Search

By hybridization and special sexual reproduction, we sequentially aggregated Zea mays, Zea perennis, and Tripsacum dactyloides in an allohexaploid, backcrossed it with maize, derived self-fertile allotetraploids of maize and Z. perennis by natural genome extraction, extended their first six selfed generations, and finally constructed amphitetraploid maize using nascent allotetraploids as a genetic bridge. Transgenerational chromosome inheritance, subgenome stability, chromosome pairings and rearrangements, and their impacts on an organism's fitness were investigated by fertility phenotyping and molecular cytogenetic techniques genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). Results showed that diversified sexual reproductive methods produced highly differentiated progenies (2n = 35–84) with varying proportions of subgenomic chromosomes, of which one individual (2n = 54, MMMPT) overcame self-incompatibility barriers and produced a self-fertile nascent near-allotetraploid by preferentially eliminating Tripsacum chromosomes. Nascent near-allotetraploid progenies showed persistent chromosome changes, intergenomic translocations, and rDNA variations for at least up to the first six selfed generations; however, the mean chromosome number preferably maintained at the near-tetraploid level (2n = 40) with full integrity of 45S rDNA pairs, and a trend of decreasing variations by advancing generations with an average of 25.53, 14.14, and 0.37 for maize, Z. perennis, and T. dactyloides chromosomes, respectively. The mechanisms for three genome stabilities and karyotype evolution for formatting new polyploid species were discussed.

By hybridization and special sexual reproduction, we sequentially aggregatedZea mays, Zea perennis,andTripsacum dactyloidesin an allohexaploid, backcrossed it with maize, derived self-fertile allotetraploids of maize andZ. perennisby natural genome extraction, extended their first six selfed generations, and finally constructed amphitetraploid maize using nascent allotetraploids as a genetic bridge. Transgenerational chromosome inheritance, subgenomes stability, chromosome pairings and rearrangements, and their impacts on an organism’s fitness were investigated by fertility phenotyping and molecular cytogenetics techniques GISH and FISH. Results showed that diversified sexual reproductive methods produced highly differentiated progenies (2n=35-84) with varying proportions of subgenomic chromosomes, of which one individual (2n=54, MMMPT) overcame self-incompatibility barriers and produced self-fertile nascent near-allotetraploid by preferentially eliminatingTripsacumchromosomes. Nascent near-allotetraploid progenies showed persistent chromosome changes, intergenomic translocations, and rDNA variations for at least up to the first six selfed generations; however, ploidy tended to stabilize at near-tetraploid level (2n=40) with full integrity of 45SrDNA pairs, and a trend of decreasing variations by advancing generations with an average of 25.53, 14.14, and 0.37 maize,Z. perennis,andT. dactyloideschromosomes, respectively. The mechanisms for three genome stabilities and karyotype evolution for formatting new polyploid species were discussed.

Summary Low-temperature chilling injury in early spring or late spring severely restricts maize production. Therefore, improving its temperature tolerance is one of the important goals of maize breeding. The wild maize relatives such as Z. perennis and T. dactyloides have strong cold tolerance and thus can be used to improve the cold tolerance of cultivated maize. In the previous study, the allohexaploid MTP ( Zea . Z. perennis and T. dactyloides ) was created as a genetic bridge material and the introgressed cross of the backbone maize inbred line to obtain the MTP-maize introgression line with strong cold tolerance. In this study, F 2:3 populations was developed of the cold-tolerant maize introgression line IB030 and the cold-sensitive maize introgression line IB021. A high-density genetic linkage map was constructed using SSR genetic markers and performed QTL mapping for related traits at 2°C, RNA-seq was performed using cold-tolerant introgression line IB030 and recurrent parent B73 (cold-sensitive) after low temperature stress at seedling stage. The key genes controlling REC, SDW and SFW of maize introgression line seedlings were located by forward genetics combined with WGCNA analysis strategy. Key genes function verification results showed that, Zm00001d037590 REC and Zm00001d012321 REC have the highest expression levels in introgression line IB030, followed by the wild type B73, and finally the EMS mutant after low temperature stress, these results initially concluded that key genes positively regulate the cold tolerance of maize, and also laid a theoretical and practical foundation for improvement of cultivated maize against low-temperature stress.

Maize ear carries paired spikelets, whereas the ear of its wild ancestor, teosinte, bears single spikelets. However, little is known about the genetic basis of the processes of transformation of single spikelets in teosinte ear to paired spikelets in maize ear. In this study, a two-ranked, paired-spikelets primitive maize and a two-ranked, single-spikelet teosinte were utilized to develop an F(2) population, and quantitative trait locus (loci) (QTL) mapping for single vs. paired spikelets (PEDS) was performed. One major QTL (qPEDS3.1) for PEDS located on chromosome 3S was identified in the 162 F(2) plants using the inclusive composite interval mapping of additive (ICIM-ADD) module, explaining 23.79% of the phenotypic variance. Out of the 409 F(2) plants, 43 plants with PEDS = 0% and 43 plants with PEDS > 20% were selected for selective genotyping, and the QTL (qPEDS3.1) was detected again. Moreover, the QTL (qPEDS3.1) was validated in three environments, which explained 31.05%, 38.94%, and 23.16% of the phenotypic variance, respectively. In addition, 50 epistatic QTLs were detected in the 162 F(2) plants using the two-locus epistatic QTL (ICIM-EPI) module; they were distributed on all 10 chromosomes and explained 94.40% of the total phenotypic variance. The results contribute to a better understanding of the genetic basis of domestication of paired spikelets and provide a genetic resource for future map-based cloning; in addition, the systematic dissection of epistatic interactions underlies a theoretical framework for overcoming epistatic effects on QTL fine mapping. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-022-01276-x.

