Junxing Li

Heritable variation and small RNAs in the progeny of chimeras of Brassica juncea and Brassica oleracea

Chimeras have been used to study the transmission of genetic material and the resulting genetic variation. In this study, two chimeras, TCC and TTC (where the origin of the outer, middle, and inner cell layers, respectively, of the shoot apical meristem is designated by a ‘T’ for tuber mustard and ‘C’ for red cabbage), as well as their asexual and sexual progeny, were used to analyse the mechanism and the inheritance of the variation induced by grafting. Asexual TCC progeny were obtained by adventitious shoot regeneration, while TTC sexual progeny were produced by self-crossing. This study observed similar morphological variations in both the asexual and sexual progeny, including changes in leaf shape and the pattern of shoot apical meristem termination. The leaf shape variation was stable, while the rate of shoot apical meristem termination in the TTC progenies decreased from 74.52% to 3.01% after three successive rounds of self-crossing. Specific red cabbage small RNAs were found in the asexually regenerated plants (rTTT) that were not present in TTT, indicating that small RNAs might be transmitted from red cabbage to tuber mustard during grafting. Moreover, in parallel with the variations in phenotype observed in the progeny, some conserved miRNAs were differentially expressed in rTTT and TTT, which correlated with changes in expression of their target genes. These results suggest that the change in small RNA expression induced by grafting may be an important factor for introducing graft-induced genetic variations, providing a basis for further investigating the mechanism of graft-induced genetic variation through epigenetics.

Heritability and Reversibility of DNA Methylation Induced by in vitro Grafting between Brassica juncea and B-oleracea

Grafting between tuber mustard and red cabbage produced a chimeric shoot apical meristem (SAM) of TTC, consisting of Layers I and II from Tuber mustard and Layer III from red Cabbage. Phenotypic variations, which mainly showed in leaf shape and SAM, were observed in selfed progenies GSn (GS = grafting-selfing, n = generations) of TTC. Here the heritability of phenotypic variation and its association with DNA methylation changes in GSn were investigated. Variation in leaf shape was found to be stably inherited to GS5, but SAM variation reverted over generations. Subsequent measurement of DNA methylation in GS1 revealed 5.29–6.59% methylation changes compared with tuber mustard (TTT), and 31.58% of these changes were stably transmitted to GS5, but the remainder reverted to the original status over generations, suggesting grafting-induced DNA methylation changes could be both heritable and reversible. Sequence analysis of differentially methylated fragments (DMFs) revealed methylation mainly changed within transposons and exon regions, which further affected the expression of genes, including flowering time- and gibberellin response-related genes. Interestingly, DMFs could match differentially expressed siRNA of GS1, GS3 and GS5, indicating that grafting-induced DNA methylation could be directed by siRNA changes. These results suggest grafting-induced DNA methylation may contribute to phenotypic variations induced by grafting.

The role of small RNAs on phenotypes in reciprocal hybrids between Solanum lycopersicum and S. pimpinellifolium.

BackgroundReciprocal hybrids showing different phenotypes have been well documented in previous studies, and many factors accounting for different phenotypes have been extensively investigated. However, less is known about whether the profiles of small RNAs differ between reciprocal hybrids and how these small RNAs affect gene expression and phenotypes. To better understand this mechanism, the role of small RNAs on phenotypes in reciprocal hybrids was analysed.ResultsReciprocal hybrids between Solanum lycopersicum cv. Micro-Tom and S. pimpinellifolium line WVa700 were generated. Significantly different phenotypes between the reciprocal hybrids were observed, including fruit shape index, single fruit weight and plant height. Then, through the high-throughput sequencing of small RNAs, we found that the expression levels of 76 known miRNAs were highly variable between the reciprocal hybrids. Subsequently, a total of 410 target genes were predicted to correspond with these differentially expressed miRNAs. Furthermore, gene ontology (GO) annotation indicated that those target genes are primarily involved in metabolic processes. Finally, differentially expressed miRNAs, such as miR156f and 171a, and their target genes were analysed by qRT-PCR, and their expression levels were well correlated with the different phenotypes.ConclusionsThis study showed that the profiles of small RNAs differed between the reciprocal hybrids, and differentially expressed genes were also observed based on the different phenotypes. The qRT-PCR results of target genes showed that differentially expressed miRNAs negatively regulated their target genes. Moreover, the expression of target genes was well correlated with the observations of different phenotypes. These findings may aid in elucidating small RNAs contribute significantly to different phenotypes through epigenetic modification during reciprocal crossing.

