental conditions. So far, a number of experiments have been conducted to reveal numbers of GWC and GDR QTL, however, very few QTL have been confirmed, and no QTL has been fine-mapped or even been cloned. RESULTS: We demonstrated that GWCs after PM were positively correlated with GWC at PM, whereas negatively with GDRs after PM. With a recombinant inbred line (RIL) population, we identified totally 31 QTL related to GWC and 17 QTL related to GDR in three field trials. Seven GWC QTL were consistently detected in at least two of the three field trials, each of which could explain 6.92-24.78% of the total GWC variation. Similarly, one GDR QTL was consistently detected, accounting for 9.44-14.46% of the total GDR variation. Three major GWC QTL were found to overlap with three GDR QTL in bins 1.05/06, 2.06/07, and 3.05, respectively. One of the consistent GWC QTL, namely qGwc1.1, was fine-mapped from a 27.22 Mb to a 2.05 Mb region by using recombinant-derived progeny test. The qGwc1.1 acted in a semi-dominant manner to reduce GWC by 1.49-3.31%. CONCLUSIONS: A number of consistent GWC and GDR QTL have been identified, and one of them, QTL-qGwc1.1, was successfully refined into a 2.05 Mb region. Hence, it is realistic to clone the genes underlying the GWC and GDR QTL and to make use of them in breeding of maize varieties with low GWC at harvest.

A helitron-induced RabGDIα variant causes quantitative recessive resistance to maize rough dwarf disease.

Maize rough dwarf disease (MRDD), caused by various species of the genus Fijivirus, threatens maize production worldwide. We previously identified a quantitative locus qMrdd1 conferring recessive resistance to one causal species, rice black-streaked dwarf virus (RBSDV). Here, we show that Rab GDP dissociation inhibitor alpha (RabGDIα) is the host susceptibility factor for RBSDV. The viral P7-1 protein binds tightly to the exon-10 and C-terminal regions of RabGDIα to recruit it for viral infection. Insertion of a helitron transposon into RabGDIα intron 10 creates alternative splicing to replace the wild-type exon 10 with a helitron-derived exon 10. The resultant splicing variant RabGDIα-hel has difficulty being recruited by P7-1, thus leading to quantitative recessive resistance to MRDD. All naturally occurring resistance alleles may have arisen from a recent single helitron insertion event. These resistance alleles are valuable to improve maize resistance to MRDD and potentially to engineer RBSDV resistance in other crops.

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.

Identification and characterization analysis of sulfotransferases (SOTs) gene family in cotton (Gossypium) and its involvement in fiber development.

BACKGROUND: Sulfotransferases (SOTs) (EC 2.8.2.-) play a crucial role in the sulphate conjugation reaction involved in plant growth, vigor, stress resistance and pathogen infection. SOTs in Arabidopsis have been carried out and divided into 8 groups. However, the systematic analysis and functional information of SOT family genes in cotton have rarely been reported. RESULTS: According to the results of BLASTP and HMMER, we isolated 46, 46, 76 and 77 SOT genes in the genome G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. A total of 170 in 245 SOTs were further classified into four groups based on the orthologous relationships comparing with Arabidopsis, and tandem replication primarily contributed to the expansion of SOT gene family in G. hirsutum. Expression profiles of the GhSOT showed that most genes exhibited a high level of expression in the stem, leaf, and the initial stage of fiber development. The localization analysis indicated that GhSOT67 expressed in cytoplasm and located in stem and leaf tissue. Additionally, the expression of GhSOT67 were induced and the length of stem and leaf hairs were shortened after gene silencing mediated by Agrobacterium, compared with the blank and negative control plants. CONCLUSIONS: Our findings indicated that SOT genes might be associated with fiber development in cotton and provided valuable information for further studies of SOT genes in Gossypium.

Mapping and Functional Analysis of a Maize Silkless Mutant sk-A7110.

The maize (Zea mays) stigma, which is commonly known as silk, is indispensable for reproduction and thus for grain yield. Here, we isolated a spontaneous mutant sk-A7110, which completely lacks silk; scanning electron microscopy showed that the sk-A7110 pistils degenerated during late floret differentiation. Genetic analysis confirmed that this trait was controlled by a recessive nuclear gene and sk-A7110 was mapped to a 74.13-kb region on chromosome 2 between the simple sequence repeat markers LA714 and L277. Sequence analysis of candidate genes in this interval identified a single-nucleotide insertion at position 569 downstream of the transcriptional start site in Zm00001d002970, which encodes a UDP-glycosyltransferase; this insertion produces a frameshift and premature translational termination. RNA-sequencing analysis of young ears identified 258 differentially expressed genes (DEGs) between sk-A7110 and the wild type (WT), including 119 up- and 139 down-regulated genes. Interestingly, most DEGs related to jasmonic acid (JA) synthesis were up-regulated in the mutant compared to WT. Consistent with this, the JA and JA-Isoleucine (JA-Ile) contents were significantly higher in sk-A7110 ears than in WT. At the same time, RNA-sequencing analysis of tassels showed that sk-A7110 could reduce the number of tassel branches in maize by down-regulating the expression of UB2 and UB3 genes. Our identification of the sk-A7110 mutant and the responsible gene will facilitate further studies on female infertility research or maize breeding.