C-type cytoplasmic male sterility (CMS-C), one of the three major CMS types in maize, has a promising application prospect in hybrid seed production. However, the complex genetic mechanism underlying the fertility restoration of CMS-C remains poorly understood. The maize inbred line A619 is one of the rare strong restorer lines carrying the restorer gene Rf4, but different fertility segregation ratios are found in several F2 populations derived from crosses between isocytoplasmic allonucleus CMS-C lines and A619. In the present study, the segregation ratios of fertile to sterile plants in the (CHuangzaosi × A619) F2 and BC1F1 populations (36.77:1 and 2.36:1, respectively) did not follow a typical monogenic model of inheritance, which suggested that some F2 and BC1F1 plants displayed restored fertility even without Rf4. To determine the hidden locus affecting fertility restoration, next-generation sequencing-based QTL-seq was performed with two specific extreme bulks consisting of 30 fertile and 30 sterile rf4rf4 individuals from the F2 population. A major QTL related to fertility restoration, designated qRf8-1, was detected on the long arm of chromosome 8 in A619. Subsequently, qRf8-1 was further validated and narrowed down to a 17.93-Mb genomic interval by insertion and deletion (InDel) and simple sequence repeat (SSR) marker-based traditional QTL mapping, explaining 12.59% (LOD = 25.06) of the phenotypic variation. Thus, using genetic analyses and molecular markers, we revealed another fertility restoration system acting in parallel with Rf4 in A619 that could rescue the male sterility of CHuangzaosi. This study not only expands the original fertility restoration system but also provides valuable insights into the complex genetic mechanisms underlying the fertility restoration of CMS-C.

A trispecific hybrid, MTP (hereafter called tripsazea), was developed from intergeneric crosses involving tetraploid Zea mays (2n = 4x = 40, genome: MMMM), tetraploid Tripsacum dactyloides (2n = 4x = 72, TTTT), and tetraploid Z. perennis (2n = 4x = 40, PPPP). On crossing maize-Tripsacum (2n = 4x = 56, MMTT) with Z. perennis, 37 progenies with varying chromosome numbers (36-74) were obtained, and a special one (i.e., tripsazea) possessing 2n = 74 chromosomes was generated. Tripsazea is perennial and expresses phenotypic characteristics affected by its progenitor parent. Flow cytometry analysis of tripsazea and its parents showed that tripsazea underwent DNA sequence elimination during allohexaploidization. Of all the chromosomes in diakinesis I, 18.42% participated in heterogenetic pairing, including 16.43% between the M- and P-genomes, 1.59% between the M- and T-genomes, and 0.39% in T- and P-genome pairing. Tripsazea is male sterile and partly female fertile. In comparison with previously synthesized trihybrids containing maize, Tripsacum and teosinte, tripsazea has a higher chromosome number, higher seed setting rate, and vegetative propagation ability of stand and stem. However, few trihybrids possess these valuable traits at the same time. The potential of tripsazea is discussed with respect to the deployment of the genetic bridge for maize improvement and forage breeding.

In this study, the chromosome number and composition of a novel perennial forage crop, 'Yucao No. 6' (Yu6), was revealed by chromosome spread and McGISH (multicolor genomic in situ hybridization) techniques to clarify its genitor origin. Cytogenetic analysis showed that Yu6, which has 56 chromosomes, is an aneuploid representing 12, 17 and 27 chromosomes from Zea mays ssp. mays L. (Zm, 2n = 2x = 20), Tripsacum dactyloides L. (Td, 2n = 4x = 72), and Z. perennis (Hitchc.) Reeves & Mangelsd. (Zp, 2n = 4x = 40), respectively. This finding indicates that Yu6 is the product of a reduced egg (n = 36 = 12Zm + 17Td + 7Zp) of MTP (a near-allohexaploid hybrid, 2n = 74 = 20Zm + 34Td + 20Zp) fertilized by a haploid sperm nucleus (n = 20Zp) of Z. perennis. Moreover, 3 translocated chromosomes consisting of the maize-genome chromosome with the segment of Z. perennis were observed. These results suggest that it is practical to develop perennial forage maize by remodeling the chromosomal architecture of MTP offspring with Z. perennis as a pollen parent. Finally, the overview of forage breeding in the Zea and Tripsacum genera was discussed.

