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Plant Genomics and Genome Editing

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Plant Sciences".

Deadline for manuscript submissions: closed (31 January 2022) | Viewed by 103995

Special Issue Editor

Special Issue Information

Dear Colleagues,

Scientists to be able to precisely edit genomes efficiently and cost-effectively thanks to the many high-quality reference genome sequences currently available. Zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system have been successfully employed to edit numerous individual genes in many organisms, and CRISPR-edited plants are on the market now. The CRISPR-Cas system has been widely used to edit genetic elements in a genome for various applications—not only crop improvement, but also many gene therapy studies. In this Special Issue, we cordially invite scientists from across the world to contribute their cutting-edge research on plant genomics and genome editing. Original research articles and short communications contributing to the advancement of genomics and genome editing, including techniques, applications, trait development, and enabling database and computational software, are welcome. We are also inviting scientists to write reviews for this Special Issue.  

 
 

Prof. Dr. Hikmet Budak
Guest Editor

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Keywords

  • next generation sequencing
  • structural and functional genomics
  • SNPs
  • miRNA
  • LncRNA
  • CRISPR/Cas9
  • zinc finger nuclease
  • TALEN
  • genome editing

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Published Papers (32 papers)

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14 pages, 2954 KiB  
Article
Genome-Wide Investigation of G6PDH Gene in Strawberry: Evolution and Expression Analysis during Development and Stress
by Diya Lei, Yuanxiu Lin, Mengwen Luo, Bing Zhao, Honglan Tang, Xuan Zhou, Wantian Yao, Yunting Zhang, Yan Wang, Mengyao Li, Qing Chen, Ya Luo, Xiaorong Wang, Haoru Tang and Yong Zhang
Int. J. Mol. Sci. 2022, 23(9), 4728; https://doi.org/10.3390/ijms23094728 - 25 Apr 2022
Cited by 4 | Viewed by 1668
Abstract
As one of the key enzymes in the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) provides NADPH and plays an important role in plant development and stress responses. However, little information was available about the G6PDH genes in strawberry (Fragaria × ananassa [...] Read more.
As one of the key enzymes in the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) provides NADPH and plays an important role in plant development and stress responses. However, little information was available about the G6PDH genes in strawberry (Fragaria × ananassa). The recent release of the whole-genome sequence of strawberry allowed us to perform a genome-wide investigation into the organization and expression profiling of strawberry G6PDH genes. In the present study, 19 strawberry G6PDH genes (FaG6PDHs) were identified from the strawberry genome database. They were designated as FaG6PDH1 to FaG6PDH19, respectively, according to the conserved domain of each subfamily and multiple sequence alignment with Arabidopsis. According to their structural and phylogenetic features, the 19 FaG6PDHs were further classified into five types: Cy, P1, P1.1, P2 and PO. The number and location of exons and introns are similar, suggesting that genes of the same type are very similar and are alleles. A cis-element analysis inferred that FaG6PDHs possessed at least one stress-responsive cis-acting element. Expression profiles derived from transcriptome data analysis exhibited distinct expression patterns of FaG6PDHs genes in different developmental stages. Real-time quantitative PCR was used to detect the expression level of five types FaG6PDHs genes and demonstrated that the genes were expressed and responded to multiple abiotic stress and hormonal treatments. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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24 pages, 16292 KiB  
Article
Combined Analysis of the Metabolome and Transcriptome to Explore Heat Stress Responses and Adaptation Mechanisms in Celery (Apium graveolens L.)
by Mengyao Li, Jie Li, Ran Zhang, Yuanxiu Lin, Aisheng Xiong, Guofei Tan, Ya Luo, Yong Zhang, Qing Chen, Yan Wang, Yunting Zhang, Xiaorong Wang and Haoru Tang
Int. J. Mol. Sci. 2022, 23(6), 3367; https://doi.org/10.3390/ijms23063367 - 20 Mar 2022
Cited by 19 | Viewed by 2753
Abstract
Celery is an important leafy vegetable that can grow during the cool season and does not tolerate high temperatures. Heat stress is widely acknowledged as one of the main abiotic stresses affecting the growth and yield of celery. The morphological and physiological indices [...] Read more.
Celery is an important leafy vegetable that can grow during the cool season and does not tolerate high temperatures. Heat stress is widely acknowledged as one of the main abiotic stresses affecting the growth and yield of celery. The morphological and physiological indices of celery were investigated in the present study to explore the physiological mechanisms in response to high temperatures. Results showed that the antioxidant enzyme activity, proline, relative conductivity, and malondialdehyde were increased, while chlorophyll and the water content of leaves decreased under high-temperature conditions. Short-term heat treatment increased the stomatal conductance to cool off the leaves by transpiration; however, long-term heat treatment led to stomatal closure to prevent leaf dehydration. In addition, high temperature caused a disordered arrangement of palisade tissue and a loose arrangement of spongy tissue in celery leaves. Combined metabolomic and transcriptomic analyses were further used to reveal the regulatory mechanisms in response to heat stress at the molecular level in celery. A total of 1003 differential metabolites were identified and significantly enriched in amino acid metabolism and the tricarboxilic acid (TCA) cycle. Transcriptome sequencing detected 24,264 different genes, including multiple transcription factor families such as HSF, WRKY, MYB, AP2, bZIP, and bHLH family members that were significantly upregulated in response to heat stress, suggesting that these genes were involved in the response to heat stress. In addition, transcriptional and metabolic pathway analyses showed that heat stress inhibited the glycolysis pathway and delayed the TCA cycle but increased the expression of most amino acid synthesis pathways such as proline, arginine, and serine, consistent with the results of physiological indicators. qRT-PCR further showed that the expression pattern was similar to the expression abundance in the transcriptome. The important metabolites and genes in celery that significantly contributed to the response to high temperatures were identified in the present study, which provided the theoretical basis for breeding heat-resistant celery. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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21 pages, 5054 KiB  
Article
The Plastome Sequences of Triticum sphaerococcum (ABD) and Triticum turgidum subsp. durum (AB) Exhibit Evolutionary Changes, Structural Characterization, Comparative Analysis, Phylogenomics and Time Divergence
by Lubna, Sajjad Asaf, Rahmatullah Jan, Abdul Latif Khan, Waqar Ahmad, Saleem Asif, Ahmed Al-Harrasi, Kyung-Min Kim and In-Jung Lee
Int. J. Mol. Sci. 2022, 23(5), 2783; https://doi.org/10.3390/ijms23052783 - 03 Mar 2022
Cited by 5 | Viewed by 1987
Abstract
The mechanism and course of Triticum plastome evolution is currently unknown; thus, it remains unclear how Triticum plastomes evolved during recent polyploidization. Here, we report the complete plastomes of two polyploid wheat species, Triticum sphaerococcum (AABBDD) and Triticum turgidum subsp. durum (AABB), and [...] Read more.
