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Combined QTL Mapping across Multiple Environments and Co-Expression Network Analysis Identified Key Genes for Embryogenic Callus Induction from Immature Maize Embryos.

ABSTRACT: The ability of immature embryos to induce embryogenic callus (EC) is crucial for genetic transformation in maize, which is highly genotype-dependent. To dissect the genetic basis of maize EC induction, we conducted QTL mapping for four EC induction-related traits, the rate of embryogenic callus induction (REC), rate of shoot formation (RSF), length of shoot (LS), and diameter of callus (DC) under three environments by using an IBM Syn10 DH population derived from a cross of B73 and Mo17. These EC induction traits showed high broad-sense heritability (>80%), and significantly negative correlations were observed between REC and each of the other traits across multiple environments. A total of 41 QTLs for EC induction were identified, among which 13, 12, 10, and 6 QTLs were responsible for DC, RSF, LS, and REC, respectively. Among them, three major QTLs accounted for >10% of the phenotypic variation, including qLS1-1 (11.54%), qLS1-3 (10.68%), and qREC4-1 (11.45%). Based on the expression data of the 215 candidate genes located in these QTL intervals, we performed a weighted gene co-expression network analysis (WGCNA). A combined use of KEGG pathway enrichment and eigengene-based connectivity (KME) values identified the EC induction-associated module and four hub genes (Zm00001d028477, Zm00001d047896, Zm00001d034388, and Zm00001d022542). Gene-based association analyses validated that the variations in Zm00001d028477 and Zm00001d034388, which were involved in tryptophan biosynthesis and metabolism, respectively, significantly affected EC induction ability among different inbred lines. Our study brings novel insights into the genetic and molecular mechanisms of EC induction and helps to promote marker-assisted selection of high-REC varieties in maize.

SUBMITTER: Long Y

PROVIDER: S-EPMC9368897 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Combined QTL Mapping across Multiple Environments and Co-Expression Network Analysis Identified Key Genes for Embryogenic Callus Induction from Immature Maize Embryos.

Long Yun Y Liang Tianhu T Ma Langlang L Liu Peng P Yang Yun Y Zhang Xiaoling X Zou Chaoying C Zhang Minyan M Ge Fei F Yuan Guangsheng G Lübberstedt Thomas T Pan Guangtang G Shen Yaou Y

International journal of molecular sciences 20220807 15

The ability of immature embryos to induce embryogenic callus (EC) is crucial for genetic transformation in maize, which is highly genotype-dependent. To dissect the genetic basis of maize EC induction, we conducted QTL mapping for four EC induction-related traits, the rate of embryogenic callus induction (REC), rate of shoot formation (RSF), length of shoot (LS), and diameter of callus (DC) under three environments by using an IBM Syn10 DH population derived from a cross of B73 and Mo17. These E ...[more]

PMID: 35955919

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Project description:BackgroundSorghum (Sorghum bicolor L.) is one of the world's most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency.ResultsWe report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5-2.0 mm in length) isolated 12-15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt-II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs.ConclusionsThis protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.

Project description:Picea mongolica is a rare tree species in China, which is of great significance in combating desertification and improving the harsh ecological environment. Due to the low rate of natural regeneration, high mortality, and susceptibility to pests and cold springs, Picea mongolica has gradually become extinct. At present, somatic embryogenesis (SE) is the most effective method of micro-proliferation in conifers, but the induction rate of embryogenic callus (EC) is low, and EC is difficult to differentiate from non-embryonic callus (NEC). Therefore, the EC and NEC of Picea mongolica were compared from the morphology, histological, physiological, and transcriptional levels, respectively. Morphological observation showed that the EC was white and transparent filamentous, while the NEC was compact and brownish-brown lumpy. Histological analyses showed that the NEC cells were large and loosely arranged; the nuclei attached to the edge of the cells were small; the cytoplasm was low; and the cell gap was large and irregular. In the EC, small cells, closely arranged cells, and a large nucleus and nucleolus were observed. Physiological studies showed significant differences in ROS-scavenging enzymes between the EC and NEC. Transcriptome profiling revealed that 13,267 differentially expressed genes (DEGs) were identified, 3682 were up-regulated, and 9585 were down-regulated. In total, 63 GO terms had significant enrichment, 32 DEGs in plant hormone signal transduction pathway were identified, and 502 different transcription factors (TFs) were characterized into 38 TF families. Meanwhile, we identified significant gene expression trends associated with somatic embryo development in plant hormones (AUX/IAA, YUCCA, LEA, etc.), stress (GST, HSP, GLP, etc.), phenylpropanoid metabolism (4CL, HCT, PAL, etc.), and transcription factors (AP2/ERF, MYB, WOX, etc.). In addition, nine genes were chosen for RT-qPCR, and the results were consistent with RNA-Seq data. This study revealed the changes in morphology, histology, physiology, and gene expression in the differentiation of NEC into EC and laid the foundation for finding the key genes to promote EC formation.

Dataset Information

Combined QTL Mapping across Multiple Environments and Co-Expression Network Analysis Identified Key Genes for Embryogenic Callus Induction from Immature Maize Embryos.

Publications

Combined QTL Mapping across Multiple Environments and Co-Expression Network Analysis Identified Key Genes for Embryogenic Callus Induction from Immature Maize Embryos.

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