Maize ear carries paired spikelets, whereas the ear of its wild ancestor, teosinte, bears single spikelets. However, little is known about the genetic basis of the processes of transformation of single spikelets in teosinte ear to paired spikelets in maize ear. In this study, a two-ranked, paired-spikelets primitive maize and a two-ranked, single-spikelet teosinte were utilized to develop an F2 population, and QTL mapping for single vs. paired spikelets (PEDS) was performed. Two QTL (qPEDS1.1 and qPEDS3.1) for PEDS located on chromosomes 1L and 3S were identified in the 162 F2 plants using the inclusive composite interval mapping of additive (ICIM-ADD) module, explaining 1.93% and 23.79% of the phenotypic variance, respectively. Out of the 409 F2 plants, 43 plants with PEDS = 0% and 43 plants with PEDS > 20% were selected for selective genotyping; the QTL (qPEDS3.1) accounting for 64.01% of the phenotypic variance for PEDS was also detected. Moreover, the QTL (qPEDS3.1) was validated in three environments, which explained 31.05%, 38.94% and 23.16% of the phenotypic variance, respectively. In addition, 50 epistatic QTLs were detected in 162 F2 plants using the two-locus epistatic QTL (ICIM-EPI) module; they were distributed on all 10 chromosomes and explained 94.40% of the total phenotypic variance. The results contribute to a better understanding of the genetic basis of domestication of paired spikelets and provide a genetic resource for future map-based cloning; in addition, the systematic dissection of epistatic interactions underlies a theoretical framework for overcoming epistatic effects on QTL fine mapping.

SUMMARY Inorganic phosphorus (Pi) is an essential element in numerous metabolic reactions and signaling pathways, but the molecular details of these pathways remain largely unknown. In this study, metabolite profiles of maize (Zea mays L.) leaves and roots were compared between six low‐Pi‐sensitive lines and six low‐Pi‐tolerant lines under Pi‐sufficient and Pi‐deficient conditions to identify pathways and genes associated with the low‐Pi stress response. Results showed that under Pi deprivation the concentrations of nucleic acids, organic acids and sugars were increased, but that the concentrations of phosphorylated metabolites, certain amino acids, lipid metabolites and nitrogenous compounds were decreased. The levels of secondary metabolites involved in plant immune reactions, including benzoxazinoids and flavonoids, were significantly different in plants grown under Pi‐deficient conditions. Among them, the 11 most stable metabolites showed significant differences under low‐ and normal‐Pi conditions based on the coefficient of variation (CV). Isoleucine and alanine were the most stable metabolites for the identification of Pi‐sensitive and Pi‐resistant maize inbred lines. With the significant correlation between morphological traits and metabolites, five low‐Pi‐responding consensus genes associated with morphological traits and simultaneously involved in metabolic pathways were mined by combining metabolites profiles and genome‐wide association study (GWAS). The consensus genes induced by Pi deficiency in maize seedlings were also validated by reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR). Moreover, these genes were further validated in a recombinant inbred line (RIL) population, in which the glucose‐6‐phosphate‐1‐epimerase encoding gene mediated yield and correlated traits to phosphorus availability. Together, our results provide a framework for understanding the metabolic processes underlying Pi‐deficient responses and give multiple insights into improving the efficiency of Pi use in maize., Significance statement Metabolite profiling and GWAS were applied to reveal maize low‐Pi response mechanisms.

Natural antisense transcripts (NATs) are a prominent and complex class of regulatory RNAs. Using strand-specific RNA sequencing, we identified 1769 sense and antisense transcript pairs (NAT pairs) in two maize inbreds with different sensitivity to drought, as well as in two derivative recombination inbred lines (RILs). A significantly higher proportion of NATs relative to non-NATs are specifically expressed under water stress (WS). Surprisingly, expression of sense and antisense transcripts produced by NAT pairs is significantly correlated, particularly under WS. We found an unexpected large proportion of NATs with protein coding potential, as estimated by ribosome release scores. Small RNAs significantly accumulate within NAT pairs, with 21 nt smRNA particularly enriched in overlapping regions of these pairs of genes. The abundance of these smRNAs is significantly altered in the leafbladeless1 mutant, suggesting that these genes may be regulated by the tasiRNA pathway. Further, NATs are significantly hypomethylated and include fewer transposable element sequences relative to non-NAT genes. NAT gene regions also exhibit higher levels of H3K36me3, H3K9ac, and H3K4me3, but lower levels of H3K27me3, indicating that NAT gene pairs generally exhibit an open chromatin configuration. Finally, NAT pairs in 368 diverse maize inbreds and 19 segregating populations were specifically enriched for polymorphisms associated with drought tolerance. Taken together, the data highlight the potential impact of that small RNAs and histone modifications have in regulation of NAT expression, and the significance of NATs in response to WS.

Gro/Tup1 proteins act as negative transcriptional regulators and play crucial roles in many growth and developmental processes in a wide range of organisms. However, our understanding of Gro/Tup1 protein functions in plants is confined to the model plant Arabidopsis. Here, 11 Gro/Tup1 genes, which were characterized by the typical LisH and WD40 repeat domains, were identified in maize through a genome-wide survey. A phylogenetic analysis revealed that maize Gro/Tup1 proteins could be divided into three subfamilies, in which members shared similar protein and gene structures. The predicted maize Gro/Tup1 genes were distributed on seven chromosomes and segmental duplication contributed to their expansion. Many predicted cis-elements associated with hormones, biotic- or abioticstress responses, meristem and seed development, and circadian rhythms, were found in their putative promoter regions. A potential associated protein analysis identified a large number of candidates, including transcription factors, chromatin-modifying enzymes, protein kinases, and ubiquitinconjugating enzymes. An expression profile derived from the RNA-seq data indicated that Gro/Tup1 genes in maize were widely expressed in various organs and tissues. Quantitative real-time PCR revealed that these genes responded to at least one hormone or abiotic stress, either in roots or in shoots. Our study provides useful information on the Gro/Tup1 genes in maize and will facilitate the further functional validation of these genes in growth and development, hormone responses, and biotic- or abiotic-stress resistance.