The mechanism and course of Triticum plastome evolution is currently unknown; thus, it remains unclear how Triticum plastomes evolved during recent polyploidization. Here, we report the complete plastomes of two polyploid wheat species, Triticum sphaerococcum (AABBDD) and Triticum turgidum subsp. durum (AABB), and compare them with 19 available and complete Triticum plastomes to create the first map of genomic structural variation. Both T. sphaerococcum and T. turgidum subsp. durum plastomes were found to have a quadripartite structure, with plastome lengths of 134,531 bp and 134,015 bp, respectively. Furthermore, diploid (AA), tetraploid (AB, AG) and hexaploid (ABD, AGAm) Triticum species plastomes displayed a conserved gene content and commonly harbored an identical set of annotated unique genes. Overall, there was a positive correlation between the number of repeats and plastome size. In all plastomes, the number of tandem repeats was higher than the number of palindromic and forward repeats. We constructed a Triticum phylogeny based on the complete plastomes and 42 shared genes from 71 plastomes. We estimated the divergence of Hordeum vulgare from wheat around 11.04–11.9 million years ago (mya) using a well-resolved plastome tree. Similarly, Sitopsis species diverged 2.8–2.9 mya before Triticum urartu (AA) and Triticum monococcum (AA). Aegilops speltoides was shown to be the maternal donor of polyploid wheat genomes and diverged ~0.2–0.9 mya. The phylogeny and divergence time estimates presented here can act as a reference framework for future studies of Triticum evolution. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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18 pages, 4679 KiB  
Article
Cold Response Transcriptome Analysis of the Alternative Splicing Events Induced by the Cold Stress in D. catenatum
by Yan Zheng, Landi Luo, Qian Chen, Danni Yang, Yuqiang Gong, Ya Yang, Xiangshi Qin, Yuhua Wang, Xiangxiang Kong and Yongping Yang
Int. J. Mol. Sci. 2022, 23(2), 981; https://doi.org/10.3390/ijms23020981 - 17 Jan 2022
Cited by 7 | Viewed by 2183
Abstract
Dendrobium catenatum Lindl is a valuable medicinal herb and gardening plant due to its ornamental value and special medical value. Low temperature is a major bottleneck restricting D. catenatum expansion towards the north, which influences the quality and yield of D. catenatum. [...] Read more.
Dendrobium catenatum Lindl is a valuable medicinal herb and gardening plant due to its ornamental value and special medical value. Low temperature is a major bottleneck restricting D. catenatum expansion towards the north, which influences the quality and yield of D. catenatum. In this study, we analysed the cold response of D. catenatum by RNA-Seq. A total of 4302 differentially expressed genes were detected under cold stress, which were mainly linked to protein kinase activity, membrane transport and the glycan biosynthesis and metabolism pathway. We also identified 4005 differential alternative events in 2319 genes significantly regulated by cold stress. Exon skipping and intron retention were the most common alternative splicing isoforms. Numerous genes were identified that differentially modulated under cold stress, including cold-induced transcription factors and splicing factors mediated by AS (alternative splicing). GO enrichment analysis found that differentially alternatively spliced genes without differential expression levels were related to RNA/mRNA processing and spliceosomes. DAS (differentially alternative splicing) genes with different expression levels were mainly enriched in protein kinase activity, plasma membrane and cellular response to stimulus. We further identified and cloned DcCBP20 in D. catenatum; we found that DcCBP20 promotes the generation of alternative splicing variants in cold-induced genes under cold stress via genetic experiments and RT–PCR. Taken together, our results identify the main cold-response pathways and alternative splicing events in D. catenatum responding to cold treatment and that DcCBP20 of D. catenatum get involved in regulating the AS and gene expression of cold-induced genes during this process. Our study will contribute to understanding the role of AS genes in regulating the cold stress response in D. catenatum. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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13 pages, 1935 KiB  
Article
Genome-Wide Identification of OSC Gene Family and Potential Function in the Synthesis of Ursane- and Oleanane-Type Triterpene in Momordica charantia
by Yutong Han, Ya Yang, Yan Li, Xin Yin, Zhiyu Chen, Danni Yang, Yongping Yang, Yunqiang Yang and Xuefei Yang
Int. J. Mol. Sci. 2022, 23(1), 196; https://doi.org/10.3390/ijms23010196 - 24 Dec 2021
Cited by 4 | Viewed by 3027
Abstract
The triterpenes in bitter gourd (Momordica charantia) show a variety of medicinal activities. Oxidosqualene cyclase (OSC) plays an indispensable role in the formation of triterpene skeletons during triterpene biosynthesis. In this study, we identified nine genes encoding OSCs from bitter gourd [...] Read more.
The triterpenes in bitter gourd (Momordica charantia) show a variety of medicinal activities. Oxidosqualene cyclase (OSC) plays an indispensable role in the formation of triterpene skeletons during triterpene biosynthesis. In this study, we identified nine genes encoding OSCs from bitter gourd (McOSC19). Analyses of their expression patterns in different tissues suggested that characteristic triterpenoids may be biosynthesized in different tissues and then transported. We constructed a hairy root system in which McOSC7 overexpression led to an increased accumulation of camaldulenic acid, enoxolone, and quinovic acid. Thus, the overexpression of McOSC7 increased the active components content in bitter gourd. Our data provide an important foundation for understanding the roles of McOSCs in triterpenoid synthesis. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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17 pages, 3280 KiB  
Article
Organelle Comparative Genome Analysis Reveals Novel Alloplasmic Male Sterility with orf112 in Brassica oleracea L.
by Li Chen, Wenjing Ren, Bin Zhang, Wendi Chen, Zhiyuan Fang, Limei Yang, Mu Zhuang, Honghao Lv, Yong Wang, Jialei Ji and Yangyong Zhang
Int. J. Mol. Sci. 2021, 22(24), 13230; https://doi.org/10.3390/ijms222413230 - 08 Dec 2021
Cited by 2 | Viewed by 2097
Abstract
B. oleracea Ogura CMS is an alloplasmic male-sterile line introduced from radish by interspecific hybridization and protoplast fusion. The introduction of alien cytoplasm resulted in many undesirable traits, which affected the yield of hybrids. Therefore, it is necessary to identify the composition and [...] Read more.
