Yonglian Zheng

Quantitative trait locus analysis of drought tolerance and yield in maize in China

Drought accounts for significant yield losses in crops. Maize (Zea mays L.) is particularly sensitive to water stress at reproductive stages, and breeding to improve drought tolerance has been a challenge. By use of a linkage map with 121 single sequence repeat (SSR) markers, quantitative trait loci (QTLs) for grain yield and yield components were characterized in the population of the cross X178×B73 under water-stressed and well-watered conditions. Under the well-watered regime, 2, 4, 4, 1, 2, 2, and 3 QTLs were identified for grain yield, 100-kernel weight, kernel number per ear, cob weight per ear, kernel weight per ear, ear weight, and ear number per plant, respectively, whereas under the water-stressed conditions, 1, 5, 2, 6, 1, 3, and 2 QTLs, respectively, were found. The significant phenotypic correlations among yield and yield components to some extent were observed under both water conditions, and some overlaps between the corresponding QTLs were also found. QTLs for grain yield and kernel weight per ear under well-watered conditions and ear weight under both well-watered and water-stressed conditions over-lapped, and all were located on chromosome 1.03 near marker bnlg176. Two other noticeable QTL regions were on chromosome 9.05 and 9.07 near markers umc1657 and bnlg1525; the first corresponded to grain yield, kernel weight per ear, and ear weight under well-watered conditions and kernel number per ear under both water conditions, and the second to grain yield and cob weight per ear under water-stressed conditions and ear number per plant under both water conditions. A comparative analysis of the QTLs herein identified with those described in previous studies for yield and yield components in different maize populations revealed a number of QTLs in common. These QTLs have potential use in molecular marker-assisted selection.

Identification of transcriptome induced in roots of maize seedlings at the late stage of waterlogging.

BackgroundPlants respond to low oxygen stress, particularly that caused by waterlogging, by altering transcription and translation. Previous studies have mostly focused on revealing the mechanism of the response at the early stage, and there is limited information about the transcriptional profile of genes in maize roots at the late stage of waterlogging. The genetic basis of waterlogging tolerance is largely unknown. In this study, the transcriptome at the late stage of waterlogging was assayed in root cells of the tolerant inbred line HZ32, using suppression subtractive hybridization (SSH). A forward SSH library using RNA populations from four time points (12 h, 16 h, 20 h and 24 h) after waterlogging treatment was constructed to reveal up-regulated genes, and transcriptional and linkage data was integrated to identify candidate genes for waterlogging tolerance.ResultsReverse Northern analysis of a set of 768 cDNA clones from the SSH library revealed a large number of genes were up-regulated by waterlogging. A total of 465 ESTs were assembled into 296 unigenes. Bioinformatic analysis revealed that the genes were involved in complex pathways, such as signal transduction, protein degradation, ion transport, carbon and amino acid metabolism, and transcriptional and translational regulation, and might play important roles at the late stage of the response to waterlogging. A significant number of unigenes were of unknown function. Approximately 67% of the unigenes could be aligned on the maize genome and 63 of them were co-located within reported QTLs.ConclusionThe late response to waterlogging in maize roots involves a broad spectrum of genes, which are mainly associated with two response processes: defense at the early stage and adaption at the late stage. Signal transduction plays a key role in activating genes related to the tolerance mechanism for survival during prolonged waterlogging. The crosstalk between carbon and amino acid metabolism reveals that amino acid metabolism performs two main roles at the late stage: the regulation of cytoplasmic pH and energy supply through breakdown of the carbon skeleton.

Changes of Antioxidative Enzymes and Lipid Peroxidation in Leaves and Roots of Waterlogging-Tolerant and Waterlogging-Sensitive Maize Genotypes at Seedling Stage