In this study, the chromosome number and composition of a novel perennial forage crop, ‘Yucao No. 6’ (Yu6), was revealed by chromosome spread and McGISH (multicolor genomic in situ hybridization) techniques to clarify its genitor origin. Cytogenetic analysis showed that Yu6, which has 56 chromosomes, is an aneuploid representing 12, 17 and 27 chromosomes from Zea mays ssp. mays L. (Zm, 2n = 2x = 20), Tripsacum dactyloides L. (Td, 2n = 4x = 72), and Z. perennis (Hitchc.) Reeves & Mangelsd. (Zp, 2n = 4x = 40), respectively. This finding indicates that Yu6 is the product of a reduced egg (n = 36 = 12Zm + 17Td + 7Zp) of MTP (a near-allohexaploid hybrid, 2n = 74 = 20Zm + 34Td + 20Zp) fertilized by a haploid sperm nucleus (n = 20Zp) of Z. perennis. Moreover, 3 translocated chromosomes consisting of the maize-genome chromosome with the segment of Z. perennis were observed. These results suggest that it is practical to develop perennial forage maize by remodeling the chromosomal architecture of MTP offspring with Z. perennis as a pollen parent. Finally, the overview of forage breeding in the Zea and Tripsacum genera was discussed.

Phosphorus (P) is required for numerous developmental processes, such as flowering and reproduction, in maize (Zea mays L.). It is also an essential nutrient in fertilizers to sustain high yields in farmlands with insufficient available phosphate, particularly in acid soils in China. Flowering time is a key factor influencing the adaptation of maize to the local environment and is associated with high yields. However, few studies have focused on the genetic characteristics of flowering time under low phosphate conditions. In this study, a recombinant inbred line (RIL) population derived from a cross between X178 and 9782 was evaluated for three flowering time traits under two phosphates (Pi) levels at four locations. We found that low phosphate significantly delayed the flowering stage of maize and proposed the hypothesis of “Phosphorus Accumulation Effect”. Mixed-model-based composite interval mapping was used to dissect the genetic basis of the flowering time traits. A total of 41 quantitative trait loci (QTL) was identified for the three flowering time traits through four procedures. Among these 41 QTL, 38 focused on four hot regions, including bin 2.02, bin 2.03, bin 5.04, and bin 6.01. These regions and linked markers should be the targets for fine mapping, map-based cloning, and marker-assisted selection of flowering time and phosphate utilization efficiency.

Maize (Zea mays L.), which is an important food crop in the word, displays large genetic diversity. Knowledge of the relationships among maize inbred lines is essential to the maize breeder because it directs the exploitation of germplasm in hybrid production. In this study, the genetic diversity, population structure, and relatedness between pairs of 157 elite maize inbred lines from the current breeding program of Sichuan province in Southwest China were assessed with 4976 polymorphic single-nucleotide polymorphisms (SNPs) developed by genotyping by sequencing (GBS). A total of 91.1% of the inbred lines were considered pure with

Stalk lodging severely limits the grain yield of maize (Zea mays L.). Mechanical stalk strength can be reflected by the traits of stalk diameter (SD), stalk bending strength (SBS), and lodging rind penetrometer resistance (RPR). To determine the genetic basis of maize stalk lodging, quantitative trait loci (QTLs) were mapped for these three traits using the IBM Syn10 DH population in three environments. The results indicated that there were strong genetic correlations among the three traits, and the analyses of phenotypic variations for SD, SBS, and RPR across the three environments showed high broad-sense heritability (0.6843, 0.5175, and 0.7379, respectively). In total, 44 significant QTLs were identified control the above traits across the 3 environments. A total of 14, 14, and 16 QTLs were identified for SD, SBS, and RPR across single-environment mapping, respectively. Notably, ten QTLs were stably expressed across multiple-environments, including two QTLs for SD, three for SBS, and five for RPR. Three major QTLs each accounting for over 10% of the phenotypic variation were qSD6-2 (10.03%), qSD8-2 (13.73%), and qSBS1-2 (11.89%). Comprehensive analysis of all QTLs in this study revealed that 5 QTL clusters including 12 QTLs were located on chromosomes 1, 3, 7, and 8, respectively. Among these 44 QTLs, 9 harbored 13 stalk lodging-associated SNPs that were detected by our recently published work, with 1 SNP successfully validated in the IBM Syn10 DH population. These chromosomal regions will be useful for marker-assisted selection and fine mapping of stalk lodging-related traits in maize.

Zea nicaraguensis , a wild relative of cultivated maize ( Zea mays subsp. mays ), is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cloning and function verification to discover waterlogging tolerance genes in Z. nicaraguensis is currently impractical, because little gene sequence information for Z. nicaraguensis is available in public databases. In this study, Z. nicaraguensis seedlings were subjected to simulated waterlogging stress and total RNAs were isolated from roots stressed and non-stressed controls. In total, 80 mol L −1 Illumina 100-bp paired-end reads were generated. De novo assembly of the reads generated 81 002 final non-redundant contigs, from which 5 261 full-length transcripts were identified. Among these full-length transcripts, 3 169 had at least one Gene Ontology (GO) annotation, 2 354 received cluster of orthologous groups (COG) terms, and 1 992 were assigned a Kyoto encyclopedia of genes and genomes (KEGG) Orthology number. These sequence data represent a valuable resource for identification of Z. nicaraguensis genes involved in waterlogging response.