Nutrients, ultrastructures, and Cd subcellular localization in the cottonseeds of three upland cotton cultivars under Cd stress

A pot experiment was performed to study the Cd-induced alterations in seed quality at maturity in two transgenic upland cotton cultivars, ZD-90 and SGK3, and the upland cotton standard line, TM-1. The results shown that Cd content in cotton kernels increased linearly with the elevation of Cd stress levels. SGK3 accumulated more Cd than others. Protein content in the kernels was increased under Cd stress generally, but those at 600 µM Cd level were lower than that under 400 µM Cd level. The changes in oil content were inversed to that of protein content. Significant ultra-structural changes in cottonseeds were induced by Cd stress, especially at higher Cd levels, which were more obvious in TM-1, followed by ZD-90 and SGK3. Plasmolytic shrinkage, disintegration of nucleus, cell wall thickening, and eventual cell collapse and disintegration were observed in the cells of cottonseeds under Cd stress. Transmission electron microscope (TEM) observation and energy dispersive X-ray analysis (EDAX) confirmed that Cd existed in the form of rings and crystals as well as electron dense granules, occurred in the intercellular space, the cytoplasm, and the cell wall. SGK3 was a tolerance cultivar to Cd stress with greater Cd accumulation and sequestration in cottonseeds.

Hybrid Technology in Cruciferous Vegetables

Use of male sterile plants has become an important technique in heterosis breeding,which simplify and reduce the cost of hybrid seed production. Cruciferous vegetables are very important crops in the world, and two types of male sterility have been mainly explored in cruciferous vegetables (1) nuclear male sterility, this kind of male sterility is controlled by the dominant or recessive nuclear genes, and its sterility is easy to be restored, but difficult to maintained; (2) cytoplasmic male sterility, in which male sterility is controlled by a particular cytoplasmic male sterile gene (S). Cytoplasmic male sterility is easy to be maintained but complicated to be recovered. Male sterility can be produced by different ways; natural mutation, wide hybridization, and protoplast fusion. Ogu CMS and Polma CMS were found in radish and B. napus respectively by natural mutations. Among the male sterile materials, most of them were obtained by the wide hybridization among varieties, species and genera. CMS cabbage was produced by the fusion of leaf protoplasts from fertile cabbage and CMS Ogura broccoli lines. The Pol CMS had been transferred from CMS B. napus to Chinese cabbage. Chinese breeders produced many cabbage varieties after introduced male sterility from other materials. A new cabbage hybrid varieties Zhonggan no.16, 17 and 18 were produced by hybridization of dominant genic male sterility line and inbred line in China. A stable CMS line of tuber mustard was developed by distant crosses and subsequent backcrosses and induced 100 % male sterility. To date, extensive efforts have been made on identification of male sterility systems and the possibilities of development of hybrids in application.

The high-throughput sequencing of small RNAs profiling in wide hybridisation and allopolyploidisation between Brassica rapa and Brassica nigra

Small RNAs play an important role in maintaining the genome reconstruction and stability in the plant. However, little is known regarding the role of small RNAs during the process of wide hybridisation and chromosome doubling. Therefore, the changes in the small RNAs were assessed during the formation of an allodiploid (genome: AB) and its allotetraploid (genome: AABB) between Brassica rapa (♀) and Brassica nigra (♂) in the present study. Here, the experimental methods described in details, RNA-seq data (available at Gene Expression Omnibus database under GSE61872) and analysis published by Ghani et al. [1]. The study showed that small RNAs play an important role in maintaining the genome stability, and regulate gene expression which induces the phenotype variation in the formation of an allotetraploid. This may play an important role in the occurrence of heterosis in the allotetraploid.