Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle-scale barcodes. Next-generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high-quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long-range PCR and sequenced using next-generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early-diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome-scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs.

Nonphotosynthetic plants possess strongly reconfigured plastomes attributable to convergent losses of photosynthesis and housekeeping genes, making them excellent systems for studying genome evolution under relaxed selective pressures. We report the complete plastomes of 10 photosynthetic and nonphotosynthetic parasites plus their nonparasitic sister from the broomrape family (Orobanchaceae). By reconstructing the history of gene losses and genome reconfigurations, we find that the establishment of obligate parasitism triggers the relaxation of selective constraints. Partly because of independent losses of one inverted repeat region, Orobanchaceae plastomes vary 3.5-fold in size, with 45 kb in American squawroot (Conopholis americana) representing the smallest plastome reported from land plants. Of the 42 to 74 retained unique genes, only 16 protein genes, 15 tRNAs, and four rRNAs are commonly found. Several holoparasites retain ATP synthase genes with intact open reading frames, suggesting a prolonged function in these plants. The loss of photosynthesis alters the chromosomal architecture in that recombinogenic factors accumulate, fostering large-scale chromosomal rearrangements as functional reduction proceeds. The retention of DNA fragments is strongly influenced by both their proximity to genes under selection and the co-occurrence with those in operons, indicating complex constraints beyond gene function that determine the evolutionary survival time of plastid regions in nonphotosynthetic plants.

https://www.researchgate.net/profile/Susann_Wicke/publication/258062410_Mechanisms_of_Functional_and_Physical_Genome_Reduction_in_Photosynthetic_and_Nonphotosynthetic_Parasitic_Plants_of_the_Broomrape_Family/links/0deec52655bf6e87b6000000.pdf

Mechanisms of Functional and Physical Genome Reduction in Photosynthetic and Nonphotosynthetic Parasitic Plants of the Broomrape Family

Rafflesia is a genus of holoparasitic plants endemic to Southeast Asia that has lost the ability to undertake photosynthesis. With short-read sequencing technology, we assembled a draft sequence of the mitochondrial genome of Rafflesia lagascae Blanco, a species endemic to the Philippine island of Luzon, with ~350 sequencing depth coverage. Using multiple approaches, however, we were only able to identify small fragments of plastid sequences at low coverage depth (<2) and could not recover any substantial portion of a chloroplast genome. The gene fragments we identified included photosynthesis and energy production genes (atp,ndh,pet,psa,psb,rbcL), ribosomal RNA genes (rrn16,rrn23), ribosomal protein genes (rps7,rps11,rps16), transfer RNA genes, as well as matK,accD,ycf2, and multiple nongenic regions from the inverted repeats. None of the identified plastid gene sequences had intact reading frames. Phylogenetic analysis suggests that ~33% of these remnant plastid genes may have been horizontally transferred from the host plant genus Tetrastigma with the rest having ambiguous phylogenetic positions (<50% bootstrap support), except for psaB that was strongly allied with the plastid homolog in Nicotiana. Our inability to identify substantial plastid genome sequences from R. lagascae using multiple approaches—despite success in identifying and developing a draft assembly of the much larger mitochondrial genome—suggests that the parasitic plant genus Rafflesia may be the first plant group for which there is no recognizable plastid genome, or if presen ti s found in cryptic form at very low levels.

/pdf/possible-loss-of-the-chloroplast-genome-in-the-parasitic-14fe7llce2.pdf

Possible Loss of the Chloroplast Genome in the Parasitic Flowering Plant Rafflesia lagascae (Rafflesiaceae)

An increasing number of studies using genetic data at the generic and species levels reveal complex patterns of rela- tionships among populations and species. Incomplete lineage sorting and reticulation are, for instance, commonly observed in low-rank phylogenies. These two processes falsify the simplifying assumptions often used when reconstructing phylogenetic relationships or when assigning specimens to species using barcodes, i.e., the absence of ancestral polymorphism or the obligate dichotomous relationship among species. We recognize seven processes that act as veils to obscure species boundaries and relationships and lead to erroneous interpretations if not diagnosed and analysed properly. These processes include the trans- fer of genetic material from plastids or mitochondria to the nuclear genome (NuPt, NuMt), hybridization, the stochasticity of lineage sorting within and among species, genome organization, species demography, selection, genetic and geographic struc- ture. Most of these processes have a direct impact on species effective population sizes, a central parameter to be considered. Failure to take the seven processes into account can affect the accuracy of barcode-based identification, in particular between closely related species. It can also bias the delimitation of species, result in inaccurate estimations of species relationships and lead to incorrect estimation of species ages. We suggest that these problems can be identified and, to some extent, mitigated by ensuring that the full spatial and genetic ranges of the species are sampled, and by the use of information from all genomes of the species. By analysing numerous loci, each as a separate entity in a coalescence framework, it is possible to take into account stochasticity as well as variation in genome effective population sizes. Such a critical approach would allow the use of genetic data to make realistic assessments of species delimitation, affinities and ages, and should promote a more biologically