Abstract To better understand the physiological and biochemical mechanisms of waterlogging tolerance, waterlogging effects on lipid peroxidation and the activity of antioxidative enzymes were investigated in leaves and roots of two maize genotypes, HZ32 (waterlogging-tolerant) and K12 (waterlogging-sensitive). Potted maize plants were waterlogged at the second leaf stage under glasshouse conditions. Leaves and roots were harvested 1 d before and 2, 4, 6, 8 and 10 d after the start of waterlogging treatment. Through comparing the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT) and guaiacol peroxidase (POD) between waterlogging-tolerant and waterlogging-sensitive genotype, we deduced that CAT was the most important H 2 O 2 scavenging enzyme in leaves, while APX seemed to play a key role in roots. POD, APX, GR and CAT activities in conjunction with SOD seem to play an essential protective role in the O 2 T and H 2 O 2 scavenging process. Lipid peroxidation was enhanced significantly only in K12 ( P P > 0.05) in HZ32 up to 6 d after waterlogging stress. These results indicated that oxidative stress may play an important role in waterlogging-stressed maize plants and that the greater protection of HZ32 leaves and roots from waterlogging-induced oxidative damage results, at least in part, through the maintenance of increased antioxidant enzyme activity.

Screening Methods for Waterlogging Tolerance at Maize (Zea mays L.) Seedling Stage

Waterlogging strongly affects agronomic performance of maize (Zea mays L.). In order to investigate the suitable selection criteria of waterflooding tolerant genotypes, and identify the most susceptible stage and the best continuous treatment time to waterlogging, 20 common maize inbred lines were subjected to successive artificial waterflooding at seedling stage, and waterlogging tolerance coefficient (WTC) was used to screen waterflooding tolerant genotypes. In addition, peroxidase (POD) activities and malondialdehyde (MDA) contents were measured for 6 of 20 lines. The results showed that the second leaf stage (V2) was the most susceptible stage, and 6 d after waterflooding was the best continuous treatment time. Dry weight (DW) of both shoots and roots of all lines were significantly reduced at 6 d time-point of waterlogging, compared to control. POD activities and MDA contents were negatively and significantly correlated, and the correlation coefficient was -0.9686 (P＜0.0001). According to the results, WTC of shoot DW can be used for practical screening as a suitable index, which is significantly different from control and waterlogged plants happened 6 d earlier. Furthermore, leaf chlorosis, MDA content and POD activities could also be used as reference index for material screening. The implications of the results for waterlogging-tolerant material screening and waterlogging-tolerant breeding have been discussed in maize.

Functional genomics of maize submergence tolerance and cloning of the related gene Sicyp51

In this study, SSH (Suppression Subtractive Hybridization) and cDNA microarray were used to identify genes associated with waterlogging response of maize roots. Mo17 and Hz32 are two maize inbred lines with differential tolerance to hypoxia. Seedlings of the inbred lines with two leaves were submerged in hypoxia buffer. SSH libraries were constructed with cDNA samples from roots. Both forward and reverse subtractions were performed for each inbred line, and 105 positive clones induced by hypoxia were selected by differential screening. The treated and control message RNA were hybridized with the cDNA microarray of Mo17, sequentially, 57 of 3-fold differentially expressed clones were obtained. A total of 162 positive clones were all sequenced. Bioinformatics analysis showed these positive clones represent 85 TUGs, including genes involved in several biochemistry pathways, such as glycolysis, protection, signal transduction, cell construction and energy metabolism and 41 EST with unknown function. Comparison between Mo17 and Hz32 indicates that genes related to hypoxia tolerance have different expression patterns in submerged roots. Several positive clones’ expression patterns were revealed by Northern or RT-PCR, and a new gene (Sicyp51), which may contribute to hypoxia tolerance, was identified.

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.

Identification of Candidate Genes Associated with Fertility Restoration in Maize S Cytoplasmic Male Sterility