Magnesium (Mg(2+)) is an essential macronutrient for plant growth and development, and the CorA/MRS2/MGT-type Mg(2+) transporters play important roles in maintaining Mg(2+) homeostasis in plants. Although the MRS2/MGT genes have been identified in two model plant species, Arabidopsis and rice, a comprehensive analysis of the MRS2/MGT gene family in other plants is lacking. In this work, 12 putative MRS2/MGT genes (ZmMGT1- ZmMGT12) were identified in maize and all of them were classified into five distinct subfamilies by phylogenetic analysis. A complementation assay in the Salmonella typhimurium MM281 strain showed that five representatives of the 12 members possess Mg(2+) transport abilities. Inhibition of ZmMGT protein activity using the hexaamminecobalt (III) (Co-Hex) inhibitor indicated that the ZmMGT protein mediated both low-affinity and high-affinity Mg(2+) transport in maize. A semi-quantitative reverse transcription-PCR (RT-PCR) analysis revealed that eight genes were constitutively expressed in all of the detected tissues, with one being specifically expressed in roots and three having no detectable expression signals. A quantitative RT-PCR analysis showed that some ZmMGT members displayed differential responses to Mg(2+) deficiency and aluminum (Al) stress. Furthermore, root growth inhibition and Mg(2+) accumulation analyses in two maize inbred lines, which conferred different levels of Al tolerance, revealed that ZmMGT proteins contributed to the Al resistance of the Al tolerance genotype. We hypothesize that ZmMGT family members function as Mg(2+) transporters and may play a role in linking Mg(2+) deficiency and Al stress responses. Our results will be valuable in a further analysis of the important biological functions of ZmMGT members in maize.

A novel male-sterile maize mutant male sterility 39 (ms39) was obtained from offspring of the commercial hybrid Chuandan No. 9 that had been carried into outer space. A previous study demonstrated that ms39 is controlled by a single recessive nuclear gene, located on the long arm of chromosome 3. Here, we used 1073 mutant individuals derived from the (ms39 × Mo17) F2 population and sequentially developed new primers to identify markers supporting the fine mapping of ms39. A 365-kb region on chromosome 3 flanked by markers L8 and M30 at a genetic distance of 0.18 and 0.47 cM, respectively, was identified. According to the reference sequence of ZmB73_Ref-Gen_v4, 12 candidate genes were identified within the 365-kb mapping region. Based on cloning and sequence BLAST analysis of the 12 candidate genes, a four-base-pair deletion was found within the exon of Zm00001d043909, which encoded callose synthase12. This four-base-pair deletion resulted in a frameshift mutation in ms39, leading to the earlier termination of the coding protein, and ultimately caused abnormal performance of the callose synthase. Additionally, cytological observation was conducted on a sister cross population (ms39/ms39 × ms39/Ms39). These observations showed that the tapetum cells of the ms39 mutant appeared abnormal from the dyad stage, and aborted microspores were observed during pollen development. These results lay the foundation for the cloning of ms39 and exploration of the molecular mechanism underlying aborted pollen development in ms39 maize.

Tripsacum dactyloides is closely related to Zea mays since Zea perennis and the MTP tri- species hybrid have four possible reproductive modes. Eastern gamagrass (Tripsacum dactyloides L.) and tetraploid perennial teosinte (Zea perennis) are well known to possess genes conferring resistance against biotic and abiotic stresses as well as adaptation to flood and aluminum toxic soils. However, plant breeders have been hampered to utilize these and other beneficial traits for maize improvement due to sterility in their hybrids. By crossing a tetraploid maize-inbred line × T. dactyloides, a female fertile hybrid was produced that was crossed with Z. perennis to yield a tri-genomic female fertile hybrid, which was backcrossed with diploid maize to produce BC1 and BC2. The tri-genomic hybrid provided a new way to transfer genetic material from both species into maize by utilizing conventional plant breeding methods. On the basis of cytogenetic observations using multi-color genomic in situ hybridization, the progenies were classified into four groups, in which chromosomes could be scaled both up and down with ease to produce material for varying breeding and genetic purposes via apomixis or sexual reproduction. In the present study, pathways were found to recover maize and to obtain specific translocations as well as a speedy recovery of the T. dactyloides–maize addition line in a second backcross generation. However, phenotypes of the recovered maize were in most cases far from maize as a result of genetic load from T. dactyloides and Z. perennis, and could not be directly used as a maize-inbred line but could serve as an intermediate material for maize improvement. A series of hybrids was produced (having varying chromosome number, constitution, and translocations) with agronomic traits from all three parental species. The present study provides an application of overcoming the initial interspecific barriers among these species. Moreover, T. dactyloides is closely related to Z. mays L. ssp. mays since Z. perennis and the MTP tri- species hybrid have four possible reproductive modes.

