Demonstration of targeted crossovers in hybrid maize using CRISPR technology

Naturally occurring chromosomal crossovers (CO) during meiosis are a key driver of genetic diversity. The ability to target CO at specific allelic loci in hybrid plants would provide an advantage to the plant breeding process by facilitating trait introgression, and potentially increasing the rate of genetic gain. We present the first demonstration of targeted CO in hybrid maize utilizing the CRISPR Cas12a system. Our experiments showed that stable and heritable targeted CO can be produced in F1 somatic cells using Cas12a at a significantly higher rate than the natural CO in the same interval. Molecular characterization of the recombinant plants demonstrated that the targeted CO were driven by the non-homologous end joining (NHEJ) or HDR repair pathways, presumably during the mitotic cell cycle. These results are a step towards the use of RNA-guided nuclease technology to simplify the creation of targeted genome combinations in progeny and accelerate breeding.

A 2.09 Mb fragment translocation on chromosome 6 causes abnormalities during meiosis and leads to less seed watermelon

Seedlessness is a valuable agronomic trait in watermelon (Citrullus lanatus) breeding. Conventional less seed watermelons are mainly triploid, which has many disadvantages due to unbalanced genome content. Less seed watermelon can be achieved at the diploid level when certain reproductive genes are mutated or by chromosome translocation, which leads to defects during meiosis. However, the formation mechanism of diploid less seed watermelons remains largely unknown. Here, we identified a spontaneous mutant line, watermelon line “148”, which can set seeds normally when self-pollinated. A total of 148 × JM F1 hybrid plants exhibited seed number reductions to 50.3% and 47.3% of those of the two parental lines, respectively, which are considered to be less seed. Examination of pollen viability and hybridization experiments revealed that F1 hybrids produce semisterile pollen and ovules. Further cytological observations indicated that semisterility was a result of a reciprocal translocation of chromosomes, which exhibited one quadrivalent ring of four chromosomes at prometaphase I during meiosis. RT-qPCR analysis indirectly confirmed that the semisterile phenotype is caused by chromosome translocation rather than disruption of specific meiotic gene expression. F2 population genetic analysis indicated that the “148” watermelon line is a homozygous translocation and that the less seed phenotype of the F1 hybrid is prompted by one chromosome fragment translocation. The translocated fragment was further fine mapped to a 2.09 Mb region on chromosome 6 by whole-genome resequencing and genetic map cloning procedures. Our work revealed that a 2.09 Mb chromosome fragment translocation on chromosome 6, causing meiotic defects at metaphase I during meiosis, leads to diploid less seed watermelon. Our findings provide a new promising method for less seed watermelon breeding at the diploid level, as well as a fragment size reference for breeding less seed watermelon through artificially induced chromosome translocation.

Identity of genes of resistance to broomrape race G in sunflower lines of different origin

Broomrape is an obligate parasite and one of the most significant biotic factors reducing sunflower yield. The main means of controlling broomrape is breeding for sunflower resistance to it. New strategies are required to develop parasite resistant breeding germplasm, such as a stacking of key genes or a combination of qualitative and quantitative resistance mechanisms. In this regard, it is necessary to keep searching for new sources of resistance and to combine existing genes in case they are not identical. Thus, it is necessary to determine whether the genes of resistance to race G of broomrape are identical in the sunflower lines bred at the V.S. Pustovoit All-Russian Research Institute of Oil Crops. The research material was the previously developed sunflower lines RG, RGP1, RGP2, RGВ, RGL1, RGL2, and RGM – donors of resistance to race G of broomrape. The F1 hybrids from pair crossbreeding of these lines were obtained in a greenhouse. The F2 hybrids were obtained in the field by self-pollination of F1 hybrid plants. Plants were tested in a greenhouse for resistance and susceptibility to broomrape using the method of early diagnosis. Mathematical processing was performed by using the χ 2 -test. 13 combinations of crossbreeding were received to complete the test for allelism. In each F1 combination 11–33 plants were evaluated for resistance to race G of broomrape. Among the tested combinations, there was no one resistant to broomrape. Despite the differences in the number of affected plants, the affection degree was small in all hybrid combinations – from 1.0 to 1.8. All F2 hybrid combinations were also affected by broomrape. For eight combinations of F2, two phenotypic classes were found. There was no segregation in three combinations. The number of plants of two combinations was too small to perform χ 2 -test. In general, the χ 2 values were higher than the acceptable ones with a probability of 0.05, which indicates that the genes of resistance to race G of broomrape in these lines are identical.

