Fazhan Qiu

Identification of Major QTL for Waterlogging Tolerance Using Genome-Wide Association and Linkage Mapping of Maize Seedlings

To investigate the genetic basis of maize seedling response to waterlogging, we performed a genome-wide association study in 144 maize inbred lines, measuring length, fresh and dry weight of roots and shoots under normal and waterlogged conditions using 45,868 SNPs. This panel was divided into three subgroups based on the population structure results and the LD decay distance was 180xa0kb. A biparental advanced backcross (AB) population was also used to detect quantitative trait loci (QTL). In a comparison of 16 different models, principal components analysis (PCA/top PC3)u2009+u2009K was found to be best for reduction of false-positive associations for further analysis. A whole-genome scan detected four strong peak signals (Pxa0<u20092.18u2009×u200910−5) significantly associated with the waterlogging response on chromosomes 5, 6 and 9. SNP4784, SNP200, SNP298, and SNP6314 showed significant association with corresponding traits under waterlogging and explained 14.99–19.36xa0%, 15.75–17.64xa0%, 16.08xa0% and 15.44xa0% of the phenotypic variation, respectively. The identified SNPs were located in GRMZM2G012046, GRMZM2G009808, GRMZM2G137108 and GRMZM2G369629 (AGPV1). SNP4784 (GRMZM2G012046) was colocalized with the major QTL that was identified with the same traits in the AB population. Forty-seven SNPs significantly associated (Pu2009<u20092.18u2009×u200910−4) with six traits in association mapping were identified and, among these, 33 SNPs were already reported in literature as waterlogging-related traits. These results will help elucidate the genetic basis of differential responses and tolerance to waterlogging stress among maize inbred lines, and provide novel loci for improvement of waterlogging tolerance of maize inbred lines using marker-assisted selection.

Comparative proteomic analysis revealing the complex network associated with waterlogging stress in maize (Zea mays L.) seedling root cells.

Soil waterlogging is one of the major abiotic stresses affecting maize grain yields. To understand the molecular mechanisms underlying waterlogging tolerance in maize, the iTRAQ LC‐MS/MS technique was employed to map the proteomes of seedling root cells of the A3237 (tolerant inbred) and A3239 (sensitive inbred) lines under control and waterlogging conditions. Among the 3318 proteins identified, 211 were differentially abundant proteins (DAPs), of which 81 were specific to A3237 and 57 were specific to A3239. These DAPs were categorized into 11 groups that were closely related to the plant stress response, including metabolism, energy, transport, and disease/defense. In the waterlogged A3237 root cells, NADP‐malic enzyme, glutamate decarboxylase, coproporphyrinogen III oxidase, GSH S‐transferase, GSH dehydrogenase, and xyloglucan endotransglycosylase 6 were specifically accumulated to manage energy consumption, maintain pH levels, and minimize oxidative damage. The evaluations of five specific physiological parameters (alcohol dehydrogenase activity and GSH, malondialdehyde, adenosine 5‐triphosphate, and nicotinamide adenine dinucleotide concentrations) were in agreement with the proteomic results. Moreover, based on the proteomic assay, eight representative genes encoding DAPs were selected for validation at the transcriptional level. qRT‐PCR revealed that the expression levels of these genes correlated with their observed protein abundance. These findings shed light on the complex mechanisms underlying waterlogging tolerance in maize. All MS data have been deposited into the ProteomeXchange with the identifier PXD001125 http://proteomecentral.proteomexchange.org/dataset/PXD001125.

Genome-Wide Identification, Evolution and Expression Analysis of mTERF Gene Family in Maize

Plant mitochondrial transcription termination factor (mTERF) genes comprise a large family with important roles in regulating organelle gene expression. In this study, a comprehensive database search yielded 31 potential mTERF genes in maize (Zea mays L.) and most of them were targeted to mitochondria or chloroplasts. Maize mTERF were divided into nine main groups based on phylogenetic analysis, and group IX represented the mitochondria and species-specific clade that diverged from other groups. Tandem and segmental duplication both contributed to the expansion of the mTERF gene family in the maize genome. Comprehensive expression analysis of these genes, using microarray data and RNA-seq data, revealed that these genes exhibit a variety of expression patterns. Environmental stimulus experiments revealed differential up or down-regulation expression of maize mTERF genes in seedlings exposed to light/dark, salts and plant hormones, respectively, suggesting various important roles of maize mTERF genes in light acclimation and stress-related responses. These results will be useful for elucidating the roles of mTERF genes in the growth, development and stress response of maize.

