Genomic analysis of a new heterotic maize group reveals key loci for pedigree breeding.

Genome-wide analyses of maize populations have clarified the genetic basis of crop domestication and improvement. However, limited information is available on how breeding improvement reshaped the genome in the process of the formation of heterotic groups. In this study, we identified a new heterotic group (X group) based on an examination of 512 Chinese maize inbred lines. The X group was clearly distinct from the other non-H&L groups, implying that X × HIL is a new heterotic pattern. We selected the core inbred lines for an analysis of yield-related traits. Almost all yield-related traits were better in the X lines than those in the parental lines, indicating that the primary genetic improvement in the X group during breeding was yield-related traits. We generated whole-genome sequences of these lines with an average coverage of 17.35× to explore genome changes further. We analyzed the identity-by-descent (IBD) segments transferred from the two parents to the X lines and identified 29 and 28 IBD conserved regions (ICRs) from the parents PH4CV and PH6WC, respectively, accounting for 28.8% and 12.8% of the genome. We also identified 103, 89, and 131 selective sweeps (SSWs) using methods that involved the π, Tajima's D, and CLR values, respectively. Notably, 96.13% of the ICRs co-localized with SSWs, indicating that SSW signals concentrated in ICRs. We identified 171 annotated genes associated with yield-related traits in maize both in ICRs and SSWs. To identify the genetic factors associated with yield improvement, we conducted QTL mapping for 240 lines from a DH population (PH4CV × PH6WC, which are the parents of X1132X) for ten key yield-related traits and identified a total of 55 QTLs. Furthermore, we detected three QTL clusters both in ICRs and SSWs. Based on the genetic evidence, we finally identified three key genes contributing to yield improvement in breeding the X group. These findings reveal key loci and genes targeted during pedigree breeding and provide new insights for future genomic breeding.

ORF355 confers enhanced salinity stress adaptability to S-type cytoplasmic male sterility maize by modulating the mitochondrial metabolic homeostasis.

Moderate stimuli in mitochondria improve wide-ranging stress adaptability in animals, but whether mitochondria play similar roles in plants is largely unknown. Here, we report the enhanced stress adaptability of S-type cytoplasmic male sterility (CMS-S) maize and its association with mild expression of sterilizing gene ORF355. A CMS-S maize line exhibited superior growth potential and higher yield than those of the near-isogenic N-type line in saline fields. Moderate expression of ORF355 induced the accumulation of reactive oxygen species and activated the cellular antioxidative defense system. This adaptive response was mediated by elevation of the nicotinamide adenine dinucleotide concentration and associated metabolic homeostasis. Metabolome analysis revealed broad metabolic changes in CMS-S lines, even in the absence of salinity stress. Metabolic products associated with amino acid metabolism and galactose metabolism were substantially changed, which underpinned the alteration of the antioxidative defense system in CMS-S plants. The results reveal the ORF355-mediated superior stress adaptability in CMS-S maize and might provide an important route to developing salt-tolerant maize varieties.

Comparative analysis of mitochondrial genomes of maize CMS-S subtypes provides new insights into male sterility stability.

BACKGROUND: Cytoplasmic male sterility (CMS) is a trait of economic importance in the production of hybrid seeds. In CMS-S maize, exerted anthers appear frequently in florets of field-grown female populations where only complete male-sterile plants were expected. It has been reported that these reversions are associated with the loss of sterility-conferring regions or other rearrangements in the mitochondrial genome. However, the relationship between mitochondrial function and sterility stability is largely unknown. RESULTS: In this study, we determined the ratio of plants carrying exerted anthers in the population of two CMS-S subtypes. The subtype with a high ratio of exerted anthers was designated as CMS-Sa, and the other with low ratio was designated as CMS-Sb. Through next-generation sequencing, we assembled and compared mitochondrial genomes of two CMS-S subtypes. Phylogenetic analyses revealed strong similarities between the two mitochondrial genomes. The sterility-associated regions, S plasmids, and terminal inverted repeats (TIRs) were intact in both genomes. The two subtypes maintained high transcript levels of the sterility gene orf355 in anther tissue. Most of the functional genes/proteins were identical at the nucleotide sequence and amino acid sequence levels in the two subtypes, except for NADH dehydrogenase subunit 1 (nad1). In the mitochondrial genome of CMS-Sb, a 3.3-kilobase sequence containing nad1-exon1 was absent from the second copy of the 17-kb repeat region. Consequently, we detected two copies of nad1-exon1 in CMS-Sa, but only one copy in CMS-Sb. During pollen development, nad1 transcription and mitochondrial biogenesis were induced in anthers of CMS-Sa, but not in those of CMS-Sb. We suggest that the impaired mitochondrial function in the anthers of CMS-Sb is associated with its more stable sterility. CONCLUSIONS: Comprehensive analyses revealed diversity in terms of the copy number of the mitochondrial gene nad1-exon1 between two subtypes of CMS-S maize. This difference in copy number affected the transcript levels of nad1 and mitochondrial biogenesis in anther tissue, and affected the reversion rate of CMS-S maize. The results of this study suggest the involvement of mitochondrial robustness in modulation of sterility stability in CMS-S maize.