/pdf/species-delimitation-and-relationships-the-dance-of-the-rmjpgydxg9.pdf

Species delimitation and relationships: The dance of the seven veils

The 23 species of mycoheterotrophic or exoparasitic land plants (from 15 genera and 6 families) studied so far all retain a minimal set of 17 of the normally 116 plastome genes. Only Rafflesia lagascae, an endoparasite concealed in its host except when flowering, has been reported as perhaps lacking a plastome, although it still possesses plastid-like compartments. We analyzed two other endoparasites, the African Apodanthaceae Pilostyles aethiopica and the Australian Pilostyles hamiltonii, both living inside Fabaceae. Illumina and 454 data and Sanger resequencing yielded circularized plastomes of 11,348 and 15,167 bp length, with both species containing five possibly functional genes (accD, rps3, rps4, rrn16, rrn23) and two/three pseudogenes (rpoC2 in P. aethiopica and rpl2 and rps12 in both species; rps12 may be functional in P. hamiltonii). Previously known smallest land plant plastomes contain 27-29 genes, making these Apodanthaceae plastomes the most reduced in size and gene content. A similar extent of divergence might have caused the plastome of Rafflesia to escape detection. The higher plastome degeneration in both these families of endoparasites, Rafflesiaceae and Apodanthaceae, of similar high age, compared with exoparasites points to a difference of plastome function between those two modes of parasitic life.

/pdf/the-plastomes-of-two-species-in-the-endoparasite-genus-5115ozjuhy.pdf

The Plastomes of Two Species in the Endoparasite Genus Pilostyles (Apodanthaceae) Each Retain Just Five or Six Possibly Functional Genes

Background 
The central function of chloroplasts is to carry out photosynthesis, and its gene content and structure are highly conserved across land plants. Parasitic plants, which have reduced photosynthetic ability, suffer gene losses from the chloroplast (cp) genome accompanied by the relaxation of selective constraints. Compared with the rapid rise in the number of cp genome sequences of photosynthetic organisms, there are limited data sets from parasitic plants.


Principal Findings/Significance 
Here we report the complete sequence of the cp genome of Cistanche deserticola, a holoparasitic desert species belonging to the family Orobanchaceae. The cp genome of C. deserticola is greatly reduced both in size (102,657 bp) and in gene content, indicating that all genes required for photosynthesis suffer from gene loss and pseudogenization, except for psbM. The striking difference from other holoparasitic plants is that it retains almost a full set of tRNA genes, and it has lower dN/dS for most genes than another close holoparasitic plant, E. virginiana, suggesting that Cistanche deserticola has undergone fewer losses, either due to a reduced level of holoparasitism, or to a recent switch to this life history. We also found that the rpoC2 gene was present in two copies within C. deserticola. Its own copy has much shortened and turned out to be a pseudogene. Another copy, which was not located in its cp genome, was a homolog of the host plant, Haloxylon ammodendron (Chenopodiaceae), suggesting that it was acquired from its host via a horizontal gene transfer.

/pdf/complete-chloroplast-genome-sequence-of-holoparasite-19yc8672ob.pdf

Complete chloroplast genome sequence of holoparasite Cistanche deserticola (Orobanchaceae) reveals gene loss and horizontal gene transfer from its host Haloxylon ammodendron (Chenopodiaceae).

Ti-Cao Zhang

Papers

Complete chloroplast genome sequence of holoparasite Cistanche deserticola (Orobanchaceae) reveals gene loss and horizontal gene transfer from its host Haloxylon ammodendron (Chenopodiaceae).

Detecting adaptive evolution and functional divergence in aminocyclopropane-1-carboxylate synthase (ACS) gene family