This study was aimed to investigate the stability of chromosomes during meiosis in autopolyploid and allopolyploid maize, as well as to determine an association of chromosomes between maize (Zeamaysssp.mays Linnaeus, 1753) andZ.perennis(Hitchcock, 1922) Reeves & Mangelsdor, 1942, by producing a series of autopolyploid and allopolyploid maize hybrids. The intra-genomic and inter-genomic meiotic pairings in these polyploids were quantified and compared using dual-color genomic in-situ hybridization. The results demonstrated higher level of chromosome stability in allopolyploid maize during meiosis as compared to autopolyploid maize. In addition, the meiotic behavior ofZ.perenniswas relatively more stable as compared to the allopolyploid maize. Moreover, ten chromosomes of "A” subgenome in maize were homologous to twenty chromosomes ofZ.perennisgenome with a higher pairing frequency and little evolutionary differentiation. At the same time, little evolutionary differentiation has been shown by chromosomes of "A” subgenome in maize, while chromosomes of "B” subgenome, had a lower pairing frequency and higher evolutionary differentiation. Furthermore, 5IM+ 5IIPP+ 5IIIMPPand 5IIMM+ 5IIPP+ 5IVMMPPwere observed in allotriploids and allotetraploids respectively, whereas homoeologous chromosomes were found between the "A” and "B” genome of maize andZ.perennis.

Allopolyploidization plays the special role in the evolution of many crops. Moreover, the evolution in early stage of some allopolyploidization events is predicted to be effected by nuclear-cytoplasmic interactions. Maize and teosintes are well model system for study of genetic recombination in allopolyploidization. In order to investigate the effects of genome organization and cytoplasm on genome evolution in newly synthesized allopolyploids (neoallopolyploids), a series of neoallopolyploids were produced by reciprocal crosses of maize and Zea perennis. By using dual-color genomic in situ hybridization, intra- and intergenomic meiosis pairing of these polyploids were quantified and compared with regard to its genome organization and cytoplasm background. In the four neoallopolyploids, the stability of maize genome is consistently lower than that of Z. perennis genome. Additional, the stability of maize genome is affected by genome ploidy. The cytoplasm, genome composition and their interaction do have the special role in chromosome paring and the meiosis behaviors in Zea allopolyploids vary significantly and showed non-diploidization. Z. perennis cytoplasm may give a relatively relaxed environment for maize genome.

Ear row number (ERN) is not only a key trait involved in maize (Zea mays L.) evolution but also an important component directly related to grain yield. In this report, 325 recombinant inbred lines (RILs, F6:7) derived from a cross between B73 with 16 rows and SICAU1212 with four rows (two-ranked with two rows per rank) were utilized to detect quantitative trait loci (QTL) associated with ERN and two-ranked versus many-ranked ears (TR). Compared to modern maize that formed approximately 8–20 rows, SICAU1212 with four rows was the extreme case. A total of 12 and 8 QTLs were associated with ERN and TR across four environments through single-environment mapping, respectively. Each QTL responsible for ERN explained 2.33–21.28 % of the phenotypic variation. And the TR variation contributed by individual TR QTL ranged from 2.09 to 12.99 %. Notably, only three QTLs, qERN2-1 (bin 2.02), qERN8-1 (bin 8.02) and qERN8-2 (bin 8.04), were consistently detected in each environment and by joint analysis among all environments, which simultaneously influenced ERN and TR. One of the three QTLs, qERN8-1 was also identified as interacting with environment. In addition, nine pairs of significant epistatic interactions (two for ERN and seven for TR) were detected among all QTLs. The epistasis between qTR2-1 and qTR8-1 was consistent in most environments. This present study may provide the understanding of the genetic basis of ERN and TR and a foundation for further fine-mapping of these common QTLs.

C-type cytoplasmic male sterile (CMS-C) line plays an important role in hybrid seed production in maize. However, mechanisms of pollen abortion and fertility restoration remain unclear. This study aimed to investigate the mechanisms of CMS-C pollen abortion and fertility restoration, particularly based on epigenetics, and to understand the relationship between male fertility performance and DNA methylation status. Methylation-sensitive amplification polymorphism (MSAP) technique was conducted to analyze DNA methylation levels and patterns of tassels at pollen-mother cell, tetrad, mononuclear, and binuclear stages among four half-sib hybrids and their parents with different fertility. Results showed that DNA methylation levels in fertility restored hybrids were higher than those in sterility maintained hybrids. Nine CCGG sites, which displayed methylation polymorphism between male-fertile and male-sterile hybrids, were screened. To validate MSAP results, we performed methylation-sensitive PCR (MS-PCR) and found consistent observations in cloned methylation sites. Interestingly, a specific site named 16–1 was discovered in the region of Rf5 gene, which is one of the restorer genes of CMS-C. Thus, DNA methylation may participate in regulating the expression of fertility restorer genes of maize CMS-C.

Hybridization, which allows for gene flow between crops, is difficult between maize and Zea perennis. In this study, we aim to initiate and study gene flow between maize and Z. perennis via a special aneuploid plant (MDT) derived from an interspecific hybrid of the two species. The chromosome constitution and morphological characters of MDT as well as certain backcross progenies were examined. Results from genomic in situ hybridization (GISH) indicate that aneuploid MDT consisted of nine maize chromosomes and 30 Z. perennis chromosomes. The backcross progenies of MDT×maize displayed significant diversity of vegetative and ear morphology; several unusual plants with specific chromosome constitution were founded in its progenies. Some special perennial progeny with several maize chromosomes were obtained by backcrossing MDT with Z. perennis , and the first whole chromosome introgression from maize to Z. perennis was detected in this study. With this novel material and method, a number of maize-tetraploid teosinte addition or substitution lines can be generated for further study, which has great significance to maize and Z. perennis genetic research, especially for promoting introgression and transferring desirable traits.

