Genetic basis of the oil biosynthesis in ultra-high-oil maize grains with an oil content exceeding 20.

Vegetable oil is an important part of the human diet and has multiple industrial uses. The rapid increase in vegetable oil consumption has necessitated the development of viable methods for optimizing the oil content of plants. The key genes regulating the biosynthesis of maize grain oil remain mostly uncharacterized. In this study, by analyzing oil contents and performing bulked segregant RNA sequencing and mapping analyses, we determined that su1 and sh2-R mediate the shrinkage of ultra-high-oil maize grains and contribute to the increase in the grain oil content. Functional kompetitive allele-specific PCR (KASP) markers developed for su1 and sh2-R detected su1su1Sh2Sh2, Su1Su1sh2sh2, and su1su1sh2sh2 mutants among 183 sweet maize inbred lines. An RNA sequencing (RNA-seq) analysis indicated that genes differentially expressed between two conventional sweet maize lines and two ultra-high-oil maize lines were significantly associated with linoleic acid metabolism, cyanoamino acid metabolism, glutathione metabolism, alanine, aspartate, and glutamate metabolism, and nitrogen metabolism. A bulk segregant analysis and sequencing (BSA-seq) analysis identified another 88 genomic intervals related to grain oil content, 16 of which overlapped previously reported maize grain oil-related QTLs. The combined analysis of BSA-seq and RNA-seq data enabled the identification of candidate genes. The KASP markers for GRMZM2G176998 (putative WD40-like beta propeller repeat family protein), GRMZM2G021339 (homeobox-transcription factor 115), and GRMZM2G167438 (3-ketoacyl-CoA synthase) were significantly related to maize grain oil content. Another candidate gene, GRMZM2G099802 (GDSL-like lipase/acylhydrolase), catalyzes the final step of the triacylglycerol synthesis pathway and was expressed at significantly higher levels in the two ultra-high-oil maize lines than in the two conventional sweet maize lines. These novel findings will help clarify the genetic basis of the increased oil production in ultra-high-oil maize lines with grain oil contents exceeding 20%. The KASP markers developed in this study may be useful for breeding new high-oil sweet maize varieties.

A newly characterized allele of ZmR1 increases anthocyanin content in whole maize plant and the regulation mechanism of different ZmR1 alleles.

KEY MESSAGE: The novel ZmR1CQ01 allele for maize anthocyanin synthesis was identified, and the biological function and regulatory molecular mechanisms of three ZmR1 alleles were unveiled. Anthocyanins in maize are valuable to human health. The R1 gene family is one of the important regulatory genes for the tissue-specific distribution of anthocyanins. R1 gene allelic variations are abundant and its biological function and regulatory molecular mechanisms are not fully understood. By exploiting genetic mapping and transgenic verification, we found that anthocyanin pigmentation in maize leaf midrib was controlled by ZmR1 on chromosome 10. Allelism test of maize zmr1 EMS mutants confirmed that anthocyanin pigmentation in leaf sheath was also controlled by ZmR1. ZmR1CQ01 was a novel ZmR1 allelic variation obtained from purple maize. Its overexpression caused the whole maize plant to turn purple. ZmR1B73 allele confers anthocyanin accumulation in near ground leaf sheath rather than in leaf midribs. The mRNA expression level of ZmR1B73 was low in leaf midribs, resulting in no anthocyanin accumulation. ZmR1B73 overexpression promoted anthocyanin accumulation in leaf midribs. Loss of exon 5 resulted in ZmR1ZN3 allele function destruction and no anthocyanin accumulation in leaf midrib and leaf sheath. DNA affinity purification sequencing revealed 1010 genes targeted by ZmR1CQ01, including the bz2 in anthocyanin synthesis pathway. RNA-seq analysis showed 55 genes targeted by ZmR1CQ01 changed the expression level significantly, and the expression of genes encoding key enzymes in flavonoid and phenylpropanoid biosynthesis pathways were significantly up-regulated. ZmR1 functional molecular marker was developed. These results revealed the effects of transcriptional regulation and sequence variation on ZmR1 function and identified the genes targeted by ZmR1CQ01 at the genome-wide level.

Molecular dissection of maize seedling salt tolerance using a genome-wide association analysis method.

Salt stress is a major devastating abiotic factor that affects the yield and quality of maize. However, knowledge of the molecular mechanisms of the responses to salt stress in maize is limited. To elucidate the genetic basis of salt tolerance traits, a genome-wide association study was performed on 348 maize inbred lines under normal and salt stress conditions using 557 894 single nucleotide polymorphisms (SNPs). The phenotypic data for 27 traits revealed coefficients of variation of >25%. In total, 149 significant SNPs explaining 6.6%-11.2% of the phenotypic variation for each SNP were identified. Of the 104 identified quantitative trait loci (QTLs), 83 were related to salt tolerance and 21 to normal traits. Additionally, 13 QTLs were associated with two to five traits. Eleven and six QTLs controlling salt tolerance traits and normal root growth, respectively, co-localized with QTL intervals reported previously. Based on functional annotations, 13 candidate genes were predicted. Expression levels analysis of 12 candidate genes revealed that they were all responsive to salt stress. The CRISPR/Cas9 technology targeting three sites was applied in maize, and its editing efficiency reached 70%. By comparing the biomass of three CRISPR/Cas9 mutants of ZmCLCg and one zmpmp3 EMS mutant with their wild-type plants under salt stress, the salt tolerance function of candidate genes ZmCLCg and ZmPMP3 were confirmed. Chloride content analysis revealed that ZmCLCg regulated chloride transport under sodium chloride stress. These results help to explain genetic variations in salt tolerance and provide novel loci for generating salt-tolerant maize lines.

Sequence polymorphism of the waxy gene in waxy maize accessions and characterization of a new waxy allele.

Waxy maize has many excellent characteristics in terms of its nutritional and economic value. In recent decades, the waxy maize germplasm has increased dramatically as a result of different selection methods. We collected 200 waxy maize inbred accessions from different origins to study their genetic diversity and phylogenetic relationships, and to identify new waxy mutations. A simple sequence repeat (SSR) analysis revealed wide genetic diversity among the 200 waxy maize accessions. The maize accessions were clustered into three groups. We sequenced the waxy gene from the first to the 14th exon. Nucleotide variation analysis of 167 waxy maize and 14 flint maize lines revealed some nucleotide differences in the waxy gene among different waxy maize groups, and much narrower nucleotide diversity in waxy maize than in flint maize. In a phylogenetic analysis, waxy maize carrying the same mutation allele clustered together, and waxy maize carrying different mutation alleles distributed in different groups; waxy maize was intermixed with flint maize in each branch, and wx-D7 waxy maize separated significantly from waxy maize lines carrying wx-D10, wx-124 and wx-hAT mutant alleles. The wx-hAT was a new waxy mutation identified in this study. It consisted of a 2286-bp transposon inserted into the middle of exon three of the waxy gene. A PCR marker specific for the wx-hAT allele was developed. These results will be useful for the utilization and preservation of the waxy maize germplasm, and the PCR marker has potential uses in waxy maize breeding programs.