Forward and Reverse Genetics in Crop Breeding

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the β-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the β-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate β-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the β-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m−2 s−1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.

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RESULTS OF COOPERATION BETWEEN BREEDERS OF DIFFERENT INSTITUTIONS IN CREATION OF NEW GENERATION APPLE TREE

Vestnik of the Russian agricultural science ◽

10.30850/vrsn/2020/4/46-49 ◽

1970 ◽

pp. 46-49

Author(s):

Е. N. Sedov ◽

T. V. Yanchuk ◽

S. А. Korneeva ◽

L. I. Dutova ◽

Е. V. Ulianovskaya

Keyword(s):

Selection Process ◽

North Caucasus ◽

Joint Work ◽

Crop Breeding ◽

Scientific Center ◽

The North ◽

State Register ◽

Russian Research Institute ◽

Apple Cultivars ◽

New Generation

The experience in cooperation of breeders of different institutions in creating cultivars is shown. It is not always when the breeding institution has the necessary initial forms for selection. In this regard, there is a need to conduct separate stages of selection in different breeding institutions. For this purpose, a provision on authorship and continuity in the integrated work of several institutions in fruit breeding has been developed (Program and methods of fruit, berry and nut crop breeding. Annex. – Orel, 1995. – pp. 492-498). Breeding work of the Russian Research Institute of Fruit Crop Breeding (VNIISPK) and North Caucasian Federal Scientific Center of Horticulture, Viticulture, Winemaking can serve as a positive experience of creating new apple cultivars by two institutions. As a result of the joint work of these two institutions, 22 apple cultivars have been created, of which 9 have already been included in the state register of breeding achievements approved for use (zoned), including three cultivars for the conditions of the Middle zone of Russia – Aleksandr Boiko, Maslovskoye and Yablochny Spas and six cultivars for the conditions of the North Caucasus – Vasilisa, Karmen, Margo, Orfey, Soyuz and Talisman. Brief economical and biological characteristics of these cultivars are given in this paper. The obtained practical results indicate that in some cases, when creating modern cultivars that meet the requirements of production, it is necessary to use the knowledge of breeders, the source material and equipment of different breeding institutions, and carry out separate stages of the selection process in different institutions.

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Peculiarities of the juvenile period of the native Chechen genotypes of willow leaf pear (Pyrus salicifolia Pall.)

POMICULTURE & SMALL FRUITS CULTURE IN RUSSIA ◽

10.31676/2073-4948-2020-62-32-38 ◽

2020 ◽

Vol 62 ◽

pp. 32-38

Author(s):

E. A. Dolmatov ◽

R. B. Borzayev ◽

A. N. Shaipov

Keyword(s):

Chestnut Soil ◽

Crop Breeding ◽

Juvenile Period ◽

Period 2 ◽

Russian Research Institute ◽

The Common ◽

One Year ◽

First Time ◽

Selection Of ◽

Creative Cooperation

The results of the study of the duration of the juvenile period of indigenous Chechen willow leaf pear genotypes (Pyrus salicifolia Pall.) are given in connection with the acceleration of the breeding process and the use of selected forms in pear breeding for high precocity. The studies were carried out in 2016-2019 at OOO “Orchards of Chechnya” in accordance with the Agreement on creative cooperation with the Russian Research Institute of Fruit Crop Breeding. The work was carried out in accordance with generally accepted programs and methods. The objects of the study were one-year and two-year-old pear seedlings obtained from sowing seeds of selected dwarf and low-growing local Chechen forms of willow pear (P. salicifolia Pall.), laying fruit buds on annual growths and seedlings of Caucasian pear (P. caucasica Fed.), 20 500 pcs. of each specie. The aim of the research was to study the potential of precocity of willow pear seedlings and to reveal of selected forms with the greatest degree of this trait. Stratified seeds were sown in the sowing department of the OOO “Orchards of Chechnya” production nursery in April, 2017. The seedlings were grown according to the common technology in dryland conditions on the plot with chestnut soil. The first fl owering of plants was noted in the spring, 2019. As a result of the research, for the first time on a large number of the experimental material it was found that in the off spring of the indigenous Chechen willow leaf pear genotypes, the selection of a little more than 2% of seedlings with a very short juvenile period (2 years) was possible. They are of great interest in accelerating the breeding process and in the selection of new pear varieties with high precocity. 20 willow leaf pear genotypes were selected for the further use in breeding for high precocity and as sources of the trait of short juvenile period.