RppM, Encoding a Typical CC-NBS-LRR Protein, Confers Resistance to Southern Corn Rust in Maize.

Southern corn rust (SCR) caused by Puccinia polysora Underw. poses a major threat to maize production worldwide. The utilization of host SCR-resistance genes and the cultivation of resistant cultivars are the most effective, economical strategies for controlling SCR. Here, we identified and cloned a new SCR resistance gene, RppM, from the elite maize inbred line Jing2416K. RppM was found to encode a typical CC-NBS-LRR protein localized in both the nucleus and cytoplasm. This gene was constitutively expressed at all developmental stages and in all tissues examined, with the strongest expression detected in leaves at the mature stage. A transcriptome analysis provided further evidence that multiple defense systems were initiated in Jing2416K, including pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity, reinforcement of cell walls, accumulation of antimicrobial compounds, and activation of phytohormone signaling pathways. Finally, we developed functional Kompetitive allele-specific PCR markers for RppM using two conserved SNP sites and successfully applied these functional markers for the detection of RppM and the cultivation of resistant maize cultivars, demonstrating their great potential utility in maize breeding.

Stalk architecture, cell wall composition, and QTL underlying high stalk flexibility for improved lodging resistance in maize.

BACKGROUND: Stalk fracture caused by strong wind can severely reduce yields in maize. Stalks with higher stiffness and flexibility will exhibit stronger lodging resistance. However, stalk flexibility is rarely studied in maize. Stalk fracture of the internode above the ear before tasseling will result in the lack of tassel and pollen, which is devastating for pollination in seed production. In this study, we focused on stalk lodging before tasseling in two maize inbred lines, JING724 and its improved line JING724A1 and their F2:3 population. RESULTS: JING724A1 showed a larger stalk fracture angle than JING724, indicating higher flexibility. In addition, compared to JING724, JING724A1 also had longer and thicker stalks, with a conical, frustum-shaped internode above the ear. Microscopy and X-ray microcomputed tomography of the internal stalk architecture revealed that JING724A1 had more vascular bundles and thicker sclerenchyma tissue. Furthermore, total soluble sugar content of JING724A1, especially the glucose component, was substantially higher than in JING724. Using an F2:3 population derived from a JING724 and JING724A1 cross, we performed bulk segregant analysis for stalk fracture angle and detected one QTL located on Chr3: 14.00-19.28 Mb. Through transcriptome data analysis and ∆ (SNP-index), we identified two candidate genes significantly associated with high stalk fracture angle, which encode a RING/U-box superfamily protein (Zm00001d039769) and a MADS-box transcription factor 54 (Zm00001d039913), respectively. Two KASP markers designed from these two candidate genes also showed significant correlations with stalk fracture angle. CONCLUSIONS: The internode shape and glucose content are possibly correlated with stalk flexibility in maize. Two genes in the detected QTL are potentially associated with stalk fracture angle. These novel phenotypes and associated loci will provide a theoretical foundation for understanding the genetic mechanisms of lodging, and facilitate the selection of maize varieties with improved flexibility and robust lodging resistance.

Identification and Fine Mapping of RppM, a Southern Corn Rust Resistance Gene in Maize.