Evaluation of ZmCCT haplotypes for genetic improvement of maize hybrids.

KEY MESSAGE: The elite ZmCCT haplotypes which have no transposable element in the promoter could enhance maize resistance to Gibberella stalk rot and improve yield-related traits, while having no or mild impact on flowering time. Therefore, they are expected to have great value in future maize breeding programs. A CCT domain-containing gene, ZmCCT, is involved in both photoperiod response and stalk rot resistance in maize. At least 15 haplotypes are present at the ZmCCT locus in maize germplasm, whereas only three of them are found in Chinese commercial maize hybrids. Here, we evaluated ZmCCT haplotypes for their potential application in corn breeding. Nine resistant ZmCCT haplotypes that have no CACTA-like transposable element in the promoter were introduced into seven elite maize inbred lines by marker-assisted backcrossing. The resultant 63 converted lines had 0.7-5.1 Mb of resistant ZmCCT donor segments with over 90% recovery rates. All converted lines tested exhibited enhanced resistance to maize stalk rot but varied in photoperiod sensitivity. There was a close correlation between the hybrids and their parental lines with respect to both resistance performance and photoperiod sensitivity. Furthermore, in a given hybrid A5302/83B28, resistant ZmCCT haplotype could largely improve yield-related traits, such as ear length and 100-kernel weight, resulting in enhanced grain yield. Of nine resistant ZmCCT haplotypes, haplotype H5 exhibited excellent performance for both flowering time and stalk rot resistance and is thus expected to have potential value in future maize breeding programs.

Genetic regulation of maize flower development and sex determination.

MAIN CONCLUSION: The determining process of pistil fate are central to maize sex determination, mainly regulated by a genetic network in which the sex-determining genes SILKLESS 1 , TASSEL SEED 1 , TASSEL SEED 2 and the paramutagenic locus Required to maintain repression 6 play pivotal roles. Maize silks, which emerge from the ear shoot and derived from the pistil, are the functional stigmas of female flowers and play a pivotal role in pollination. Previous studies on sex-related mutants have revealed that sex-determining genes and phytohormones play an important role in the regulation of flower organogenesis. The processes determining pistil fate are central to flower development, where a silk identified gene SILKLESS 1 (SK1) is required to protect pistil primordia from a cell death signal produced by two commonly known genes, TASSEL SEED 1 (TS1) and TASSEL SEED 2 (TS2). In this review, maize flower developmental process is presented together with a focus on important sex-determining mutants and hormonal signaling affecting pistil development. The role of sex-determining genes, microRNAs, phytohormones, and the paramutagenic locus Required to maintain repression 6 (Rmr6), in forming a regulatory network that determines pistil fate, is discussed. Cloning SK1 and clarifying its function were crucial in understanding the regulation network of sex determination. The signaling mechanisms of phytohormones in sex determination are also an important research focus.

[Functional haplotypes of INCENP affect promoter activity and bovine semen quality].

To explore the association between single nucleotide polymorphisms (SNPs) in the promoter region of the inner centromere protein (INCENP) gene and bovine semen quality, the haplotypes in 250 Chinese Holstein bulls were detected using PCR-RFLP method in this study. Two SNPs (g.-556 G>T, rs 136823901 and g.-692 C>T, rs 211010999) and three haplotypes (CG, TT, TG) were identified in the promoter region of INCENP. The genotype frequency and allele frequency of these two SNPs as well as the correlation between different SNP haplotype combinations and bovine semen quality were then analyzed. Our results showed that fresh sperm motility of the GT genotype was significantly higher than that of the GG genotype (P<0.05) at the SNP site g.-556 G>T, while fresh and frozen-thawed sperm motilities of the haplotype combinations H1H1(CCGG), H1H3(CTGT), H2H3(TTGT) and H3H3(TTTT) were significantly higher than that of H1H2 (P<0.05). To further study the possible mechanisms by which g.-556 G>T and g.-692 C>T affect semen quality, three haplotype plasmids were respectively transfected into MLTC-1 cells. The TG haplotype demonstrated the highest luciferase activity, suggesting that g.-556 G>T and g.-692 C>T are functional mutations which could regulate INCENP gene expression by affecting promoter activity and thus affect semen quality.

Identification and fine-mapping of a QTL, qMrdd1, that confers recessive resistance to maize rough dwarf disease.

