Heterozygosity analysis of spontaneous 2n female gametes and centromere mapping of the diploid Hevea brasiliensis based on full-sib triploid populations.

KEY MESSAGE: Unreduced megagametophytes via second-division restitution were confirmed through heterozygosity analysis, and four candidate physical centromeres of rubber were located for the first time. The evaluation of maternal heterozygosity restitution (MHR) is vital in identifying the mechanism of 2n gametogenesis and assessing the utilization value of 2n gametes. In this study, three full-sib triploid populations were employed to evaluate the MHR of 2n female gametes of rubber tree clone GT1 and to confirm their genetic derivation. The 2n female gametes of GT1 were derived from second-division restitution (SDR) and transmitted more than half of the parental heterozygosity. In addition, low recombination frequency markers were developed, and four candidate physical centromeres of rubber tree were located for the first time. The confirmation that 2n female gametes of rubber tree clone GT1 are derived from SDR provides insights into the molecular mechanisms of 2n gametogenesis. In addition, the identified centromere location will aid in the development of centromeric markers for the rapid identification of the 2n gametogenesis mechanism.

Characterization of the complete chloroplast genome of Hevea pauciflora (Euphorbiaceae), an important wild relative of the rubber tree.

Hevea pauciflora belongs to the Euphorbiaceae family, an important wild relative of the rubber tree. This study sequenced, assembled, and annotated the complete chloroplast genome of H. pauciflora. The complete chloroplast genome is 161,123 bp with a canonical quadripartite structure containing a large single-copy (LSC) region (89,109 bp), a small single-copy (SSC) region (18,376 bp), and two inverted repeat regions (IRa and IRb) (26,819 bp, each). A total of 134 genes were annotated, including 86 protein-coding genes, four pseudogenes, 36 tRNA genes, and eight rRNA genes. The 134 genes include four major groups: 'self-replication', 'photosynthesis', 'unknown function', and 'others'. A phylogenetic analysis clustered H. pauciflora, H. brasiliensis, H. camargoana, and H. benthamiana into one clade, consistent with traditional taxonomy. This study provides useful data for further studies of Hevea genus and the phylogenetic relationships of Euphorbiaceae species.

[Construction of a methylation filtration library in Hevea brasiliensis.].

In order to enrich gene encoding region of Hevea brasiliensis, a methylation filtration library was constructed using Escherichia coli McrBC restriction-modification system. The titers of the non-amplified library and the amplified library were 2.6×106 pfu/ml and 9.0×109, respectively. The rate of positive clones was 86.4%. The lengths of inserted DNA sequence ranged from 1 kb to 2.5 kb and the average size of inserts was 1.2 kb. One hundred clones were selected randomly for sequencing, resulting in splicing out of 81 non-redundant sequences, including 6 contigs and 75 singlets. The redundancy was 17.35%. Blast analysis showed that 39.5% of non-redundant sequences were homologous with the Nr database, 14.81% with the EST database, and 32.1% were unknown sequences. Some sequences were related genes for flowering, insect and disease resistance. Therefore, the rubber tree methylation library is helpful for discovery and cloning of functional genes.

[Prokaryotic expression and CRT-binding activity analysis of Hevea HbCBF1 protein].

CBF pathway is the most important pathway during the process of cold acclimation in plants. In previous study, we cloned a CBF like gene HbCBF1 from Hevea brasiliensis, which implied that CBF pathway might exist in this kind of tropical oriented tree. However, it was still unknown if this gene functioned as C-repeat binding factor. Therefore it was very important to analyze the C-repeat binding activity of HbCBF1. Using a prokaryotic expression system, the HbCBF1 was expressed and the soluble fusion protein was extracted under the optimized conditions. The C-repeat binding activity of HbCBF1 protein was analyzed by gel shift assay, which indicated that the fusion protein could bind to the COR15a probe efficiently, and this binding activity could be com-peted with by overloading non-labeled COR15a probe. An unlabeled mutated probe M1, which did not include the C-repeat sequence, could not compete with the binding activity of HbCBF1 to COR15a probe. These results indicated HbCBF1 fusion protein could bind to the C-repeat containing DNA fragment specifically in vitro.