B. oleracea Ogura CMS is an alloplasmic male-sterile line introduced from radish by interspecific hybridization and protoplast fusion. The introduction of alien cytoplasm resulted in many undesirable traits, which affected the yield of hybrids. Therefore, it is necessary to identify the composition and reduce the content of alien cytoplasm in B. oleracea Ogura CMS. In the present study, we sequenced, assembled, and compared the organelle genomes of Ogura CMS cabbage and its maintainer line. The chloroplast genome of Ogura-type cabbage was completely derived from normal-type cabbage, whereas the mitochondrial genome was recombined from normal-type cabbage and Ogura-type radish. Nine unique regions derived from radish were identified in the mitochondrial genome of Ogura-type cabbage, and the total length of these nine regions was 35,618 bp, accounting for 13.84% of the mitochondrial genome. Using 32 alloplasmic markers designed according to the sequences of these nine regions, one novel sterile source with less alien cytoplasm was discovered among 305 materials and named Bel CMS. The size of the alien cytoplasm in Bel CMS was 21,587 bp, accounting for 8.93% of its mtDNA, which was much less than that in Ogura CMS. Most importantly, the sterility gene orf138 was replaced by orf112, which had a 78-bp deletion, in Bel CMS. Interestingly, Bel CMS cabbage also maintained 100% sterility, although orf112 had 26 fewer amino acids than orf138. Field phenotypic observation showed that Bel CMS was an excellent sterile source with stable 100% sterility and no withered buds at the early flowering stage, which could replace Ogura CMS in cabbage heterosis utilization. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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17 pages, 3023 KiB  
Article
Transcriptional Responses and GCMS Analysis for the Biosynthesis of Pyrethrins and Volatile Terpenes in Tanacetum coccineum
by Tuo Zeng, Jia-Wen Li, Li Zhou, Zhi-Zhuo Xu, Jin-Jin Li, Hao Hu, Jing Luo, Ri-Ru Zheng, Yuan-Yuan Wang and Cai-Yun Wang
Int. J. Mol. Sci. 2021, 22(23), 13005; https://doi.org/10.3390/ijms222313005 - 30 Nov 2021
Cited by 9 | Viewed by 1849
Abstract
Natural pyrethrins have been widely used as natural pesticides due to their low mammalian toxicity and environmental friendliness. Previous studies have mainly focused on Tanacetumcinerariifolium, which contains high levels of pyrethrins and volatile terpenes that play significant roles in plant defense [...] Read more.
Natural pyrethrins have been widely used as natural pesticides due to their low mammalian toxicity and environmental friendliness. Previous studies have mainly focused on Tanacetumcinerariifolium, which contains high levels of pyrethrins and volatile terpenes that play significant roles in plant defense and pollination. However, there is little information on T. coccineum due to its lower pyrethrin content and low commercial value. In this study, we measured the transcriptome and metabolites of the leaves (L), flower buds (S1), and fully blossomed flowers (S4) of T. coccineum. The results show that the expression of pyrethrins and precursor terpene backbone genes was low in the leaves, and then rapidly increased in the S1 stage before decreasing again in the S4 stage. The results also show that pyrethrins primarily accumulated at the S4 stage. However, the content of volatile terpenes was consistently low. This perhaps suggests that, despite T. coccineum and T. cinerariifolium having similar gene expression patterns and accumulation of pyrethrins, T. coccineum attracts pollinators via its large and colorful flowers rather than via inefficient and metabolically expensive volatile terpenes, as in T. cinerariifolium. This is the first instance of de novo transcriptome sequencing reported for T. coccineum. The present results could provide insights into pyrethrin biosynthetic pathways and will be helpful for further understanding how plants balance the cost–benefit relationship between plant defense and pollination. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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20 pages, 2840 KiB  
Article
Root-to-Shoot Long-Distance Mobile miRNAs Identified from Nicotiana Rootstocks
by Zhuying Deng, Huiyan Wu, Dongyi Li, Luping Li, Zhipeng Wang, Wenya Yuan, Yongzhong Xing, Chengdao Li and Dacheng Liang
Int. J. Mol. Sci. 2021, 22(23), 12821; https://doi.org/10.3390/ijms222312821 - 26 Nov 2021
Cited by 11 | Viewed by 2173
Abstract
Root-derived mobile signals play critical roles in coordinating a shoot’s response to underground conditions. However, the identification of root-to-shoot long-distance mobile signals has been scant. In this study, we aimed to characterize root-to-shoot endogenous mobile miRNAs by using an Arabidopsis/Nicotiana interfamilial heterograft in [...] Read more.
Root-derived mobile signals play critical roles in coordinating a shoot’s response to underground conditions. However, the identification of root-to-shoot long-distance mobile signals has been scant. In this study, we aimed to characterize root-to-shoot endogenous mobile miRNAs by using an Arabidopsis/Nicotiana interfamilial heterograft in which these two taxonomically distant species with clear genetic backgrounds had sufficient diversity in differentiating miRNA sources. Small RNA deep sequencing analysis revealed that 82 miRNAs from the Arabidopsis scion could travel through the graft union to reach the rootstock, whereas only a very small subset of miRNA (6 miRNAs) preferred the root-to-shoot movement. We demonstrated in an ex vivo RNA imaging experiment that the root-to-shoot mobile Nb-miR164, Nb-miR395 and Nb-miR397 were targeted to plasmodesmata using the bacteriophage coat protein MS2 system. Furthermore, the Nb-miR164 was shown to move from the roots to the shoots to induce phenotypic changes when its overexpressing line was used as rootstock, strongly supporting that root-derived Nb-miR164 was able to modify the scion trait via its long-distance movement. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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23 pages, 5220 KiB  
Article
Plastidial Expression of 3β-Hydroxysteroid Dehydrogenase and Progesterone 5β-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco
by Muhammad Sameeullah, Muhammet Yildirim, Noreen Aslam, Mehmet Cengiz Baloğlu, Buhara Yucesan, Andreas G. Lössl, Kiran Saba, Mohammad Tahir Waheed and Ekrem Gurel
Int. J. Mol. Sci. 2021, 22(21), 11736; https://doi.org/10.3390/ijms222111736 - 29 Oct 2021
Cited by 4 | Viewed by 2934
Abstract
The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3β-hydroxysteroid dehydrogenase (3β-HSD) and progesterone 5-β-reductases (P5βR1, P5βR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, [...] Read more.
The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3β-hydroxysteroid dehydrogenase (3β-HSD) and progesterone 5-β-reductases (P5βR1, P5βR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 3β-HSD, P5βR1 and P5βR2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end-to-end PCR, qRT-PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3β-HSD and P5βR1 are cytoplasmic, and P5βR2 is tonoplast-localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt-tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+/K+ ratio in transplastomic lines was also observed. The salt tolerance, mediated by plastidial expression of the 3β-HSD, P5βR1 and P5βR2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid-based expression of the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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17 pages, 4436 KiB  
Article
Genome-Wide Identification and Transcriptional Analysis of Arabidopsis DUF506 Gene Family
by Sheng Ying
Int. J. Mol. Sci. 2021, 22(21), 11442; https://doi.org/10.3390/ijms222111442 - 23 Oct 2021
Cited by 8 | Viewed by 3397
Abstract
The Domain of unknown function 506 (DUF506) family, which belongs to the PD-(D/E)XK nuclease superfamily, has not been functionally characterized. In this study, 266 DUF506 domain-containing genes were identified from algae, mosses, and land plants showing their wide occurrence in photosynthetic [...] Read more.