Efficiency of nutrient solutions with different concentration in the cultivation of white cabbage seedlings

Fertilizers help to fill the missing nutritional elements for plants and affect their growth and the future crop. The paper presents the results of conducted studies on the effect of nutrient solution concentration on morphological and biochemical indicators of white cabbage seedlings. The seedlings of two early-ripe cabbage hybrids – Mirror F1 and Tiara F1 – were grown in peat substrate trays. A nutrient solution of 50 ppm (No.1) and 150 ppm (No.2) was used to feed the seedlings twice during the growing period. The obtained results showed the differences in experimental variants in terms of the height of seedling plants, the number of leaves and leaf-area duration, the weight and volume of the root system, total solids, content of sugar, ascorbic acid. The varietal reaction of cabbage seedlings to the introduction of root feeds with different concentrations was revealed. The nutrient solution No. 2 of hybrid plants Mirror F1 stimulated the development of roots, their weight exceeded the control indicators (without fertilizers) by 37.3%, and the volume – by 37.8%, contributed to the increase of the content of solids (8.27%), general sugar (0.53%), ascorbic acid (78.79 mg%). When fed with nutrient solution No. 2 the seedlings of Tiara F1 hybrid were characterized by a large leaf-area duration, which exceeds the control by 1.3 times, and the indicators of the root system. The plants of this seedlings consisted of 49.5% of the root system. The chemical analysis of water in greenhouse farming for irrigation of plants showed that it contains various macro- and microelements that are necessary for the growth of vegetable seedlings. The concentration of the nutrient solution did not significantly affect the content of macroelements in the seedlings. When using nutrient solution No. 2, an increase in the plants of the two studied phosphorus hybrids was noted, a deficit of which is very often observed when growing seedlings.

Cross-compatibility of cultivated cowpea varieties and their wild relatives: Implications for crop improvement

Hybridization programmes that potentially exploit the variability existing in the wild germplasm of Vigna unguiculata L. Walpers could be of great potential for the future of plant breeding. Bearing this in mind, four cultivated cowpea varieties (Achi shuru, Ife Brown, Kanannado and Zebra bean) were crossed to two of their wild relatives: subsp. dekindtiana var. pubescens and subsp. unguiculata var. spontanea to ascertain the cross compatibility, reproductive potential and possible heterosis in the F1 generations. Results showed that the cultivated varieties hybridized relatively well with their wild relatives showing pod set range of 42.9% to 52.3% in crosses with subsp. dekindtiana var. pubescens and 40.0% to 52.0% in crosses with subsp. unguiculata var. spontanea. The F1 hybrid plants showed high heterosis in plant height, number of leaves per plant, number of flowers per plant, number of pods per plant and percentage pod set. They also produced viable seeds for F2 generations. These results are indications of a good reproductive potential of the hybrids thus making the wild relatives, good sources of important gene pool for the improvement of the cultivated populations.

The Assessment of Agrobiological and Disease Resistance Traits of Grapevine Hybrid Populations (Vitis vinifera L. × Muscadinia rotundifolia Michx.) in the Climatic Conditions of Crimea

The Crimean autochthonous grape varieties are unique by their origin and serve as a valuable source for breeding new cultivars with increased salt and frost resistance, as well as high-quality berries. However, they suffer from fungal pathogens, as the dry and hot summer months contribute to the epiphytotic course of diseases. An increase in the resistance of Crimean grape varieties is currently achieved through interspecific hybridization. In this study, we describe the genetic and agrobiological diversity of three hybrid populations obtained using the Vitis interspecific hybrid ‘Magarach 31-77-10′ as a female parent and Muscadinia rotundifolia × Vitis vinifera BC5 hybrid plants as male parents. The hybrid nature of the populations was assessed using RADseq high-throughput genotyping. We discovered 12,734 SNPs, which were common to all three hybrid populations. We also proved with the SSR markers that the strong powdery and downy mildew resistance of the paternal genotypes is determined by the dominant Run1/Rpv1 locus inherited from M. rotundifolia. As a result, the disease development score (R, %) for both mildew diseases in the female parent ‘Magarach 31-77-10’ was three times higher than in male parents 2000-305-143 and 2000-305-163 over two years of phytopathological assessment. The highest values of yield-contributing traits (average bunch weight ~197 g and 1.3 kg as yield per plant) were detected in the population 4-11 (♀M. No. 31-77-10 × 2000-305-163). Despite the epiphytotic development of PM, the spread of oidium to the vegetative organs of hybrids 4-11 did not exceed 20%. Some hybrid genotypes with high productivity and resistance to pathogens were selected for further assessment as promising candidates for new varieties.

