Moringa oleifera is a promising plant species for oil and forage, but its genetic improvement is limited. Our current breeding program in this species focuses on exploiting the functional genes associated with important agronomical traits. Here, we screened reliable reference genes for accurately quantifying the expression of target genes using the technique of real-time quantitative polymerase chain reaction (RT-qPCR) in M. oleifera. Eighteen candidate reference genes were selected from a transcriptome database, and their expression stabilities were examined in 90 samples collected from the pods in different developmental stages, various tissues, and the roots and leaves under different conditions (low or high temperature, sodium chloride (NaCl)- or polyethyleneglycol (PEG)- simulated water stress). Analyses with geNorm, NormFinder and BestKeeper algorithms revealed that the reliable reference genes differed across sample designs and that ribosomal protein L1 (RPL1) and acyl carrier protein 2 (ACP2) were the most suitable reference genes in all tested samples. The experiment results demonstrated the significance of using the properly validated reference genes and suggested the use of more than one reference gene to achieve reliable expression profiles. In addition, we applied three isotypes of the superoxide dismutase (SOD) gene that are associated with plant adaptation to abiotic stress to confirm the efficacy of the validated reference genes under NaCl and PEG water stresses. Our results provide a valuable reference for future studies on identifying important functional genes from their transcriptional expressions via RT-qPCR technique in M. oleifera.

Alternation of generations in plant life cycle provides a biological basis for natural selection occurring in either the gametophyte or the sporophyte phase or in both. Divergent biphasic selection could yield distinct evolutionary rates for phase-specific or pleiotropic genes. Here, we analyze models that deal with antagonistic and synergistic selection between alternative generations in terms of the ratio of nonsynonymous to synonymous divergence (K_a/K_s). Effects of biphasic selection are opposite under antagonistic selection but cumulative under synergistic selection for pleiotropic genes. Under the additive and comparable strengths of biphasic allelic selection, the absolute K_a/K_s for the gametophyte gene is equal to in outcrossing but smaller than, in a mixed mating system, that for the sporophyte gene under antagonistic selection. The same pattern is predicted for K_a/K_s under synergistic selection. Selfing reduces efficacy of gametophytic selection. Other processes, including pollen and seed flow and genetic drift, reduce selection efficacy. The polymorphism (π_a) at a nonsynonymous site is affected by the joint effects of selfing with gametophytic or sporophytic selection. Likewise, the ratio of nonsynonymous to synonymous polymorphism (π_a/π_s) is also affected by the same joint effects. Gene flow and genetic drift have opposite effects on π_a or π_a/π_s in interacting with gametophytic and sporophytic selection. We discuss implications of this theory for detecting natural selection in terms of K_a/K_s and for interpreting the evolutionary divergence among gametophyte-specific, sporophyte-specific, and pleiotropic genes.

Toona ciliata is an important timber species but is recognized as an endangered species at level II in China. Its genetic conservation is of increasing concern. Provenance trials and other breeding programs were conducted to develop seed transfer rules and multiplications. Here, we investigated twenty-nine populations sampled across the natural distribution of the T. ciliata complex using mtDNA and nrDNA ITS (ribosomal internal transcribed spacer) markers. Haplotype diversity was h = 0.190 ± 0.202 and nucleotide diversity was π = 0.000383 ± 0.000536 for mtDNA marker. Nucleotide diversity for ITS sequences was 0.00837 ± 0.000783. Haplotypes exhibited phylogeographic structure in spatial distribution. The extent of genetic differentiation was significant (F_st = 0.6994 ± 0.0079 for ITS and 0.8870 ± 0.0077 for mtDNA marker). Isolation by distance (IBD) and by elevation (IBE) occurred among populations. Phylogenetic relationships from mtDNA marker indicated three genetically distinct regions, each without IBD effects. Compared with pollen flow, seed flow was strongly impeded in the western region, but extensive in the central region, and less impeded in the eastern region. Most populations did not exhibit expansion, with only a few populations showing expansion after bottleneck effects. We discussed a strategy of region-based genetic conservation and proposed to conserve multiple populations in the western and eastern regions and a few populations in the central region.

Neolamarckia cadamba is an important tropical and subtropical tree for timber industry in southern China and is also a medicinal plant because of the secondary product cadambine. N. cadamba belongs to Rubiaceae family and its taxonomic relationships with other species are not fully evaluated based on genome sequences. Here, we report the complete sequences of mitochondrial genome of N. cadamba, which is 414,980 bp in length and successfully assembled in two genome circles (109,836 bp and 305,144 bp). The mtDNA harbors 83 genes in total, including 40 protein-coding genes (PCGs), 31 transfer RNA genes, 6 ribosomal RNA genes, and 6 other genes. The base composition of the whole genome is estimated as 27.26% for base A, 22.63% for C, 22.53% for G, and 27.56% for T, with the A + T content of 54.82% (54.45% in the small circle and 54.79% in the large circle). Repetitive sequences account for ~ 0.14% of the whole genome. A maximum likelihood (ML) tree based on DNA sequences of 24 PCGs supports that N. cadamba belongs to order Gentianales. A ML tree based on rps3 gene of 60 species in family Rubiaceae shows that N. cadamba is more related to Cephalanthus accidentalis and Hymenodictyon parvifolium and belongs to the Cinchonoideae subfamily. The result indicates that N. cadamba is genetically distant from the species and genera of Rubiaceae in systematic position. As the first sequence of mitochondrial genome of N. cadamba, it will provide a useful resource to investigate genetic variation and develop molecular markers for genetic breeding in the future.

Most initially perfect flowers of Toona ciliata Roem subsequently develop into functionally unisexual flowers and their relative positions in the same inflorescence could enhance the outcrossing system in this species. Here we investigated the mating system of this species. We used eight nuclear microsatellite markers and investigated the progeny of 125 mother trees from six populations naturally distributed in South China, with sample sizes ranging from 64 to 300 seeds. The multilocus outcrossing rate was 0.970 ± 0.063, and the single locus outcrossing rate was 0.859 ± 0.106, indicating the pattern of predominant outcrossing. Selfing was present in one population, but biparental inbreeding occurred in five populations. Inbreeding was absent in maternal parents, and correlations of selfing among families or among loci were generally insignificant. Positive correlation of paternity at multiple loci was significant in four populations, but was not consistent with the results at single loci. Population substructure occurred in male similarity between outcrosses only in one population. Population genetic differentaitaion was significant (F_st = 34.5%) and the effects of isolation-by-distance at the eight loci were significant among the six populations. These results provide evidence that self-comptability and inbreeding naturally occur in T. ciliata and indicate that inbreeding avoidance is necessary during genetic improvement and breeding of this endangered tree species.