BACKGROUND: Maize rough dwarf disease (MRDD) is a devastating viral disease that results in considerable yield losses worldwide. Three major strains of virus cause MRDD, including maize rough dwarf virus in Europe, Mal de Río Cuarto virus in South America, and rice black-streaked dwarf virus in East Asia. These viral pathogens belong to the genus fijivirus in the family Reoviridae. Resistance against MRDD is a complex trait that involves a number of quantitative trait loci (QTL). The primary approach used to minimize yield losses from these viruses is to breed and deploy resistant maize hybrids. RESULTS: Of the 50 heterogeneous inbred families (HIFs), 24 showed consistent responses to MRDD across different years and locations, in which 9 were resistant and 15 were susceptible. We performed trait-marker association analysis on the 24 HIFs and found six chromosomal regions which were putatively associated with MRDD resistance. We then conducted QTL analysis and detected a major resistance QTL, qMrdd1, on chromosome 8. By applying recombinant-derived progeny testing to self-pollinated backcrossed families, we fine-mapped the qMrdd1 locus into a 1.2-Mb region flanked by markers M103-4 and M105-3. The qMrdd1 locus acted in a recessive manner to reduce the disease-severity index (DSI) by 24.2-39.3%. The genetic effect of qMrdd1 was validated using another F6 recombinant inbred line (RIL) population in which MRDD resistance was segregating and two genotypes at the qMrdd1 locus differed significantly in DSI values. CONCLUSIONS: The qMrdd1 locus is a major resistance QTL, acting in a recessive manner to increase maize resistance to MRDD. We mapped qMrdd1 to a 1.2-Mb region, which will enable the introgression of qMrdd1-based resistance into elite maize hybrids and reduce MRDD-related crop losses.

Genetic structure of rice black-streaked dwarf virus populations in China.

Rice black-streaked dwarf virus (RBSDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Fijivirus in the family Reoviridae. The genome of RBSDV consists of ten dsRNA segments. Although RBSDV has caused significant economic losses to rice and maize production in the past few years in China, its molecular diversity and evolution remain largely unknown. To elucidate the factor(s) underlying the evolution of RBSDV, we determined segment 8 (S8; carrying ORF8 encoding the minor core capsid protein) sequences of 101 samples and segment 10 (S10; carrying ORF10 encoding the major capsid protein) sequences of 103 samples. The results show that both ORF8 and ORF10 are under negative selection. The S8 of three isolates and S10 of two isolates are recombinants. The RBSDV population in China can be classified into three groups according to S8 sequences or into two groups according to S10 sequences, irrespective of host or geographical origin. Of the RBSDV isolates with both S8 and S10 sequences available, 17 are between-group reassortants and 30 are between-subgroup reassortants. The RBSDV subpopulations from different geographical regions and hosts show frequent gene flow within or between subpopulations. The RBSDV population from maize is in a state of expansion. In this study, no new emergent population was detected. Taken together, the results indicate that, in addition to recombination and negative selection, reassortment and gene flow are important factors that drive evolution of RBSDV in China.

Characterization and fine mapping of a necrotic leaf mutant in maize (Zea mays L.).

Maize (Zea mays L.) is a commercially important crop. Its yield can be reduced by mutations in biosynthetic and degradative pathways that cause death. In this paper, we describe the necrotic leaf (nec-t) mutant, which was obtained from an inbred line, 81647. The nec-t mutant plants had yellow leaves with necrotic spots, reduced chlorophyll content, and the etiolated seedlings died under normal growth conditions. Transmission electron microscopy revealed scattered thylakoids, and reduced numbers of grana lamellae and chloroplasts per cell. Histochemical staining suggested that spot formation of nec-t leaves might be due to cell death. Genetic analysis showed that necrosis was caused by the mutation of a recessive locus. Using simple sequence repeat markers, the Nec-t gene was mapped between mmc0111 and bnlg2277 on the short arm of chromosome 2. A total of 1287 individuals with the mutant phenotype from a F2 population were used for physical mapping. The Nec-t gene was located between markers T31 and H8 within a physical region of 131.7 kb.

Molecular characterization of segments S7 to S10 of a southern rice black-streaked dwarf virus isolate from maize in northern China.

Southern rice black-streaked dwarf virus (SRBSDV) is a novel Fijivirus prevalent in rice in southern and central China, and northern Vietnam. Its genome has 10 segments of double-stranded RNA named S1 to S10 according to their size. An isolate of SRBSDV, JNi4, was obtained from naturally infected maize plants from Ji'ning, Shandong province, in the 2008 maize season. Segments S7 to S10 of JNi4 share nucleotide identities of 72.6%-73.1%, 72.3%-73%, 73.9%-74.5% and 77.3%-79%, respectively, with corresponding segments of Rice black-streaked dwarf virus isolates, and identities of 99.7%, 99.1%-99.7%, 98.9%-99.5%, and 98.6%-99.2% with those of SRBSDV isolates HN and GD. JNi4 forms a separate branch with GD and HN in the phylogenetic trees constructed with genomic sequences of S7 to S10. These results confirm the proposed taxonomic status of SRBSDV as a distinct species of the genus Fijivirus and indicate that JNi4 is an isolate of SRBSDV. Shandong is so far the northernmost region where SRBSDV is found in China.