Analysis Of Genetic Background And Mechanism For The Altered Traits Of The Granulation Variant Of Citrus

Somatic hybridization via protoplast fusion is a new alternative for circumventing some reproductive barriers in citrus traditional breeding, such as sexual incompatibility, female and/or male sterility and polyembryony. Progress in somatic hybridization in citrus over the past 20 years has produced > 250 different inter-generic and inter-specific somatic hybrids. Protoplasts from embryogenic calli have the capacity to undergo embryogenesis and to develop into mature plants, while mesophyll protoplasts themselves are not totipotent. Thus, the regenerated plants from such a fusion event are primarily composed of hybrids, cybrids, and embryogenic calli parent regenerates. And somaclonal variation is commonly observed in protoplast-derived and protoplast fusion-regenerated plants, which will bring some special germ plasm. In this study, we describe six plants with an early juice sac granulation trait derived from the inter-specific protoplast fusions between embryogenic calli of 'Bonanza' navel orange {Citrus sinensis [L.] Osbeck) and mesophyll protoplasts of 'Dahongpao' Red tangerine (C. reticulata Blanco.); analyzed their genetic background; studied the relationship between cell wall metabolism and granulation, the texture, hydrolases activity and gene expressions in juice sacs in order to studying the mechanism of granulation in juice sacs. To identify potential important or novel genes involved in somaclonal variation and juice sac granulation, suppression subtractive hybridization (SSH) and reverse Northern were performed to decipher this variation during fruit development.Six plants were analyzed by flow cytometry and using molecular markers including simple sequence repeats (SSR) and restriction fragment length polymorphism (RFLP). The results indicated that all the six plants are diploids and inherited their nuclear DNA from the embryogenic calli parent 'Bonanza' navel orange. However, analysis of morphological and fruit characteristics and measurements of the components of the cell walls in the juice sacs showed that they are not true-to-type for 'Bonanza' navel orange, especially for fruit traits such as juice sac granulation and navel structure. We found that the leaves and fruits of these plants were different from their parents. For example, their fruit rinds were rougher, easier to peel than navel orange, and did not have a navel structure. The fruits were seedless, with an orange colour instead of being seedy with a deep red skin of 'Dahongpao Red' tangerine. In addition, they exhibited serious juice sac granulation before maturityThese results confirmed that these plants were not hybrids and are more likely to be somaclonal variants arising during the regeneration of navel orange protoplasts.The texture, hydrolases activity in juice sacs of six somaclonal variation plants (BH) and the control (B) Skagg's Bonanza navel orange (Citrus sinensis [L.] Osbeck) were characterized at six developmental stages. Results showed that the contents of water soluble pectin (WSP), propectin, total pectin (TP), cellulose (CEL), hemicellulose (HC), lignin, and water between granulated juice sacs in BH and the normal sacs in Bonanza navel orange were significantly different. High and positive correlation coefficients were observed between polygalacturonase (PG) activity and content of WSP, while the contents of CEL, HC, and lignin were negatively correlated with levels of cellulase (Cx).Moreover the expression of PG and Cx genes was the same as enzyme activities. They behaved a high level in B and much low in BH fruit juice sacs. The pectinmethylesterase (PME) showed little different expression in two samples. In addition, the expression of polygalacruronase inhibiting protein (PGIP) gene was much higher in BH versus in its control B. These results suggested that the granulation of juice sacs during fruit development might partially be explained by the hydrolases activity and the transcriptional level of genes directing the cell wall metabolism pathway.After sequencing of the differentially expressed clones, a total of 357 non-redundant transcripts were obtained and 64.4% (207 single sequences and 23 contigs) of them shared homology (E-value≤1×10^-10) with known gene products. These genes were involved in many metabolic pathways such as pyruvate metabolism, starch and sucrose metabolism, citrate cycle and glycolysis pathways. Moreover, 12 genes: Pectinmethylesterase inhibiting protein (PMEIP), Pectate lyase (PL), Chitinase, Endo-beta-1, 4-glucanase,β-galactosidase (Gal), Expansins (Exp), Peroxidase (POD), Superoxide dismutase (SOD), Glutathione peroxidase (GPX), Gibberellin regulated protein (GRP), retrotransposon and Ca²⁺-binding protein(CaBP) transcripts were studied by real time PCR in six different sample times of BH fruits and B fruits. The results showed that they may associate with granulation.Through this research, the content of cell wall structure materials, the dynamic changes of relevant synthesis and hydrolases during the different development times were studied, and then combined with suppression subtractive hybridization (SSH), reverse Northern hybridization, the follow-up of bio-informatics analysis and real-time quantitative fluorescence PCR technology, for the first time analysis the level of geneexpression which may involved in changes of granulation. The gene expression,regulation and control has revealed the possible molecular mechanism of the formation ofgranulation, therefore make a firm foundation to solve this problem for the future incitrus.