Southern corn rust (SCR) caused by Puccinia polysora Underw. is a major disease causing severe yield losses during maize production. Here, we identified and mapped the SCR resistance gene RppM from the near-isogenic line Kangxiujing2416 (Jing2416K), which harbors RppM in the genetic background of the susceptible inbred line Jing2416. In this study, the inheritance of SCR resistance was investigated in F2 and F3 populations derived from a cross between Jing2416K and Jing2416. The observed 3:1 segregation ratio of resistant to susceptible plants indicated that the SCR resistance is controlled by a single dominant gene. Using an F2 population, we performed bulked segregant analysis (BSA) sequencing and mapped RppM to a 3.69-Mb region on chromosome arm 10S. To further narrow down the region harboring RppM, we developed 13 insertion/deletion (InDel) markers based on the sequencing data. Finally, RppM was mapped to a region spanning 110-kb using susceptible individuals from a large F2 population. Two genes (Zm00001d023265 and Zm00001d023267) encoding putative CC-NBS-LRR (coiled-coiled, nucleotide-binding site, and leucine-rich repeat) proteins, a common characteristic of R genes, were located in this region (B73 RefGen_v4 reference genome). Sequencing and comparison of the two genes cloned from Jing2416K and Jing2416 revealed sequence variations in their coding regions. The relative expression levels of these two genes in Jing2416K were found to be significantly higher than those in Jing2416. Zm00001d023265 and Zm00001d023267 are thus potential RppM candidates.

Activation of Mitochondrial orf355 Gene Expression by a Nuclear-Encoded DREB Transcription Factor Causes Cytoplasmic Male Sterility in Maize.

Coordination between mitochondria and the nucleus is crucial for fertility determination in plants with cytoplasmic male sterility (CMS). Using yeast one-hybrid screening, we identified a transcription factor, ZmDREB1.7, that is highly expressed in sterile microspores at the large vacuole stage and activates the expression of mitochondria-encoded CMS gene orf355. Δpro, a weak allele of ZmDREB1.7 with the loss of a key unfolded protein response (UPR) motif in the promoter, partially restores male fertility of CMS-S maize. ZmDREB1.7 expression increases rapidly in response to antimycin A treatment, but this response is attenuated in the Δpro allele. Furthermore, we found that expression of orf355 in mitochondria activates mitochondrial retrograde signaling, which in turn induces ZmDREB1.7 expression. Taken together, these findings demonstrate that positive-feedback transcriptional regulation between a nuclear regulator and a mitochondrial CMS gene determines male sterility in maize, providing new insights into nucleus-mitochondria communication in plants.

The HuangZaoSi Maize Genome Provides Insights into Genomic Variation and Improvement History of Maize.

Maize is a globally important crop that was a classic model plant for genetic studies. Here, we report a 2.2 Gb draft genome sequence of an elite maize line, HuangZaoSi (HZS). Hybrids bred from HZS-improved lines (HILs) are planted in more than 60% of maize fields in China. Proteome clustering of six completed sequenced maize genomes show that 638 proteins fall into 264 HZS-specific gene families with the majority of contributions from tandem duplication events. Resequencing and comparative analysis of 40 HZS-related lines reveals the breeding history of HILs. More than 60% of identified selective sweeps were clustered in identity-by-descent conserved regions, and yield-related genes/QTLs were enriched in HZS characteristic selected regions. Furthermore, we demonstrated that HZS-specific family genes were not uniformly distributed in the genome but enriched in improvement/function-related genomic regions. This study provides an important and novel resource for maize genome research and expands our knowledge on the breadth of genomic variation and improvement history of maize.

Identification of Genes Potentially Associated with the Fertility Instability of S-Type Cytoplasmic Male Sterility in Maize via Bulked Segregant RNA-Seq.

S-type cytoplasmic male sterility (CMS-S) is the largest group among the three major types of CMS in maize. CMS-S exhibits fertility instability as a partial fertility restoration in a specific nuclear genetic background, which impedes its commercial application in hybrid breeding programs. The fertility instability phenomenon of CMS-S is controlled by several minor quantitative trait locus (QTLs), but not the major nuclear fertility restorer (Rf3). However, the gene mapping of these minor QTLs and the molecular mechanism of the genetic modifications are still unclear. Using completely sterile and partially rescued plants of fertility instable line (FIL)-B, we performed bulk segregant RNA-Seq and identified six potential associated genes in minor effect QTLs contributing to fertility instability. Analyses demonstrate that these potential associated genes may be involved in biological processes, such as floral organ differentiation and development regulation, energy metabolism and carbohydrates biosynthesis, which results in a partial anther exsertion and pollen fertility restoration in the partially rescued plants. The single nucleotide polymorphisms (SNPs) identified in two potential associated genes were validated to be related to the fertility restoration phenotype by KASP marker assays. This novel knowledge contributes to the understanding of the molecular mechanism of the partial fertility restoration of CMS-S in maize and thus helps to guide the breeding programs.

The complete mitochondrial genome of Gossypium hirsutum and evolutionary analysis of higher plant mitochondrial genomes.

BACKGROUND: Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes. METHODOLOGY/PRINCIPAL FINDINGS: We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes. CONCLUSION: The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species.