Genetic Analysis Reveals Relationships Among Populations of Puccinia striiformis f. sp. tritici from the Longnan, Longdong and Central Shaanxi Regions of China

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is one of the most important diseases of wheat worldwide. In China, Longnan (LN) and Longdong (LD), in the south and east of Gansu province, are important PST over-summering areas and are a source of PST inoculum for the major wheat-growing regions in eastern China. Central Shaanxi (CS) is a wheat-growing region that acts as an important bridge zone for stripe rust epidemic development between LN and LD in the west, and the Huanghuai wheat-growing region in the east, and thus plays an essential role in PST epidemics in China. To study the relationships among PST populations in the three regions (LN, LD and CS), we sampled 284 isolates from different geographic locations. Based on 10 SSR markers, the results demonstrated high genetic diversity in all three regions although diversity did vary between region with LN > LD > CS. Genetic differentiation was lower with more extensive gene flow between LD and CS. PST populations in the CS region were genetically closer to those from LD than from LN, which may be due to geographical proximity and topography. A positive and significant correlation existed between linearized FST and the log of geographical distances among all subpopulations. Linkage disequilibrium analysis showed that subpopulations of PST from Qinzhou, Qincheng, Beidao, and Maiji from LN, and Qianyang and Longxian from CS were in equilibrium (P > 0.05), suggesting somatic hybridization and/or sexual reproduction may exist in these subpopulations.

Asymmetric Somatic Hybridization Affects Synonymous Codon Usage Bias in Wheat

Asymmetric somatic hybridization is an efficient strategy for crop breeding by introducing exogenous chromatin fragments, which leads to whole genomic shock and local chromosomal shock that induces genome-wide genetic variation including indel (insertion and deletion) and nucleotide substitution. Nucleotide substitution causes synonymous codon usage bias (SCUB), an indicator of genomic mutation and natural selection. However, how asymmetric somatic hybridization affects SCUB has not been addressed. Here, we explored this issue by comparing expressed sequence tags of a common wheat cultivar and its asymmetric somatic hybrid line. Asymmetric somatic hybridization affected SCUB and promoted the bias to A- and T-ending synonymous codon (SCs). SCUB frequencies in chromosomes introgressed with exogenous fragments were comparable to those in chromosomes without exogenous fragments, showing that exogenous fragments had no local chromosomal effect. Asymmetric somatic hybridization affected SCUB frequencies in indel-flanking sequences more strongly than in non-flanking sequences, and this stronger effect was present in both chromosomes with and without exogenous fragments. DNA methylation-driven SCUB shift was more pronounced than other SC pairs. SCUB shift was similar among seven groups of allelic chromosomes as well as three sub-genomes. Our work demonstrates that the SCUB shift induced by asymmetric somatic hybridization is attributed to the whole genomic shock, and DNA methylation is a putative force of SCUB shift during asymmetric somatic hybridization. Asymmetric somatic hybridization provides an available method for deepening the nature of SCUB shift and genetic variation induced by genomic shock.

Analysis of Cytosine Methylation in Genomic DNA of Solanum × michoacanum (+) S. tuberosum Somatic Hybrids

Interspecific somatic hybridization is a noteworthy breeding strategy that allows the production of novel genetic variability when crossing barriers exist between two parental species. Although the genetic consequences of somatic hybridization have been well documented, little is known on its impact at the epigenetic level. The objective of our research was to investigate the epigenetic changes, in particular DNA methylation, occurring in a population of potato somatic hybrids. The analysis of 96 Solanum × michoacanum (+) S. tuberosum somatic hybrids from five fusion combinations and their parents was carried out by methylation-sensitive amplified polymorphism (MSAP) and high-performance liquid chromatography (HPLC) methods. Six MSAP primer combinations generated 622 unique bands, of which 295 were fully methylated. HPLC analysis showed from 15.5% to 16.9% total cytosine methylation within the parental forms. Overall, the MSAP and HPLC methods indicated an increase in DNA methylation in the somatic hybrids in comparison to their parents. Among the latter, a lower degree of DNA methylation in the wild S. × michoacanum species than S. tuberosum was found. Our findings indicated that somatic hybridization changed the level of cytosine methylation in the studied potato somatic hybrids.

Interspecific Potato Somatic Hybrids Between Solanum Malmeanum and S. Tuberosum Provide Valuable Resources for Freezing-Tolerance Breeding

Freezing stress affects the geographic distribution, growth, and development of potato, resulting in loss of its yield. Solanum malmeanum , a diploid wild species with strong freezing tolerance, was fused with a freezing sensitive dihaploid S. tuberosum by somatic hybridization. In our study, 980 calli were obtained, and 248 differentiate shoots from the calli. Parental-specific SSR markers were used to analyze the chromosome composition of the randomly selected 80 regenerated plants, resulting in 51 somatic hybrids. Among them, 44 somatic hybrids were tested with ploidy analysis in the years 2016 and 2020. During subculture, the genomic ploidy levels changed due to the composition of the unstable chromosome in 56.82% of the somatic hybrids. Compared with the cultivated parent, somatic hybrids showed better freezing tolerance. After freezing-tolerant somatic hybrids were selected to backcross with cultivars, we obtained some valuable breeding resources with enhanced freezing tolerance while similar tuberization capacity close to cultivars. The correlation analysis shows that freezing tolerance has no relation with tuberization capacity, which indicates that they are controlled by independent genetic loci. In all, we successfully conducted the protoplast fusion between S. malmeanum and S. tuberosum for the first time, which provided valuable resources for freezing-tolerant breeding.

