ZbAGL11, a class D MADS-box transcription factor of Zanthoxylum bungeanum, is involved in sporophytic apomixis

Apomixis is a reproductive model that bypasses sexual reproduction, so it does not require the combination of paternal and maternal gametes but instead results in the production of offspring directly from maternal tissues. This reproductive mode results in the same genetic material in the mother and the offspring and has significant applications in agricultural breeding. Molecular and cytological methods were used to identify the reproductive type of Zanthoxylum bungeanum (ZB). Fluorescence detection of the amplified products of 12 pairs of polymorphic SSR primers showed consistent fluorescence signals for mother and offspring, indicating that no trait separation occurred during reproduction. In addition, the cytological observation results showed differentiation of ZB embryos (2n) from nucellar cells (2n) to form indefinite embryonic primordia and then form adventitious embryos (2n), indicating that the apomictic type of ZB is sporophytic apomixis. The MADS-box transcription factor ZbAGL11 was highly expressed during the critical period of nucellar embryo development in ZB. Unpollinated ZbAGL11-OE Arabidopsis produced fertile offspring and exhibited an apomictic phenotype. The overexpression of ZbAGL11 increased the callus induction rate of ZB tissue. In addition, the results of the yeast two-hybrid experiment showed that ZbAGL11 could interact with the ZbCYP450 and ZbCAD11 proteins. Our results demonstrate that ZbAGL11 can cause developmental disorders of Arabidopsis flower organs and result in apomixis-like phenotypes.

Identification of ‘Haryejosaeng’ mandarin by multiplex SNP genotyping

BackgroundMost of the satsuma mandarin (Citrus unshiu Marc.) cultivars grown on Jeju Island farms, Korea, are difficult to improve through hybridization because of polyembryony and male sterility. Therefore, their improvement has mostly been based on the selection of nucellar embryo and bud mutation. These cultivars are supplied to breeders and farms at the seedling stage, which renders their identification based only on morphological traits. In addition, because these seedlings originate from nucellar embryo and bud mutation selection, they are genetically very similar. Therefore, the present study was carried out to develop markers that can specifically and rapidly distinguish ‘Haryejosaeng,’ which is generally supplied to breeders, from other satsuma mandarin cultivars that are planted on farms.ResultsPolymerase chain reaction (PCR) was performed to distinguish ‘Haryejosaeng’ from other 8 cultivars (‘Haryejosaeng’- breeder’s stock, ‘Miyagawa wase,’ ‘Okitsu wase,’ ‘Yura wase,’ ‘Miyamoto wase,’ ‘Ueno wase,’ ‘Yonezawa wase,’ and ‘Nichinan 1 gou’) using 6 single nucleotide polymorphism (SNP) markers specific for ‘Haryejosaeng’ and one SNP primer pair, which was used as the negative control. Using a multiplex PCR, SNP markers P1 (HL-SNP-SCAF_2-23997586-F and HL-SNP-SCAF_2-23997586-R), P2 (HL-SNP-SCAF_2-36059523-F and HL-SNP-SCAF_2-36059523-R), and P5 (HL-SNP-SCAF_9-30793978-F and HL-SNP-SCAF_9-30793978-R) simultaneously yielded 165, 150, and 526 bp amplicons, respectively, for Haryejosaeng only. The SNP markers were further validated by high-resolution melting analysis. The multiplex PCR based on P1/P5 and P2/P5 was also used to identify ‘Haryejosaeng’ on a farm growing 17 different cultivars of satsuma mandarin.ConclusionsWe developed specific molecular markers for accurate identification of ‘Haryejosaeng,’ which can be performed by multiplex PCR to save time and cost associated with the supply of ‘Haryejosaeng’ to farms.

Anther, ovule, seed, and nucellar embryo development in Citrus sinensis cv. Valencia

'Valencia' orange, a commercially important cultivar of Citrus, forms polyembryonic seeds by an apomictic process called nucellar embryony in which many embryos initiate directly from nucellar cells surrounding the sexual embryo sac. We observed anther, ovule, seed, and fruit development in relation to nucellar embryo development in seeds and unfertilized ovules of 'Valencia'. Pollination and fertilization are required to set fruit in 'Valencia', and low seed set was found to be related to defects in both male and female gametogenesis. Nucellar embryo initial cells were evident histologically in ovules of flowers just prior to anthesis. However, in vitro culture of ovules from flowers at different prepollination stages showed that embryos could develop from ovules cultured as early as the binucleate stage of megagametogenesis in which nucellar initial cells were absent histologically. During fruit development, the timing and sequence of the early events of nucellar embryo formation were synchronous in seeds and unfertilized ovules, indicating a co-ordinated control of embryo development in spatially and developmentally distinct structures. In both developing seeds and unfertilized ovules, embryo initial cells first formed thick walls, which isolated them from surrounding maternal tissue. In later stages, the cell walls thinned in some initial cells and embryogenesis became asynchronous. Cleavage of embryogénie cells coincided with degenerative processes linked to embryo sac expansion in seeds and to a previously unreported, localized degeneration in the central portion of the nucellus in unfertilized ovules. Some initial cells never divided. Nucellar embryo development was restricted to the central portion of unfertilized ovules and to the micropylar region of seeds. Only fertilized ovules had the capacity to form mature polyembryonic seeds. In unfertilized ovules a specialized vascular structure formed linking developing embryos to the chalazal vasculature of the ovule. Embryo development arrested at the globular stage in unfertilized ovules and the integuments differentiated to form a seed coat. The timing of reproductive events described was linked to floral and fruit morphological characteristics to facilitate molecular characterization of nucellar embryogenesis and seed formation in this cultivar. Key words: Citrus, nucellar embryony, seed, ovule, apomixis.