The Domain of unknown function 506 (DUF506) family, which belongs to the PD-(D/E)XK nuclease superfamily, has not been functionally characterized. In this study, 266 DUF506 domain-containing genes were identified from algae, mosses, and land plants showing their wide occurrence in photosynthetic organisms. Bioinformatics analysis identified 211 high-confidence DUF506 genes across 17 representative land plant species. Phylogenetic modeling classified three groups of plant DUF506 genes that suggested functional preservation among the groups based on conserved gene structure and motifs. Gene duplication and Ka/Ks evolutionary rates revealed that DUF506 genes are under purifying positive selection pressure. Subcellular protein localization analysis revealed that DUF506 proteins were present in different organelles. Transcript analyses showed that 13 of the Arabidopsis DUF506 genes are ubiquitously expressed in various tissues and respond to different abiotic stresses and ABA treatment. Protein-protein interaction network analysis using the STRING-DB, AtPIN (Arabidopsis thaliana Protein Interaction Network), and AI-1 (Arabidopsis Interactome-1) tools indicated that AtDUF506s potentially interact with iron-deficiency response proteins, salt-inducible transcription factors, or calcium sensors (calmodulins), implying that DUF506 genes have distinct biological functions including responses to environmental stimuli, nutrient-deficiencies, and participate in Ca(2+) signaling. Current results provide insightful information regarding the molecular features of the DUF506 family in plants, to support further functional characterizations. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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15 pages, 1771 KiB  
Article
Two TGA Transcription Factor Members from Hyper-Susceptible Soybean Exhibiting Significant Basal Resistance to Soybean mosaic virus
by Hua Jiang, Shengyu Gu, Kai Li and Junyi Gai
Int. J. Mol. Sci. 2021, 22(21), 11329; https://doi.org/10.3390/ijms222111329 - 20 Oct 2021
Cited by 4 | Viewed by 1747
Abstract
TGA transcription factors (TFs) exhibit basal resistance in Arabidopsis, but susceptibility to a pathogen attack in tomatoes; however, their roles in soybean (Glycine max) to Soybean mosaic virus (SMV) are unknown. In this study, 27 TGA genes were isolated from [...] Read more.
TGA transcription factors (TFs) exhibit basal resistance in Arabidopsis, but susceptibility to a pathogen attack in tomatoes; however, their roles in soybean (Glycine max) to Soybean mosaic virus (SMV) are unknown. In this study, 27 TGA genes were isolated from a SMV hyper-susceptible soybean NN1138-2, designated GmTGA1~GmTGA27, which were clustered into seven phylogenetic groups. The expression profiles of GmTGAs showed that the highly expressed genes were mainly in Groups I, II, and VII under non-induction conditions, while out of the 27 GmTGAs, 19 responded to SMV-induction. Interestingly, in further transient N. benthamiana-SMV pathosystem assay, all the 19 GmTGAs overexpressed did not promote SMV infection in inoculated leaves, but they exhibited basal resistance except one without function. Among the 18 functional ones, GmTGA8 and GmTGA19, with similar motif distribution, nuclear localization sequence and interaction proteins, showed a rapid response to SMV infection and performed better than the others in inhibiting SMV multiplication. This finding suggested that GmTGA TFs may support basal resistance to SMV even from a hyper-susceptible source. What the mechanism of the genes (GmTGA8, GmTGA19, etc.) with basal resistance to SMV is and what their potential for the future improvement of resistance to SMV in soybeans is, are to be explored. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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11 pages, 2464 KiB  
Article
Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts
by Magdalena Klimek-Chodacka, Miron Gieniec and Rafal Baranski
Int. J. Mol. Sci. 2021, 22(19), 10740; https://doi.org/10.3390/ijms221910740 - 04 Oct 2021
Cited by 4 | Viewed by 2661
Abstract
The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in [...] Read more.
The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33–152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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18 pages, 10653 KiB  
Article
Genome-Wide Identification and Expression Analysis of the Aux/IAA and Auxin Response Factor Gene Family in Medicago truncatula
by Rui Liu, Zhenfei Guo and Shaoyun Lu
Int. J. Mol. Sci. 2021, 22(19), 10494; https://doi.org/10.3390/ijms221910494 - 28 Sep 2021
Cited by 16 | Viewed by 2090
Abstract
Aux/IAA and auxin response transcription factor (ARF) genes are key regulators of auxin responses in plants. A total of 25 MtIAA and 40 MtARF genes were identified based on the latest updated Medicago truncatula reference genome sequence. They were clustered into [...] Read more.
Aux/IAA and auxin response transcription factor (ARF) genes are key regulators of auxin responses in plants. A total of 25 MtIAA and 40 MtARF genes were identified based on the latest updated Medicago truncatula reference genome sequence. They were clustered into 10 and 8 major groups, respectively. The homologs among M. truncatula, soybean, and Arabidopsis thaliana shared close relationships based on phylogenetic analysis. Gene structure analysis revealed that MtIAA and MtARF genes contained one to four concern motifs and they are localized to eight chromosomes, except chromosome 6 without MtARFs. In addition, some MtIAA and MtARF genes were expressed in all tissues, while others were specifically expressed in specific tissues. Analysis of cis-acting elements in promoter region and expression profiles revealed the potential response of MtIAA and MtARF genes to hormones and abiotic stresses. The prediction protein–protein interaction network showed that some ARF proteins could interact with multiple Aux/IAA proteins, and the reverse is also true. The investigation provides valuable, basic information for further studies on the biological functions of MtIAA and MtARF genes in the regulation of auxin-related pathways in M. truncatula. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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12 pages, 2393 KiB  
Article
Global Survey of the Full-Length Cabbage Transcriptome (Brassica oleracea Var. capitata L.) Reveals Key Alternative Splicing Events Involved in Growth and Disease Response
by Yong Wang, Jialei Ji, Long Tong, Zhiyuan Fang, Limei Yang, Mu Zhuang, Yangyong Zhang and Honghao Lv
Int. J. Mol. Sci. 2021, 22(19), 10443; https://doi.org/10.3390/ijms221910443 - 28 Sep 2021
Cited by 5 | Viewed by 1822
Abstract
Cabbage (Brassica oleracea L. var. capitata L.) is an important vegetable crop cultivated around the world. Previous studies of cabbage gene transcripts were primarily based on next-generation sequencing (NGS) technology which cannot provide accurate information concerning transcript assembly and structure analysis. To [...] Read more.
Cabbage (Brassica oleracea L. var. capitata L.) is an important vegetable crop cultivated around the world. Previous studies of cabbage gene transcripts were primarily based on next-generation sequencing (NGS) technology which cannot provide accurate information concerning transcript assembly and structure analysis. To overcome these issues and analyze the whole cabbage transcriptome at the isoform level, PacBio RS II Single-Molecule Real-Time (SMRT) sequencing technology was used for a global survey of the full-length transcriptomes of five cabbage tissue types (root, stem, leaf, flower, and silique). A total of 77,048 isoforms, capturing 18,183 annotated genes, were discovered from the sequencing data generated through SMRT. The patterns of both alternative splicing (AS) and alternative polyadenylation (APA) were comprehensively analyzed. In total, we detected 13,468 genes which had isoforms containing APA sites and 8978 genes which underwent AS events. Moreover, 5272 long non-coding RNAs (lncRNAs) were discovered, and most exhibited tissue-specific expression. In total, 3147 transcription factors (TFs) were detected and 10 significant gene co-expression network modules were identified. In addition, we found that Fusarium wilt, black rot and clubroot infection significantly influenced AS in resistant cabbage. In summary, this study provides abundant cabbage isoform transcriptome data, which promotes reannotation of the cabbage genome, deepens our understanding of their post-transcriptional regulation mechanisms, and can be used for future functional genomic research. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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15 pages, 2186 KiB  
Article
Multiple Variant Calling Pipelines in Wheat Whole Exome Sequencing
by H. Busra Cagirici, Bala Ani Akpinar, Taner Z. Sen and Hikmet Budak
Int. J. Mol. Sci. 2021, 22(19), 10400; https://doi.org/10.3390/ijms221910400 - 27 Sep 2021
Cited by 2 | Viewed by 3120
Abstract
The highly challenging hexaploid wheat (Triticum aestivum) genome is becoming ever more accessible due to the continued development of multiple reference genomes, a factor which aids in the plight to better understand variation in important traits. Although the process of variant [...] Read more.