Somatic Embryogenesis in Olive

The olive is a fruit tree species economically very important in countries of the Mediterranean basin. Somatic embryogenesis is a powerful in vitro technique with multiple applications in different fields, including breeding programs performed by both classical and innovative procedures. This editorial paper presents a special issue focused on “Somatic embryogenesis in olive”. In this manuscript, the conceptual framework of the special issue is established and the contributions are summarized and put into context. Finally, the main bottlenecks limiting the practical applicability of somatic embryogenesis are identified and the future research prospects are discussed.

Development of a Yellow-Seeded Stable Allohexaploid Brassica Through Inter-Generic Somatic Hybridization With a High Degree of Fertility and Resistance to Sclerotinia sclerotiorum

The Brassica coenospeceis have treasure troves of genes that could be beneficial if introgressed into cultivated Brassicas to combat the current conditions of climate change. Introducing genetic variability through plant speciation with polyploidization is well documented, where ploidy augmentation of inter-generic allohexaploids using somatic hybridization has significantly contributed to genetic base broadening. Sinapis alba is a member of the Brassicaceae family that possesses valuable genes, including genes conferring resistance to Sclerotinia sclerotiorum, Alternaria brassicae, pod shattering, heat, and drought stress. This work aimed to synthesize stable allohexaploid (AABBSS) Brassica while incorporating the yellow-seed trait and resistance to S. sclerotiorum stem rot. The two fertile and stable allohexaploids were developed by polyethylene glycol mediated protoplast fusions between Brassica juncea (AABB) and S. alba (SS) and named as JS1 and JS2. These symmetric hybrids (2n = 60) were validated using morphological and molecular cytology techniques and were found to be stable over consecutive generations. The complete chromosome constitution of the three genomes was determined through genomic in situ hybridization of mitotic cells probed with S. alba genomic DNA labeled with fluorescein isothiocyanate. These two allohexaploids showed 24 hybridization signals demonstrating the presence of complete diploid chromosomes from S. alba and 36 chromosomes from B. juncea. The meiotic pollen mother cell showed 30 bivalent sets of all the 60 chromosomes and none of univalent or trivalent observed during meiosis. Moreover, the backcross progeny 1 plant revealed 12 hybridization signals out of a total of 48 chromosome counts. Proper pairing and separation were recorded at the meiotic metaphase and anaphase, which proved the stability of the allohexaploid and their backcross progeny. When screening, the allohexaploid (JS2) of B. juncea and S. alba displayed a high degree of resistance to S. sclerotiorum rot along with a half-yellow and half-brown (mosaic) seed coat color, while the B. juncea and S. alba allohexaplopid1 (JS1) displayed a yellow seed coat color with the same degree of resistance to Sclerotinia rot.

Advancement in CMS Based Hybrid Development in Cauliflower (Brassica oleracea var. Botrytis)

Among the different mechanism of male sterility operated in the Brassica group crop. Cytoplasmic male sterility mechanism is most suitable for hybrid development in cauliflower because here the curd (intermediate stage) is an edible part of the cauliflower. Further, there is no requirement of restorer line in this case as required in other seed crop. For the multiplication and maintenance of the different lines (A line and B line), sib mating and selfing is not always desirable. In fact, in such situation doubled haploid production through microspore culture is a more appropriate mechanism. Apart from this, the undesirable effect of integration of male sterile cytoplasm can be mitigated by adopting the repeated back crossing, through chloroplast substitution or somatic hybridization mechanism.

Electrofusion of mesophyll protoplasts from two varieties of sugar beet, (Beta vulgaris L.)

Somatic hybridization between different plants through protoplast fusion represent an efficient experimental approach to produce genetically transformed plant species. Electrofution of mesophyll protoplasts in sugar beet was occurred to overcome the barriers faced breeding program of this economically industrial crop Protoplasts were successfully isolated from leave's mesophyll of two varieties of sugar beet (Beta vulgaris L.). Various enzyme solutions were assessed for the cell wall degrading ability. They express different efficiency in isolation of mesophyll protoplasts of var. Baraka. The protoplasts yield was 18 × 104 cell ml-1 using the mixture consisting of 0.5% Cellulase RS, 1.0% Hemicellulase and 0.1% Pectolyase Y-23 with 13% mannitol. A total of 16 hrs. for cell wall digestion, and protoplast viability approached 93%. Protoplasts were isolated from leaf mesophyll of var. Carola using the same enzymatic mixtures. High protoplasts yield 20 × 104 cell ml-1 was obtained, requiring the same period 16 hrs. to approach viability 96%. The protoplasts were spherical in shape, varied in chloroplast distribution, having size ranged 12 – 52 µm. The present study succeeded in electrofusion between Baraka × Carola mesophyll protoplasts, producing somatic hybrid cells under conditions of 1MHz, 1000 Vcm-1, 2 pulses, 1.5 msec./pulse with fusion percent of 73%.