In CMS-S of maize (Zea mays L.), mitochondrial orf355-orf77 and the nuclear restorer of fertility designated as Rf3/rf3 interact to control fertility of gametophytes. Genes expressed differentially in young leaves and pollen were screened using complementary DNA amplified fragment length polymorphism (cDNA-AFLP) combined with a set of Rf3/rf3 near isogenic lines. Sixty-four combinations of AFLP primers produced 2,450 observed transcript-derived fragments (TDFs). The differentially expressed TDFs were assorted into seven different expressed types, of which, 33 TDFs expressed only in fertile pollen and three expressed in fertile materials specifically were cloned and sequenced. Sequencing analysis indicated that 33 tentative unique genes were obtained and assigned into eight different categories including protein synthesis and fate, post-transcript regulation, signal transduction, and so on. TDF-E1 was indicated representing a gene coding a protein containing pentatricopeptide repeat (PPR) motif by BLASTx analysis, which temporarily denominated as PPRE1. Real-time polymerase chain reaction analysis revealed that PPRE1 expressed far more strongly in pollen of S-Mo17Rf3Rf3 than in other materials investigated. Polymorphism analysis of PPRE1 in genomes showed difference between S-Mo17Rf3Rf3 and S-Mo17rf3rf3. Taken together, we proposed that PPRE1 may be a promising candidate gene for restoration of fertility in maize S-CMS, as a set of near-isogenic lines had identical cytoplasm and eliminated most noise from nuclear genetic backgrounds. Another gene, which corresponds to TDF-A5 and codes 26S protease regulatory particle non-ATPase subunit5 was also discussed about its potential role in participating in the repression of mitochondrial programmed cell death associated with sterile pollen in S-type cytoplasm.

Fertility restoration mechanisms in S-type cytoplasmic male sterility of maize (Zea mays L.) revealed through expression differences identified by cDNA microarray and suppression subtractive hybridization

Cytoplasmic male sterility (CMS) is thought to be due to an incompatibility of 2 genomes that results in pollen abortion. In CMS-S of maize (Zea mays L.), mitochondrialorf355-orf77 and the nuclear restorer of fertility interact to control fertility of gametophytes. Numerous studies demonstrated thatRf3 can regulate nuclear and mitochondrial gene expression and shows pleiotropic effects on the transcriptional level. Little is known, however, about the alteration of the global expression profile caused byRf3 substitution ofrf3 under S cytoplasm or about molecular fertility restoration mechanisms. In this study, cDNA microarray and suppression subtractive hybridization were used to reveal differentially expressed genes during pollen development by comparing a set ofRf3/rf3 near isogenic lines. A total of 137 tentative unique genes (TUGs) were identified at the transcriptional level. On the basis of functional category analysis, these TUGs were involved in a broad range of cellular and biochemical activities, including metabolism, cell structure, cell defense, and apoptosis, as well as signal transduction pathways. Northern blot analysis using 5 representative clones as probes confirmed differential expression among S-(Rf3) and S-(rf3). Especially in S-(Rf3), the expression patterns of genes associated with electron or H+ conduction and antiapoptosis genes (e.g., VADC2, BI-1, cystatin, 14-3-3) are distinctly different from in S-(rf3). Together with normalization of cellular and biochemical activities in S-(Rf3), we proposed thatRf3 might regulate accumulation of nuclear and mitochondrial gene transcripts directly or indirectly to inhibit multiple programmed cell death pathways in S-type cytoplasm allowing the normal developmental pathways to unfold.

Characterization of Zm26Sub5 and Its Potential Roles in Regulating Pollen Fertility of S-CMS in Maize

Mitochondrial orf355-orf77 and the nuclear fertility restorer locus Rf3/rf3 mutually control the fertility of male gametes in CMS-S in maize (Zea mays L.). A fragment of gene Zm26Sub5 was identified through cDNA-AFLP from a set of Rf3/rf3 near-isogenic lines in a previous study. In the present study, real-time PCR analysis revealed that Zm26Sub5 expression levels were much higher in pollen of S-Mo17Rf3Rf3 than in pollen of S-Mo17rf3rf3, and also higher than in fresh leaves, mature leaves, and roots of both S-Mo17Rf3Rf3 and S-Mo17rf3rf3. In silico cloning of full-length cDNA was successfully implemented. Gene Zm26Sub5 was 1 451 bp in size, of which 1 329 bp encoded a protein with 443 amino acids. The structure of this gene was analyzed by comparing its full length cDNA to homologous genomic DNA sequence (GenBank accession: Ac209463.3). Subsequent sequence analysis led to sub cellular localization of protein ZM26SUB5, and construction of a phylogenetic tree. In silico mapping indicated that Zm26Sub5 was located on chromosome 5 and closed to a reported starch-filled pollen ratio QTL. ZM26SUB5, therefore might have potential roles in repressing mitochondrial PCD which is associated with sterile activity in pollen in S-type cytoplasm.