The highly challenging hexaploid wheat (Triticum aestivum) genome is becoming ever more accessible due to the continued development of multiple reference genomes, a factor which aids in the plight to better understand variation in important traits. Although the process of variant calling is relatively straightforward, selection of the best combination of the computational tools for read alignment and variant calling stages of the analysis and efficient filtering of the false variant calls are not always easy tasks. Previous studies have analyzed the impact of methods on the quality metrics in diploid organisms. Given that variant identification in wheat largely relies on accurate mining of exome data, there is a critical need to better understand how different methods affect the analysis of whole exome sequencing (WES) data in polyploid species. This study aims to address this by performing whole exome sequencing of 48 wheat cultivars and assessing the performance of various variant calling pipelines at their suggested settings. The results show that all the pipelines require filtering to eliminate false-positive calls. The high consensus among the reference SNPs called by the best-performing pipelines suggests that filtering provides accurate and reproducible results. This study also provides detailed comparisons for high sensitivity and precision at individual and population levels for the raw and filtered SNP calls. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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28 pages, 7305 KiB  
Article
Genome-Wide Identification, Evolution, and Comparative Analysis of B-Box Genes in Brassica rapa, B. oleracea, and B. napus and Their Expression Profiling in B. rapa in Response to Multiple Hormones and Abiotic Stresses
by Sonam Singh, Sushil Satish Chhapekar, Yinbo Ma, Jana Jeevan Rameneni, Sang Heon Oh, Jusang Kim, Yong Pyo Lim and Su Ryun Choi
Int. J. Mol. Sci. 2021, 22(19), 10367; https://doi.org/10.3390/ijms221910367 - 26 Sep 2021
Cited by 8 | Viewed by 2717
Abstract
The B-box zinc-finger transcription factors are important for plant growth, development, and various physiological processes such as photomorphogenesis, light signaling, and flowering, as well as for several biotic and abiotic stress responses. However, there is relatively little information available regarding Brassica B-box genes [...] Read more.
The B-box zinc-finger transcription factors are important for plant growth, development, and various physiological processes such as photomorphogenesis, light signaling, and flowering, as well as for several biotic and abiotic stress responses. However, there is relatively little information available regarding Brassica B-box genes and their expression. In this study, we identified 51, 52, and 101 non-redundant genes encoding B-box proteins in Brassica rapa (BrBBX genes), B. oleracea (BoBBX genes), and B. napus (BnBBX genes), respectively. A whole-genome identification, characterization, and evolutionary analysis (synteny and orthology) of the B-box gene families in the diploid species B. rapa (A genome) and B. oleracea (C genome) and in the allotetraploid species B. napus (AC genome) revealed segmental duplications were the major contributors to the expansion of the BrassicaBBX gene families. The BrassicaBBX genes were classified into five subgroups according to phylogenetic relationships, gene structures, and conserved domains. Light-responsive cis-regulatory elements were detected in many of the BBX gene promoters. Additionally, BrBBX expression profiles in different tissues and in response to various abiotic stresses (heat, cold, salt, and drought) or hormones (abscisic acid, methyl jasmonate, and gibberellic acid) were analyzed by qRT-PCR. The data indicated that many B-box genes (e.g., BrBBX13, BrBBX15, and BrBBX17) may contribute to plant development and growth as well as abiotic stress tolerance. Overall, the identified BBX genes may be useful as functional genetic markers for multiple stress responses and plant developmental processes. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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11 pages, 2836 KiB  
Article
Genome-Wide Analysis of the TCP Transcription Factor Genes in Dendrobium catenatum Lindl.
by Li Zhang, Cheng Li, Danni Yang, Yuhua Wang, Yongping Yang and Xudong Sun
Int. J. Mol. Sci. 2021, 22(19), 10269; https://doi.org/10.3390/ijms221910269 - 24 Sep 2021
Cited by 15 | Viewed by 2200
Abstract
Teosinte branched1/cycloidea/proliferating cell factor (TCP) gene family members are plant-specific transcription factors that regulate plant growth and development by controlling cell proliferation and differentiation. However, there are no reported studies on the TCP gene family in Dendrobium catenatum Lindl. Here, a [...] Read more.
Teosinte branched1/cycloidea/proliferating cell factor (TCP) gene family members are plant-specific transcription factors that regulate plant growth and development by controlling cell proliferation and differentiation. However, there are no reported studies on the TCP gene family in Dendrobium catenatum Lindl. Here, a genome-wide analysis of TCP genes was performed in D. catenatum, and 25 TCP genes were identified. A phylogenetic analysis classified the family into two clades: Class I and Class II. Genes in the same clade share similar conserved motifs. The GFP signals of the DcaTCP-GFPs were detected in the nuclei of tobacco leaf epidermal cells. The activity of DcaTCP4, which contains the miR319a-binding sequence, was reduced when combined with miR319a. A transient activity assay revealed antagonistic functions of Class I and Class II of the TCP proteins in controlling leaf development through the jasmonate-signaling pathway. After different phytohormone treatments, the DcaTCP genes showed varied expression patterns. In particular, DcaTCP4 and DcaTCP9 showed opposite trends after 3 h treatment with jasmonate. This comprehensive analysis provides a foundation for further studies on the roles of TCP genes in D. catenatum. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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17 pages, 6130 KiB  
Article
Genome-Wide Identification and Analysis of the Metallothionein Genes in Oryza Genus
by Mingxing Cheng, Huanran Yuan, Ruihua Wang, Jianing Zou, Ting Liang, Fang Yang and Shaoqing Li
Int. J. Mol. Sci. 2021, 22(17), 9651; https://doi.org/10.3390/ijms22179651 - 06 Sep 2021
Cited by 7 | Viewed by 2690
Abstract
Metallothionein (MT) proteins are low molecular mass, cysteine-rich, and metal-binding proteins that play an important role in maintaining metal homeostasis and stress response. However, the evolutionary relationships and functional differentiation of MT in the Oryza genus remain unclear. Here we identified 53 MT [...] Read more.