Genetic architecture of maize kernel row number and whole genome prediction

Key messageMaize kernel row number might be dominated by a set of large additive or partially dominant loci and several small dominant loci and can be accurately predicted by fewer than 300 top KRN-associated SNPs.AbstractKernel row number (KRN) is an important yield component in maize and directly affects grain yield. In this study, we combined linkage and association mapping to uncover the genetic architecture of maize KRN and to evaluate the phenotypic predictability using these detected loci. A genome-wide association study revealed 31 associated single nucleotide polymorphisms (SNPs) representing 17 genomic loci with an effect in at least one of five individual environments and the best linear unbiased prediction (BLUP) over all environments. Linkage mapping in three F2:3 populations identified 33 KRN quantitative trait loci (QTLs) representing 21 QTLs common to several population/environments. The majority of these common QTLs that displayed a large effect were additive or partially dominant. We found 70xa0% KRN-associated genomic loci were mapped in KRN QTLs identified in this study, KRN-associated SNP hotspots detected in NAM population and/or previous identified KRN QTL hotspots. Furthermore, the KRN of inbred lines and hybrids could be predicted by the additive effect of the SNPs, which was estimated using inbred lines as a training set. The prediction accuracy using the top KRN-associated tag SNPs was obviously higher than that of the randomly selected SNPs, and approximately 300 top KRN-associated tag SNPs were sufficient for predicting the KRN of the inbred lines and hybrids. The results suggest that the KRN-associated loci and QTLs that were detected in this study show great potential for improving the KRN with genomic selection in maize breeding.

Emp10 encodes a mitochondrial PPR protein that affects the cis-splicing of nad2 intron 1 and seed development in maize

In higher plants, many mitochondrial genes contain group II-type introns that are removed from RNAs by splicing to produce mature transcripts that are then translated into functional proteins. However, the factors involved in the splicing of mitochondrial introns and their biological functions are not well understood in maize. Here, we isolated an empty pericarp 10 (emp10) mutant and identified the underlying gene by map-based cloning. Emp10 encodes a P-type mitochondria-targeted pentatricopeptide repeat (PPR) protein with 10 PPR motifs. Loss of Emp10 function results in splicing defect of the first intron of nad2, a gene encoding subunit 2 of NADH dehydrogenase (also called complex I). The emp10 mutant has undetectable activity of complex I and has arrested development of embryo and endosperm, and thus defective seeds with empty pericarp. Additionally, the basal endosperm transfer layer cells were severely affected, indicating the deficiency of cell wall ingrowths in the emp10 kernels. Moreover, the alternative respiratory pathway involving alternative oxidase was significantly induced in the emp10 mutant. These results suggest that EMP10 is specifically required for the cis-splicing of mitochondrial nad2 intron 1, embryogenesis and endosperm development in maize.

Genome-wide analysis of maize cytoplasmic male sterility-S based on QTL mapping

Three types of sterile cytoplasm in cytoplasmic-male-sterility (CMS) maize, T, C and S, can be identified according to their fertility-restoration and mitochondrial DNA RFLP patterns. CMS-S, which is the least stable among the three types of CMS, is controlled by sterile cytoplasm interactions with certain nuclear-encoded factors. We constructed a high-resolution map of loci associated with male-restoration of CMS-S in BC1 populations of maize. The map covers 1730.29 cM, including 32 RFLP, 51 SSR 62 RAPD and 21 AFLP markers. Genome-wide QTL analysis detected 6 QTLs with significant effects on male fertility as assessed by their starch-filled pollen ratios. Four QTLs out of six were located between the SSR markers MSbnlg1633-Mrasg20, MSbnlg1662-Msume1126, MSume1230-MSumc1525, and RAPD marker MraopQ07-2-MraopK06-2 on chromosome 2. Two other minor loci were mapped between MraopK16-1-Mraopi4-1, on chromosome 9, and between Msuncbnlg1139-MraopR10-2, on chromosome 6. The Rf3 nuclear restoring gene was precisely located on the chromosome 2, 2.29 cM to the left of umc1525 and 8.9 cM to the right of umc1230. The results provide important markers for marker-assisted selection of stable CMS-S maize.

EMPTY PERICARP11 serves as a factor for splicing of mitochondrial nad1 intron and is required to ensure proper seed development in maize

A new pentatricopeptide repeat (PPR) gene in maize differs from previously reported genes, as it is required for splicing of all four introns of nad1.