Analysis for grain yield and some quality traits in Bulgarian bread wheat (Triticum Aestivum L.)

A major priority of the breeding programs is the development of varieties and hybrid plants combining high productivity and a level of qualitative parameters. Equally topical is the problem of achieving their stable level of expression under uncertain climatic environments. Within three vegetative growth periods (2018, 2019 and 2020) characterized with different levels of abiotic stress, 20 varieties of common winter wheat were investigated. The criteria for setting up of the working collection were the genotypes to be included in the Varietal List of Republic of Bulgaria and to cover the groups’ quality levels. The trials were carried out at two locations: the Institute of Agriculture – Karnobat and Dobrudzha Agricultural Institute – General Toshevo. These regions have specific soil and climatic conditions and are characterized with different risk factors for the development of the cereal crops. The variance analysis showed that the environmental conditions had a significant percent in the total variation of grain yield, the number of productive tillers, 1000 kernel weight and test weight. Concerning these traits, a genotype x environment interaction was also established. The cultivars with high mean yield from the two locations were Rada, Kristalina, Kalina, Katarzhina and Bozhana. The correlation between productivity and quality was successfully broken in Lazarka, Pchelina and Merilin, which belong to the group of strong wheat genotypes. Under the specific technology for conducting the field experiments we used, cultivars Pchelina and Merilin from group A were with expectedly high levels of wet gluten content and W index. The values determined for Lazarka were lower but less variable. In the group of medium wheat genotypes with increased strength, worth mentioning are Enola, Kristalina, Rada and Katarzhina.

Transgene behavior in genetically modified teosinte hybrid plants: transcriptome expression, insecticidal protein production and bioactivity against a target insect pest

Background In 2009, Spanish farmers reported a novel weed, now identified as a relative of maize’s ancestor, teosinte, in their maize fields. Introgression of the insect resistance transgene cry1Ab from genetically modified (GM) maize into populations of this weedy Spanish teosinte could endow it with additional defense mechanisms. The aims of this study were: (1) to test if hybridization between GM maize and weedy plants from Spain is possible; (2) to understand the relationship between transgene transcription activity, concentrations of the expected transgene product (Cry1Ab protein) and the bioactivity of the latter on target insect pests following transgene flow from GM maize into Spanish teosinte plants. Results We demonstrated that hybridization between GM maize and the weedy Spanish teosinte is possible, with no observable barrier to the formation of crop/weed hybrids when teosinte served as pollen donor. When GM maize plants were used as pollen donors, significant crossing incompatibility was observed: hybrid plants produced only few “normal” seeds. Nevertheless, viable F1 seeds from GM pollen crossed onto teosinte were indeed obtained. The cry1Ab transgene was stably expressed as mRNA in all crossings and backgrounds. Similarly, toxicity on neonate Ostrinia nubilalis, presumably due to Cry1Ab protein, was consistently expressed in teosinte hybrids, with mortality rates 95% or higher after only 4 days exposure, similar to rates on parental GM maize plants. Nevertheless, no strong correlations were observed between transgene transcription levels and Cry1Ab concentrations, nor between Cry1Ab concentrations and insect mortality rates across all of the different genetic backgrounds. Conclusions Our results establish fundamental parameters for environmental risk assessments in the European context: first, we show that crop/weed hybridization in fields where maize and teosinte exist sympatrically can lead to potentially catastrophic transfer of resistance traits into an already noxious weed; second, our results question the viability of using gene dosage to model and predict ecological performance in either the intended crop plant or the undesired teosinte weed. Significant questions remain that should be addressed in order to provide a scientific, sound approach to the management of this novel weed.