Metallothionein (MT) proteins are low molecular mass, cysteine-rich, and metal-binding proteins that play an important role in maintaining metal homeostasis and stress response. However, the evolutionary relationships and functional differentiation of MT in the Oryza genus remain unclear. Here we identified 53 MT genes from six Oryza genera, including O. sativa ssp. japonica, O. rufipogon, O. sativa ssp. indica, O. nivara, O. glumaepatula, and O. barthii. The MT genes were clustered into four groups based on phylogenetic analysis. MT genes are unevenly distributed on chromosomes; almost half of the MT genes were clustered on chromosome 12, which may result from a fragment duplication containing the MT genes on chromosome 12. Five pairs of segmental duplication events and ten pairs of tandem duplication events were found in the rice MT family. The Ka/Ks values of the fifteen duplicated MT genes indicated that the duplicated MT genes were under a strong negative selection during evolution. Next, combining the promoter activity assay with gene expression analysis revealed different expression patterns of MT genes. In addition, the expression of OsMT genes was induced under different stresses, including NaCl, CdCl2, ABA, and MeJ treatments. Additionally, we found that OsMT genes were mainly located in chloroplasts. These results imply that OsMT genes play different roles in response to these stresses. All results provide important insights into the evolution of the MT gene family in the Oryza genus, and will be helpful to further study the function of MT genes. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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13 pages, 1451 KiB  
Article
Genome-Wide Identification of ARF Transcription Factor Gene Family and Their Expression Analysis in Sweet Potato
by Isaac Seth Pratt and Baohong Zhang
Int. J. Mol. Sci. 2021, 22(17), 9391; https://doi.org/10.3390/ijms22179391 - 30 Aug 2021
Cited by 8 | Viewed by 2290
Abstract
Auxin response factors (ARFs) are a family of transcription factors that play an important role of auxin regulation through their binding with auxin response elements. ARF genes are represented by a large multigene family in plants; however, to our knowledge, the ARF gene [...] Read more.
Auxin response factors (ARFs) are a family of transcription factors that play an important role of auxin regulation through their binding with auxin response elements. ARF genes are represented by a large multigene family in plants; however, to our knowledge, the ARF gene family has not been well studied and characterized in sweet potatoes. In this study, a total of 25 ARF genes were identified in Ipomea trifida. The identified ItrARF genes’ conserved motifs, chromosomal locations, phylogenetic relationships, and their protein characteristics were systemically investigated using different bioinformatics tools. The expression patterns of ItfARF genes were analyzed within the storage roots and normal roots at an early stage of development. ItfARF16b and ItfARF16c were both highly expressed in the storage root, with minimal to no expression in the normal root. ItfARF6a and ItfARF10a exhibited higher expression in the normal root but not in the storage root. Subsequently, ItfARF1a, ItfARF2b, ItfARF3a, ItfARF6b, ItfARF8a, ItfARF8b, and ItfARF10b were expressed in both root types with moderate to high expression for each. All ten of these ARF genes and their prominent expression signify their importance within the development of each respective root type. This study provides comprehensive information regarding the ARF family in sweet potatoes, which will be useful for future research to discover further functional verification of these ItfARF genes. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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13 pages, 3590 KiB  
Article
Genome-Wide Identification and Expression Analysis of BBX Transcription Factors in Iris germanica L.
by Yinjie Wang, Yongxia Zhang, Qingquan Liu, Ting Zhang, Xinran Chong and Haiyan Yuan
Int. J. Mol. Sci. 2021, 22(16), 8793; https://doi.org/10.3390/ijms22168793 - 17 Aug 2021
Cited by 7 | Viewed by 2351
Abstract
The family of B-box (BBX) transcription factors contains one or two B-BOX domains and sometimes also features a highly conserved CCT domain, which plays important roles in plant growth, development and stress response. Nevertheless, no systematic study of the BBX gene family in [...] Read more.
The family of B-box (BBX) transcription factors contains one or two B-BOX domains and sometimes also features a highly conserved CCT domain, which plays important roles in plant growth, development and stress response. Nevertheless, no systematic study of the BBX gene family in Iris germanica L. has been undertaken. In this study, a set of six BBX TF family genes from I. germanica was identified based on transcriptomic sequences, and clustered into three clades according to phylogenetic analysis. A transient expression analysis revealed that all six BBX proteins were localized in the nucleus. A yeast one-hybrid assay demonstrated that IgBBX3 has transactivational activity, while IgBBX1, IgBBX2, IgBBX4, and IgBBX5 have no transcriptional activation ability. The transcript abundance of IgBBXs in different tissues was divided into two major groups. The expression of IgBBX1, IgBBX2, IgBBX3 and IgBBX5 was higher in leaves, whereas IgBBX4 and IgBBX6 was higher in roots. The stress response patterns of six IgBBX were detected under phytohormone treatments and abiotic stresses. The results of this study lay the basis for further research on the functions of BBX gene family members in plant hormone and stress responses, which will promote their application in I. germanica breeding. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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24 pages, 11608 KiB  
Article
Transcriptome Analysis of Flower Development and Mining of Genes Related to Flowering Time in Tomato (Solanum lycopersicum)
by Hexuan Wang, Yahui Yang, Yiyao Zhang, Tingting Zhao, Jingbin Jiang, Jingfu Li, Xiangyang Xu and Huanhuan Yang
Int. J. Mol. Sci. 2021, 22(15), 8128; https://doi.org/10.3390/ijms22158128 - 29 Jul 2021
Cited by 12 | Viewed by 3070
Abstract
Flowering is a morphogenetic process in which angiosperms shift from vegetative growth to reproductive growth. Flowering time has a strong influence on fruit growth, which is closely related to productivity. Therefore, research on crop flowering time is particularly important. To better understand the [...] Read more.
Flowering is a morphogenetic process in which angiosperms shift from vegetative growth to reproductive growth. Flowering time has a strong influence on fruit growth, which is closely related to productivity. Therefore, research on crop flowering time is particularly important. To better understand the flowering period of the tomato, we performed transcriptome sequencing of early flower buds and flowers during the extension period in the later-flowering “Moneymaker” material and the earlier-flowering “20965” homozygous inbred line, and we analyzed the obtained data. At least 43.92 million clean reads were obtained from 12 datasets, and the similarity with the tomato internal reference genome was 92.86–94.57%. Based on gene expression and background annotations, 49 candidate genes related to flowering time and flower development were initially screened, among which the greatest number belong to the photoperiod pathway. According to the expression pattern of candidate genes, the cause of early flowering of “20965” is predicted. The modes of action of the differentially expressed genes were classified, and the results show that they are closely related to hormone regulation and participated in a variety of life activities in crops. The candidate genes we screened and the analysis of their expression patterns provide a basis for future functional verification, helping to explore the molecular mechanism of tomato flowering time more comprehensively. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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15 pages, 25760 KiB  
Article
Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci
by Krzysztof Michalski, Christian Hertig, Dariusz R. Mańkowski, Jochen Kumlehn, Janusz Zimny and Anna M. Linkiewicz
Int. J. Mol. Sci. 2021, 22(13), 7038; https://doi.org/10.3390/ijms22137038 - 29 Jun 2021
Cited by 7 | Viewed by 2538
Abstract
Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, [...] Read more.
Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8′-HYDROXYLASE 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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14 pages, 3580 KiB  
Article
Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9
by Yuya Liang, Sudip Biswas, Backki Kim, Julia Bailey-Serres and Endang M. Septiningsih
Int. J. Mol. Sci. 2021, 22(13), 6989; https://doi.org/10.3390/ijms22136989 - 29 Jun 2021
Cited by 16 | Viewed by 4221
Abstract
Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery [...] Read more.
Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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24 pages, 6067 KiB  
Article
Small RNA, Transcriptome and Degradome Analysis of the Transgenerational Heat Stress Response Network in Durum Wheat
by Haipei Liu, Amanda J. Able and Jason A. Able
Int. J. Mol. Sci. 2021, 22(11), 5532; https://doi.org/10.3390/ijms22115532 - 24 May 2021
Cited by 10 | Viewed by 2631
Abstract
Heat stress is a major limiting factor of grain yield and quality in crops. Abiotic stresses have a transgenerational impact and the mechanistic basis is associated with epigenetic regulation. The current study presents the first systematic analysis of the transgenerational effects of post-anthesis [...] Read more.
Heat stress is a major limiting factor of grain yield and quality in crops. Abiotic stresses have a transgenerational impact and the mechanistic basis is associated with epigenetic regulation. The current study presents the first systematic analysis of the transgenerational effects of post-anthesis heat stress in tetraploid wheat. Leaf physiological traits, harvest components and grain quality traits were characterized under the impact of parental and progeny heat stress. The parental heat stress treatment had a positive influence on the offspring for traits including chlorophyll content, grain weight, grain number and grain total starch content. Integrated sequencing analysis of the small RNAome, mRNA transcriptome and degradome provided the first description of the molecular networks mediating heat stress adaptation under transgenerational influence. The expression profile of 1771 microRNAs (733 being novel) and 66,559 genes was provided, with differentially expressed microRNAs and genes characterized subject to the progeny treatment, parental treatment and tissue-type factors. Gene Ontology and KEGG pathway analysis of stress responsive microRNAs-mRNA modules provided further information on their functional roles in biological processes such as hormone homeostasis, signal transduction and protein stabilization. Our results provide new insights on the molecular basis of transgenerational heat stress adaptation, which can be used for improving thermo-tolerance in breeding. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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12 pages, 1923 KiB  
Article
An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.)
by Fernando Perez Rojo, Sumedha Seth, William Erskine and Parwinder Kaur
Int. J. Mol. Sci. 2021, 22(8), 4181; https://doi.org/10.3390/ijms22084181 - 17 Apr 2021
Cited by 6 | Viewed by 2974
Abstract
Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the [...] Read more.
Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L−1 hygromycin, 100 mg L−1 kanamycin and 200 mg L−1 cefotaxime. Additionally, 200 mg L−1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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Review

Jump to: Research

33 pages, 1495 KiB  
Review
Applications of Genomic Tools in Plant Breeding: Crop Biofortification
by Inés Medina-Lozano and Aurora Díaz
Int. J. Mol. Sci. 2022, 23(6), 3086; https://doi.org/10.3390/ijms23063086 - 13 Mar 2022
Cited by 5 | Viewed by 3706
Abstract
Crop breeding has mainly been focused on increasing productivity, either directly or by decreasing the losses caused by biotic and abiotic stresses (that is, incorporating resistance to diseases and enhancing tolerance to adverse conditions, respectively). Quite the opposite, little attention has been paid [...] Read more.
Crop breeding has mainly been focused on increasing productivity, either directly or by decreasing the losses caused by biotic and abiotic stresses (that is, incorporating resistance to diseases and enhancing tolerance to adverse conditions, respectively). Quite the opposite, little attention has been paid to improve the nutritional value of crops. It has not been until recently that crop biofortification has become an objective within breeding programs, through either conventional methods or genetic engineering. There are many steps along this long path, from the initial evaluation of germplasm for the content of nutrients and health-promoting compounds to the development of biofortified varieties, with the available and future genomic tools assisting scientists and breeders in reaching their objectives as well as speeding up the process. This review offers a compendium of the genomic technologies used to explore and create biodiversity, to associate the traits of interest to the genome, and to transfer the genomic regions responsible for the desirable characteristics into potential new varieties. Finally, a glimpse of future perspectives and challenges in this emerging area is offered by taking the present scenario and the slow progress of the regulatory framework as the starting point. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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17 pages, 969 KiB  
Review
Expanding Gene-Editing Potential in Crop Improvement with Pangenomes
by Cassandria G. Tay Fernandez, Benjamin J. Nestor, Monica F. Danilevicz, Jacob I. Marsh, Jakob Petereit, Philipp E. Bayer, Jacqueline Batley and David Edwards
Int. J. Mol. Sci. 2022, 23(4), 2276; https://doi.org/10.3390/ijms23042276 - 18 Feb 2022
Cited by 11 | Viewed by 3151
Abstract
Pangenomes aim to represent the complete repertoire of the genome diversity present within a species or cohort of species, capturing the genomic structural variance between individuals. This genomic information coupled with phenotypic data can be applied to identify genes and alleles involved with [...] Read more.
Pangenomes aim to represent the complete repertoire of the genome diversity present within a species or cohort of species, capturing the genomic structural variance between individuals. This genomic information coupled with phenotypic data can be applied to identify genes and alleles involved with abiotic stress tolerance, disease resistance, and other desirable traits. The characterisation of novel structural variants from pangenomes can support genome editing approaches such as Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein Cas (CRISPR-Cas), providing functional information on gene sequences and new target sites in variant-specific genes with increased efficiency. This review discusses the application of pangenomes in genome editing and crop improvement, focusing on the potential of pangenomes to accurately identify target genes for CRISPR-Cas editing of plant genomes while avoiding adverse off-target effects. We consider the limitations of applying CRISPR-Cas editing with pangenome references and potential solutions to overcome these limitations. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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35 pages, 1236 KiB  
Review
Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants
by Maninder Kaur, Pooja Manchanda, Anu Kalia, Farah K. Ahmed, Eugenie Nepovimova, Kamil Kuca and Kamel A. Abd-Elsalam
Int. J. Mol. Sci. 2021, 22(19), 10882; https://doi.org/10.3390/ijms221910882 - 08 Oct 2021
Cited by 17 | Viewed by 7007
Abstract
Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome [...] Read more.
Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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26 pages, 1573 KiB  
Review
Anthocyanin Biosynthesis Genes as Model Genes for Genome Editing in Plants
by Emil Khusnutdinov, Anna Sukhareva, Maria Panfilova and Elena Mikhaylova
Int. J. Mol. Sci. 2021, 22(16), 8752; https://doi.org/10.3390/ijms22168752 - 15 Aug 2021
Cited by 38 | Viewed by 7491
Abstract
CRISPR/Cas, one of the most rapidly developing technologies in the world, has been applied successfully in plant science. To test new nucleases, gRNA expression systems and other inventions in this field, several plant genes with visible phenotypic effects have been constantly used as [...] Read more.