Ear row number (ERN) is not only a key trait involved in maize (Zea mays L.) evolution but is also an important component that is directly related to grain yield. In this study, quantitative trait loci (QTLs) for ERN were detected across two F2 populations that were derived from a same cross between B73 with 16 rows (N = 233) and SICAU1212 with four rows (N = 231). As a result, 33 QTLs were associated with 12 agronomic traits: three plant traits, four ear-related traits, and five kernel-related traits. The total phenotypic variation explained by the QTLs for each trait ranged from 8.60 to 72.67%, and four QTLs were identified for ERN in the two populations. Each QTL explained between 6.78 and 36.76% of the ERN variation. Notably, three of the four QTLs (qERN2-1, qERN4-2, and qERN8-1) were associated with ERN, and qERN8-1 simultaneously influenced grain yield, plant diameter, ear diameter, and kernel length. In addition, only one significant epistatic interaction was detected in all 33 QTLs. This study should provide a foundation for further fine-mapping and map-based cloning of these consistent QTLs, and for controlling maize ERN by marker-assisted breeding.

To develop genetic markers associated with tolerance to low phosphorus, we identified candidate phosphate starvation responsive (PSR) genes and developed their intron length polymorphism (ILP) markers in maize on a genome-wide scale. Based on the known plant PSR genes, 161 candidate PSR genes were identified. Of these genes, 138 genes contained at least one intron and were then used to develop 606 PSR-ILP markers by designing PCR primers to the intron-flanking exonic regions. PCR evaluation was performed on 43 randomly selected PSR-ILP markers in 30 maize inbred lines. Of the primers, 88.4% amplified stable and pure products in all maize inbred lines, and 53.5% of the markers showed ILP, with PIC values ranging from 0.06 to 0.80. The result of clustering analysis of the 30 maize inbred lines, based on the polymorphism of 23 ILP markers, suggests that the ILP markers developed in this study are not only efficient for genetic diversity analysis but also potentially useful for marker assisted selection for tolerance to low phosphorus.

Background Maize was originally domesticated in a tropical environment but is now widely cultivated at temperate latitudes. Temperate and tropical maize populations have diverged both genotypically and phenotypically. Tropical maize lines grown in temperate environments usually exhibit delayed flowering, pollination, and seed set, which reduces their grain yield relative to temperate adapted maize lines. One potential mechanism by which temperate maize may have adapted to a new environment is novel transposable element insertions, which can influence gene regulation. Recent advances in sequencing technology have made it possible to study variation in transposon content and insertion location in large sets of maize lines. Results In total, 274,408 non-redundant TEs (NRTEs) were identified using resequencing data generated from 83 maize inbred lines. The locations of DNA TEs and copia-superfamily retrotransposons showed significant positive correlations with gene density and genetic recombination rates, whereas gypsy-superfamily retrotransposons showed a negative correlation with these two parameters. Compared to tropical maize, temperate maize had fewer unique NRTEs but higher insertion frequency, lower background recombination rates, and higher linkage disequilibrium, with more NRTEs close to flowering and stress-related genes in the genome. Association mapping demonstrated that the presence/absence of 48 NRTEs was associated with flowering time and that expression of neighboring genes differed between haplotypes where a NRTE was present or absent. Conclusions This study suggests that NRTEs may have played an important role in creating the variation in gene regulation that enabled the rapid adaptation of maize to diverse environments. Electronic supplementary material The online version of this article (10.1186/s12864-017-4103-x) contains supplementary material, which is available to authorized users.

Characterization of genetic diversity is of great value to assist breeders in parental line selection and breeding system design. We screened 770 maize inbred lines with 1,034 single nucleotide polymorphism (SNP) markers and identified 449 high-quality markers with no germplasm-specific biasing effects. Pairwise comparisons across three distinct sets of germplasm, CIMMYT (394), China (282), and Brazil (94), showed that the elite lines from these diverse breeding pools have been developed with only limited utilization of genetic diversity existing in the center of origin. Temperate and tropical/subtropical germplasm clearly clustered into two separate groups. The temperate germplasm could be further divided into six groups consistent with known heterotic patterns. The greatest genetic divergence was observed between temperate and tropical/subtropical lines, followed by the divergence between yellow and white kernel lines, whereas the least divergence was observed between dent and flint lines. Long-term selection for hybrid performance has contributed to significant allele differentiation between heterotic groups at 20% of the SNP loci. There appeared to be substantial levels of genetic variation between different breeding pools as revealed by missing and unique alleles. Two SNPs developed from the same candidate gene were associated with the divergence between two opposite Chinese heterotic groups. Associated allele frequency change at two SNPs and their allele missing in Brazilian germplasm indicated a linkage disequilibrium block of 142 kb. These results confirm the power of SNP markers for diversity analysis and provide a feasible approach to unique allele discovery and use in maize breeding programs.

