Molecular marker-assisted selection (MAS) can significantly accelerate and improve the efficiency of the breeding process in seedless grape cultivars. In this study, we developed the KASP_VviAGL11 and VviAGL11_410 markers based on a single nucleotide polymorphism (SNP) site (Chr18: 26889437 (A/C)) of the VviAGL11 gene, and compared them with previously reported SSR markers p3_VvAGL11 and 5U_VviAGL11 by testing 101 cultivars and 81 F1 hybrid progenies. The results showed that both of the proposed markers obtained 100% accuracy rates in detecting allele A, which was closely associated with the seedless trait in grapes, while p3_VvAGL11 and 5U_VviAGL11 had lower accuracy rates due to their tendency to produce false positives. After careful evaluation of the technical advantages and disadvantages associated with these markers, we concluded that KASP_VviAGL11 was superior in terms of simplicity, cost-effectiveness, efficiency, and accuracy. Thus, we optimized the process of molecular MAS for seedless grapes, focusing on the KASP_VviAGL11 marker as a central component, to provide key technical support for the development of new seedless grape cultivars.
Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes. Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P24h=0.31, P48h=0.96, P72h=0.24, P96h=0.17, and P120h=0.38) on cell proliferation of PK15 cells. These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment
The border effect (BE) is widely observed in crop field experiments, and it has been extensively studied in many crops. However, only limited attention has been paid to the BE of ratoon rice. We conducted field experiments on ratoon rice in Qichun County, Hubei Province, Central China in 2018 and 2019 to compare the BE in the main and ratoon crops, and to quantify the contribution of BE in the main crop to that in the ratoon crop. The BE of two hybrid varieties was measured for the outermost, second outermost, and third outermost rows in each plot of both crops. To determine the contribution of BE between the two crops, portions of hills in the outermost and second outermost rows were uprooted during the harvest of the main crop so that the second and third outermost rows then became the outermost rows in the ratoon crop. Overall, the BE on grain yield was greater in the main crop than in the ratoon crop. In the main crop, the BE on grain yield was 98.3% in the outermost row, which was explained by the BE on panicles m–2, spikelets/panicle, spikelets m–2, and total dry weight. In the ratoon crop, the BE on grain yield was reduced to 60.9 and 27.6% with and without the contribution of the BE in the main crop, respectively. Consequently, 55.1% of the BE on grain yield in the ratoon crop was contributed from the main crop. High stubble dry weight and non-structural carbohydrate (NSC) accumulation at the harvest of the main crop were responsible for the contribution of BE in the main crop to that in the ratoon crop. Our results suggest that increases in stubble dry weight and NSC accumulation at the harvest of the main crop could be important strategies for developing high-yielding cropping practices in the rice ratooning system.
Brassica napus is an important cash crop broadly grown for the vegetable and oil values. Yellow-seeded B. napus is preferred by breeders due to its improved oil and protein quality, less pigments and lignin compared with the black-seeded counterpart. This study compared the differences in flavonoid and fatty acid contents between yellow rapeseed from the progenies of B. napus–Sinapis alba somatic hybrids and the black-seeded counterpart using RNA-seq analysis. Through HPLC-PDA-ESI(−)/MS2 analysis, it was found that phenylpropanoids and flavonoids (i.e., isorhamnetin, epicatechin, kaempferol, and other derivatives) in yellow seed were significantly lower than those in black seed. The fatty acid (FA) content in yellow rapeseed was higher than that in black rapeseed due to the variation of C16:0, C18:0, C18:1, C18:2, and C18:3 contents. RNA-seq analysis of seeds at four and five weeks after flowering (WAF) indicated that differentially expressed genes (DEGs) between black and yellow rapeseeds were enriched in flavonoid and FA biosynthesis, including BnTT3, BnTT4, BnTT18, and BnFAD2. Also, genes related to FA biosynthesis, desaturation and elongation (FAD3, LEC1, FUS3, and LPAT2) in yellow seed were up-regulated compared to those in black seed, while genes involved in beta-oxidation cycle (AIM1 and KAT2) of yellow seed were down-regulated compared to those in black seed. The DEGs related to the variation of flavonoids, phenylpropanoids, and FAs would help improve the knowledge of yellow seed character in B. napus and promote rapeseed improvement.
Embryo rescue technology plays an important role in seedless grape breeding. However, the efficiency of embryo rescue, including the embryo formation, germination, and seedling rates, is closely related to the parental genotypes, degree of abortion, growth medium, and plant growth regulators. In this study, we investigated the effects of different concentrations of paclobutrazol (PAC), a plant growth regulator, and embryo collection times on the embryo formation, germination, and seedling rates for different hybrid combinations of grape breeding varieties used for their aroma and cold-resistance traits. The results showed that the different PAC concentrations had varying impacts on the development of ovules and embryos from the different grape varieties. The embryo formation rates of the ‘Sultanina Rose’בBeibinghong’ and ‘Kunxiang Seedless’בTaishan-2’ crosses were the highest under the 5.1 μmol L–1 PAC treatment. The 1.0 μmol L–1 PAC treatment was optimal for the germination and seedling development of the ‘Sultanina Rose’בBeibinghong’ embryos, whereas the 0.2 μmol L–1 PAC treatment induced the highest germination rate for the ‘Sultanina Rose’בKunxiang Seedless’ cross. The optimal sampling times for each cross varied as 39 d after pollination (DAP) for the ‘Flame Seedless’בMuscat Hamburg’ cross, 46 DAP for the ‘Kunxiang Seedless’בBeibinghong’ cross, and 41 DAP for the ‘Ruby Seedless’בBeibinghong’ and ‘Fantasy Seedless’בShuangyou’ crosses. Moreover, the medium modified with 0.5 g L–1 of indole-3-butyric acid allowed the malformed seedlings to develop into plantlets and achieve larger progenies. This study provides a useful basis for further studies into grape embryo rescue and could improve breeding efforts for new seedless grape varieties.