Transgenic maize lines expressing a cry1C* gene are resistant to insect pests

The lepidopteran Ostrinia furnacalis is one of the most serious pests of maize production. The Cry1C proteins are group of Bacillus thuringiensis (Bt) proteins that are toxic to the intestine of insects. Overexpression of Cry1C protein has led to increased resistance to lepidopteran pests in several crops. In the present study, the synthetic cry1C* gene that was previously tested in rice was introduced into maize Hi-II genotypes via biolistic gun-mediated transformation. A total of nine independent putative callus were obtained and 87 transgenic plants were positive with cry1C* according to polymerase chain reaction (PCR) analysis. Three highly insect-resistant transgenic plants, ZmKc-2-3, ZmKc-3-2, and ZmKc-3-5, were further confirmed by PCR analysis, field assessment, and genomic southern blotting in the T3 generations. Insect bioassays were conducted in both the field and the laboratory, and showed that progeny of the three transgenic lines were significantly resistant to lepidopteran maize pests during the whole development and growth period. The stable integration and expression of the cry1C* in the three transgenic plants’ progeny were confirmed by reverse transcription-PCR (RT-PCR) and enzyme-linked immuno sorbent assay (ELISA) methods. Hybrids were produced by crossing transgenic line ZmKc-2-3 with the elite inbred line Zheng58. There was small variation among the hybrids and backcross offspring, indicating that these cry1C* transgenic lines can be used to produce insect-resistant hybrids and served as insect-resistant sources for the development of Bt maize.

Functional divergence and origin of the DAG-like gene family in plants

The nuclear-encoded DAG-like (DAL) gene family plays critical roles in organelle C-to-U RNA editing in Arabidopsis thaliana. However, the origin, diversification and functional divergence of DAL genes remain unclear. Here, we analyzed the genomes of diverse plant species and found that: DAL genes are specific to spermatophytes, all DAL genes share a conserved gene structure and protein similarity with the inhibitor I9 domain of subtilisin genes found in ferns and mosses, suggesting that DAL genes likely arose from I9-containing proproteases via exon shuffling. Based on phylogenetic inference, DAL genes can be divided into five subfamilies, each composed of putatively orthologous and paralogous genes from different species, suggesting that all DAL genes originated from a common ancestor in early seed plants. Significant type I functional divergence was observed in 6 of 10 pairwise comparisons, indicating that shifting functional constraints have contributed to the evolution of DAL genes. This inference is supported by the finding that functionally divergent amino acids between subfamilies are predominantly located in the DAL domain, a critical part of the RNA editosome. Overall, these findings shed light on the origin of DAL genes in spermatophytes and outline functionally important residues involved in the complexity of the RNA editosome.

Dissecting the genetic architecture of waterlogging stress-related traits uncovers a key waterlogging tolerance gene in maize

Key messageA key candidate gene, GRMZM2G110141, which could be used in marker-assisted selection in maize breeding programs, was detected among the 16 genetic loci associated with waterlogging tolerance identified through genome-wide association study.AbstractWaterlogging stress seriously affects the growth and development of upland crops such as maize (Zea mays L.). However, the genetic basis of waterlogging tolerance in crop plants is largely unknown. Here, we identified genetic loci for waterlogging tolerance-related traits by conducting a genome-wide association study using maize phenotypes evaluated in the greenhouse under waterlogging stress and normal conditions. A total of 110 trait-single nucleotide polymorphism associations spanning 16 genomic regions were identified; single associations explained 2.88–10.67% of the phenotypic variance. Among the genomic regions identified, 14 co-localized with previously detected waterlogging tolerance-related quantitative trail loci. Furthermore, 33 candidate genes involved in a wide range of stress-response pathways were predicted. We resequenced a key candidate gene (GRMZM2G110141) in 138 randomly selected inbred lines and found that variations in the 5ʹ-UTR and in the mRNA abundance of this gene under waterlogging conditions were significantly associated with leaf injury. Furthermore, we detected favorable alleles of this gene and validated the favorable alleles in two different recombinant inbred line populations. These alleles enhanced waterlogging tolerance in segregating populations, strongly suggesting that GRMZM2G110141 is a key waterlogging tolerance gene. The set of waterlogging tolerance-related genomic regions and associated markers identified here could be valuable for isolating waterlogging tolerance genes and improving this trait in maize.