CRISPR/Cas, one of the most rapidly developing technologies in the world, has been applied successfully in plant science. To test new nucleases, gRNA expression systems and other inventions in this field, several plant genes with visible phenotypic effects have been constantly used as targets. Anthocyanin pigmentation is one of the most easily identified traits, that does not require any additional treatment. It is also associated with stress resistance, therefore plants with edited anthocyanin genes might be of interest for agriculture. Phenotypic effect of CRISPR/Cas editing of PAP1 and its homologs, DFR, F3H and F3′H genes have been confirmed in several distinct plant species. DFR appears to be a key structural gene of anthocyanin biosynthesis, controlled by various transcription factors. There are still many promising potential model genes that have not been edited yet. Some of them, such as Delila, MYB60, HAT1, UGT79B2, UGT79B3 and miR156, have been shown to regulate drought tolerance in addition to anthocyanin biosynthesis. Genes, also involved in trichome development, such as TTG1, GLABRA2, MYBL2 and CPC, can provide increased visibility. In this review successful events of CRISPR/Cas editing of anthocyanin genes are summarized, and new model genes are proposed. It can be useful for molecular biologists and genetic engineers, crop scientists, plant genetics and physiologists. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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51 pages, 4200 KiB  
Review
Integrating Omics and Gene Editing Tools for Rapid Improvement of Traditional Food Plants for Diversified and Sustainable Food Security
by Ajay Kumar, Thattantavide Anju, Sushil Kumar, Sushil Satish Chhapekar, Sajana Sreedharan, Sonam Singh, Su Ryun Choi, Nirala Ramchiary and Yong Pyo Lim
Int. J. Mol. Sci. 2021, 22(15), 8093; https://doi.org/10.3390/ijms22158093 - 28 Jul 2021
Cited by 25 | Viewed by 6116
Abstract
Indigenous communities across the globe, especially in rural areas, consume locally available plants known as Traditional Food Plants (TFPs) for their nutritional and health-related needs. Recent research shows that many TFPs are highly nutritious as they contain health beneficial metabolites, vitamins, mineral elements [...] Read more.
Indigenous communities across the globe, especially in rural areas, consume locally available plants known as Traditional Food Plants (TFPs) for their nutritional and health-related needs. Recent research shows that many TFPs are highly nutritious as they contain health beneficial metabolites, vitamins, mineral elements and other nutrients. Excessive reliance on the mainstream staple crops has its own disadvantages. Traditional food plants are nowadays considered important crops of the future and can act as supplementary foods for the burgeoning global population. They can also act as emergency foods in situations such as COVID-19 and in times of other pandemics. The current situation necessitates locally available alternative nutritious TFPs for sustainable food production. To increase the cultivation or improve the traits in TFPs, it is essential to understand the molecular basis of the genes that regulate some important traits such as nutritional components and resilience to biotic and abiotic stresses. The integrated use of modern omics and gene editing technologies provide great opportunities to better understand the genetic and molecular basis of superior nutrient content, climate-resilient traits and adaptation to local agroclimatic zones. Recently, realizing the importance and benefits of TFPs, scientists have shown interest in the prospection and sequencing of TFPs for their improvements, cultivation and mainstreaming. Integrated omics such as genomics, transcriptomics, proteomics, metabolomics and ionomics are successfully used in plants and have provided a comprehensive understanding of gene-protein-metabolite networks. Combined use of omics and editing tools has led to successful editing of beneficial traits in several TFPs. This suggests that there is ample scope for improvement of TFPs for sustainable food production. In this article, we highlight the importance, scope and progress towards improvement of TFPs for valuable traits by integrated use of omics and gene editing techniques. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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27 pages, 2138 KiB  
Review
Evolution and Application of Genome Editing Techniques for Achieving Food and Nutritional Security
by Sajid Fiaz, Sunny Ahmar, Sajjad Saeed, Aamir Riaz, Freddy Mora-Poblete and Ki-Hung Jung
Int. J. Mol. Sci. 2021, 22(11), 5585; https://doi.org/10.3390/ijms22115585 - 25 May 2021
Cited by 19 | Viewed by 5411
Abstract
A world with zero hunger is possible only through a sustainable increase in food production and distribution and the elimination of poverty. Scientific, logistical, and humanitarian approaches must be employed simultaneously to ensure food security, starting with farmers and breeders and extending to [...] Read more.
A world with zero hunger is possible only through a sustainable increase in food production and distribution and the elimination of poverty. Scientific, logistical, and humanitarian approaches must be employed simultaneously to ensure food security, starting with farmers and breeders and extending to policy makers and governments. The current agricultural production system is facing the challenge of sustainably increasing grain quality and yield and enhancing resistance to biotic and abiotic stress under the intensifying pressure of climate change. Under present circumstances, conventional breeding techniques are not sufficient. Innovation in plant breeding is critical in managing agricultural challenges and achieving sustainable crop production. Novel plant breeding techniques, involving a series of developments from genome editing techniques to speed breeding and the integration of omics technology, offer relevant, versatile, cost-effective, and less time-consuming ways of achieving precision in plant breeding. Opportunities to edit agriculturally significant genes now exist as a result of new genome editing techniques. These range from random (physical and chemical mutagens) to non-random meganucleases (MegaN), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein system 9 (CRISPR/Cas9), the CRISPR system from Prevotella and Francisella1 (Cpf1), base editing (BE), and prime editing (PE). Genome editing techniques that promote crop improvement through hybrid seed production, induced apomixis, and resistance to biotic and abiotic stress are prioritized when selecting for genetic gain in a restricted timeframe. The novel CRISPR-associated protein system 9 variants, namely BE and PE, can generate transgene-free plants with more frequency and are therefore being used for knocking out of genes of interest. We provide a comprehensive review of the evolution of genome editing technologies, especially the application of the third-generation genome editing technologies to achieve various plant breeding objectives within the regulatory regimes adopted by various countries. Future development and the optimization of forward and reverse genetics to achieve food security are evaluated. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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15 pages, 2842 KiB  
Review
Beyond Arabidopsis: BBX Regulators in Crop Plants
by Urszula Talar and Agnieszka Kiełbowicz-Matuk
Int. J. Mol. Sci. 2021, 22(6), 2906; https://doi.org/10.3390/ijms22062906 - 12 Mar 2021
Cited by 23 | Viewed by 3661
Abstract
B-box proteins represent diverse zinc finger transcription factors and regulators forming large families in various plants. A unique domain structure defines them—besides the highly conserved B-box domains, some B-box (BBX) proteins also possess CCT domain and VP motif. Based on the presence of [...] Read more.
B-box proteins represent diverse zinc finger transcription factors and regulators forming large families in various plants. A unique domain structure defines them—besides the highly conserved B-box domains, some B-box (BBX) proteins also possess CCT domain and VP motif. Based on the presence of these specific domains, they are mostly classified into five structural groups. The particular members widely differ in structure and fulfill distinct functions in regulating plant growth and development, including seedling photomorphogenesis, the anthocyanins biosynthesis, photoperiodic regulation of flowering, and hormonal pathways. Several BBX proteins are additionally involved in biotic and abiotic stress response. Overexpression of some BBX genes stimulates various stress-related genes and enhanced tolerance to different stresses. Moreover, there is evidence of interplay between B-box and the circadian clock mechanism. This review highlights the role of BBX proteins as a part of a broad regulatory network in crop plants, considering their participation in development, physiology, defense, and environmental constraints. A description is also provided of how various BBX regulators involved in stress tolerance were applied in genetic engineering to obtain stress tolerance in transgenic crops. Full article
(This article belongs to the Special Issue Plant Genomics and Genome Editing)
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