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Laboratory Exercise on the Segregation of Flower Color and Related Genes Using Salpiglossis sinuata

HortScience ◽

10.21273/hortsci.33.3.555b ◽

1998 ◽

Vol 33 (3) ◽

pp. 555b-555

Author(s):

Chiwon W. Lee

Keyword(s):

Phenotypic Expression ◽

Pollen Grains ◽

Flower Color ◽

Segregation Ratio ◽

Crop Breeding ◽

Flower Opening ◽

Pigmentation Pattern ◽

Laboratory Exercise ◽

Demonstration Plant ◽

Corolla Color

Velvet flower (Salpiglossis sinuata, Solanaceae) can be used as an excellent demonstration plant for horticultural crop breeding classes. Salpiglossis produces large trumpet-like flowers exhibiting an assortment of corolla color and pigmentation pattern. The pistil is large (3 to 4 cm long) with a sticky stigmatal tip and anthers can be easily emasculated prior to anthesis. The large pollen grains are shed in tetrads, which can be separated and individually placed on the stigma. It takes 8 to 9 weeks from seeding to blooming, with a prolific flowering cycle repeated in flushes. Numerous seeds (about 750/capsule) are obtained in 3 weeks after self- or cross-pollination. The influences of three genes that control flower color and pigmentation pattern can be conveniently demonstrated with their dominant and recessive alleles. The R gene controls flower color with red (RR or Rr) being dominant over yellow (rr) flower color. The D gene controls the density of pigmentation with solid (DD or Dd) color being dominant over dilute (dd) color. Corolla color striping is controlled by the St gene with striped (stst) being recessive to non-striped (StSt or Stst) pattern. For example, by using diploid lines of genotypes RRDD (red, solid), RRdd (red, dilute), or rrdd (yellow, dilute) and their crosses, students can easily learn a dominant phenotypic expression in the F1 hybrid and the digenic 9:3:3:1 segregation ratio in the F2 progeny. Another gene (C) that controls flower opening can also be used to show its influence on cleistogamous (closed, self-pollinated, CC or Cc) versus normal chasmogamous (open-pollinated, cc) corolla development. In addition, the induction and use of polyploid (4X, 3X) plants in plant breeding can be effectively demonstrated using this species.

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Reverse Genetics in Plants

Current Genomics ◽

10.2174/1389202013350779 ◽

2001 ◽

Vol 2 (3) ◽

pp. 269-284 ◽

Cited By ~ 3

Author(s):

Alain Tisser ◽

Patrice Bourgeois

Keyword(s):

Reverse Genetics

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High-throughput phenotyping: breaking through the bottleneck in future crop breeding

The Crop Journal ◽

10.1016/j.cj.2021.03.015 ◽

2021 ◽

Author(s):

Peng Song ◽

Jinglu Wang ◽

Xinyu Guo ◽

Wanneng Yang ◽

Chunjiang Zhao

Keyword(s):

High Throughput ◽

Crop Breeding ◽

High Throughput Phenotyping

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Editorial overview: Virus reverse genetics approaches for the development of preventive and therapeutic vaccines

Current Opinion in Virology ◽

10.1016/j.coviro.2020.11.003 ◽

2020 ◽

Vol 44 ◽

pp. iii-iv

Author(s):

Luis Martinez-Sobrido ◽

Aitor Nogales

Keyword(s):

Reverse Genetics ◽

Therapeutic Vaccines

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A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽

10.3390/v13020289 ◽

2021 ◽

Vol 13 (2) ◽

pp. 289

Author(s):

Kathleen K. M. Glover ◽

Danica M. Sutherland ◽

Terence S. Dermody ◽

Kevin M. Coombs

Keyword(s):

Amino Acid ◽

Temperature Sensitivity ◽

Reverse Genetics ◽

Core Protein ◽

Single Point ◽

Single Amino Acid ◽

Single Point Mutation ◽

Temperature Sensitive ◽

Amino Acid Substitutions ◽

Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

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Investigation of Antifungal Mechanisms of Thymol in the Human Fungal Pathogen, Cryptococcus neoformans

Molecules ◽

10.3390/molecules26113476 ◽

2021 ◽

Vol 26 (11) ◽

pp. 3476

Author(s):

Kwang-Woo Jung ◽

Moon-Soo Chung ◽

Hyoung-Woo Bai ◽

Byung Yeoup Chung ◽

Sungbeom Lee

Keyword(s):

Cryptococcus Neoformans ◽

Fungal Pathogens ◽

Reverse Genetics ◽

Mapk Pathway ◽

Global Climate ◽

Mitogen Activated Protein Kinase ◽

Ergosterol Biosynthesis ◽

Thymus Vulgaris ◽

Dependent Manner ◽

Expression Levels

Due to lifespan extension and changes in global climate, the increase in mycoses caused by primary and opportunistic fungal pathogens is now a global concern. Despite increasing attention, limited options are available for the treatment of systematic and invasive mycoses, owing to the evolutionary similarity between humans and fungi. Although plants produce a diversity of chemicals to protect themselves from pathogens, the molecular targets and modes of action of these plant-derived chemicals have not been well characterized. Using a reverse genetics approach, the present study revealed that thymol, a monoterpene alcohol from Thymus vulgaris L., (Lamiaceae), exhibits antifungal activity against Cryptococcus neoformans by regulating multiple signaling pathways including calcineurin, unfolded protein response, and HOG (high-osmolarity glycerol) MAPK (mitogen-activated protein kinase) pathways. Thymol treatment reduced the intracellular concentration of Ca2+ by controlling the expression levels of calcium transporter genes in a calcineurin-dependent manner. We demonstrated that thymol decreased N-glycosylation by regulating the expression levels of genes involved in glycan-mediated post-translational modifications. Furthermore, thymol treatment reduced endogenous ergosterol content by decreasing the expression of ergosterol biosynthesis genes in a HOG MAPK pathway-dependent manner. Collectively, this study sheds light on the antifungal mechanisms of thymol against C. neoformans.

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Recent Progress in Torovirus Molecular Biology

Viruses ◽

10.3390/v13030435 ◽

2021 ◽

Vol 13 (3) ◽

pp. 435

Author(s):

Makoto Ujike ◽

Fumihiro Taguchi

Keyword(s):

Molecular Biology ◽

Recent Progress ◽

Reverse Genetics ◽

Cultured Cells ◽

Host Adaptation ◽

Economic Losses ◽

New Family ◽

Enteric Diseases ◽

Long Time ◽

Medical Importance

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. ToVs are associated with enteric diseases in animals and humans. In contrast to CoVs, which are recognised as pathogens of veterinary and medical importance, little attention has been paid to ToVs because their infections are usually asymptomatic or not severe; for a long time, only one equine ToV could be propagated in cultured cells. However, bovine ToVs, which predominantly cause diarrhoea in calves, have been detected worldwide, leading to economic losses. Porcine ToVs have also spread globally; although they have not caused serious economic losses, coinfections with other pathogens can exacerbate their symptoms. In addition, frequent inter- or intra-recombination among ToVs can increase pathogenesis or unpredicted host adaptation. These findings have highlighted the importance of ToVs as pathogens and the need for basic ToV research. Here, we review recent progress in the study of ToV molecular biology including reverse genetics, focusing on the similarities and differences between ToVs and CoVs.

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