Phosphorus (P) is one of the three primary macronutrients that are required in large amounts for plant growth and development. To better understand molecular mechanism of maize and identify relevant genes in response to phosphorus deficiency, we used Solexa/Illumina's digital gene expression (DGE) technology to investigate six genome-wide expression profiles of seedling roots of the low-P tolerant maize inbred line 178. DGE studies were conducted at 6, 24 and 72 h under both phosphorus deficient and sufficient conditions. Approximately 3.93 million raw reads for each sample were sequenced and 6 816 genes exhibited significant levels of differential expressions in at least one of three time points in response to P starvation. The number of genes with increased expression increased over time from 6 to 24 h, whereas genes with decreased expression were more abundant at 72 h, suggesting a gradual response process for P deficiency at different stages. Gene annotations illustrated that most of differentially expressed genes (DEGs) are involved in different cellular and molecular processes such as environmental adaptation and carbohydrate metabolism. The expression of some known genes identified in other plants, such as those involved in root architecture, P metabolism and transport were found to be altered at least two folds, indicating that the mechanisms of molecular and morphological adaptation to P starvation are conserved in plants. This study provides insight into the general molecular mechanisms underlying plant adaptation to low-P stress and thus may facilitate molecular breeding for improving P utilization in maize.

Phosphorus (P) deficiency is one of the major limiting factors in maize production in many developing countries. This experiment was conducted to evaluate multiple low-P tolerance criteria and identify the suitable maize germplasm for our future low-P tolerance breeding. A total of 456 diverse maize inbreds were evaluated for low-P tolerance at seedling stage using four shoot-related traits and six root-related traits measured under applied phosphorus (AP) and non-applied phosphorus (NAP) conditions. Analysis of variance revealed significant genetic variation among genotypes for all tested traits. Medium-to-high heritability estimates were obtained for most traits. Total dry weight (DW) was highly inheritable while the widely used root/shoot ratio had only an intermediate level of heritability. Based on the synthetic index (SI), the tested inbreds were classified into three groups representing low, moderate and high tolerance to low-P stress. Regression model built based on selection criteria for low-P tolerance explained 67.8 and 76.8 % of variation for DW under NAP and AP conditions, respectively. Using low-P tolerance index for DW and SI as selection criteria, 23 and 109 maize inbreds were identified as germplasm resources that were extremely tolerant and sensitive to low-P stress, respectively, which could be further used for genetic improvement of low-P tolerance.

Maize (Zea mays L.) populations are potential sources of favorable alleles absent in parental inbred lines to improve elite hybrids. The maize hybrid Zhengdan 958 has been hampered by the lack of favorable new alleles for improving yield and commodity quality. In the present study, 16 testcrosses made by using eight synthetic populations as the donors and the two parental lines of Zhengdan 958 as the receptors were evaluated in 2009 and 2010 at Shunyi, Beijing and Xinxiang, Henan Province for grain yield and test weight. Four genetic parameters were used to determine the breeding potential of eight synthetic populations as the donors to improve the target hybrid. Several synthetic populations were identified as the potential sources of favorable alleles absent in the target hybrid for each trait evaluated. The two most promising germplasms, WBMC-4 and Shanxi Syn3, had the potential for simultaneously improving grain yield and test weight of the target hybrid, which could be used to improve the parental lines Zheng 58 and Chang 7-2, respectively, and further broaden the germplasm base of Chinese heterotic groups PA and Sipingtou.

Vitamin A deficiency has become a worldwide problem. Biofortified foods can potentially be an inexpensive, locally adaptable, and long-term solution to dietary-nutrient deficiency. In order to improve the β-carotene content in maize grain by breeding and minimize vitamin A deficiency, a complete diallel cross was designed with eight inbred lines of maize, and 64 combinations were obtained in this study. The experimental combinations were planted in Yunnan and Sichuan provinces, respectively, with a random complete block design. The β-carotene contents in the grains of the experimental materials were analyzed by high-performance liquid chromatography. Among the tested materials, the effect difference of general combining ability of the β-carotene content was significant; however, the effect difference of the special combining ability and the reciprocal effect were not significant. The β-carotene content of maize grain was not influenced significantly by the cross and the reciprocal cross. There was a significant correlation about the β-carotene content in the maize grains between the F1 and their parents. The combinations with high β-carotene content were obviously influenced by the environment, and the mean value of β-carotene content for the experimental materials planted in Ya'an of Sichuan was higher than that planted in Yuanjiang of Yunnan, with the results being significant at the 0.01 level.

Large-scale selective genotyping and high-throughput analysis are two important strategies for low-cost and high-effective genetic mapping. In this study, selective genotyping was applied to four maize F2 populations. Thirty plants were selected from each of the two tails of the original F2 populations to represent extreme resistant and susceptible plants to root lodging, and genotyped individually with 1536 single nucleotide polymorphisms (SNPs). A quantitative trait locus (QTL) was declared when at least three closely linked SNPs showed significant allele frequency difference between the two tails. Nine QTL were identified for root lodging across the four populations, which were located on chromosomes 2, 4, 5, 7, 8 and 10 and one of them was shared between two populations. A total of 20 segregation distortion regions (SDRs) were identified across the four populations, one of which was co-localized with a QTL on chromosome 4. The tightly linked SNPs identified in this study can be used for marker-assisted selection for root lodging. Selective genotyping, when combined with pooled DNA analysis, can be used to develop strategies for high-throughput genetic mapping for all crops.