Development and validation of a custom panel including 114 InDels using massively parallel sequencing for forensic application.

Insertion/deletion polymorphisms (InDels) have particular characteristics, such as a relatively low mutation rate, small amplicon size, and no stutter artifacts when genotyped via the capillary electrophoresis platform. It would be an important complementary tool for individual identification and certain kinship analyses. At present, massively parallel sequencing (MPS) has shown excellent application value in forensic studies. Therefore, in this study, we developed a custom MPS InDel panel that contains 114 InDels [77 autosomal InDels (A-InDels), 32 X-chromosomal InDels (X-InDels), and 5 Y-chromosomal InDels) based on previous studies. To assess this panel's performance, several validation experiments were performed, including sensitivity, inhibitor, degraded DNA testing, species specificity, concordance, repeatability, case-type samples, and population studies. The results showed that the lowest DNA input was 0.25 ng. All genotypes were obtained in the presence of 80 ng/µL humic acid, 2000 µmol/L calcium, 3000 µmol/L EDTA and indigo. In degraded DNA testing, 90% of loci could be detected for 16-day-old formalin-fixed hearts. In addition, this panel has good species specificity. The values of combined power of discrimination and the combined power of exclusion for 77 A-InDels were 1-3.9951 × 10-32 and 1-4.2956 × 10-7 , respectively. The combined mean exclusion chance for 32 X-InDels was 0.99999 in trios and 0.99904 in duos. The validation results indicate that this newly developed MPS multiplex system is a robust tool for forensic applications.

Transcriptome Characterization and Identification of Molecular Markers (SNP, SSR, and Indels) in the Medicinal Plant Sarcandra glabra spp.

Sarcandra glabra has significant metabolically active bioingredients of pharmaceutical importance. The deficiency of molecular markers for S. glabra is a hindrance in molecular breeding for genetic improvement. In this study, 57.756 million pair-end reads were generated by transcriptome sequencing in S. glabra (Thunb.) Nakai and its subspecies S. glabra ssp. brachystachys. A total of 141,954 unigenes with 646.63 bp average length were assembled. A total of 25,620 simple sequence repeats, 726,476 single nucleotide polymorphisms, and 42,939 insertions and deletions were identified, and the associated unigenes and differentially expressed genes were characterized. This work enhanced the molecular marker resources and will facilitate molecular breeding and gene mining in S. glabra spp.

Development of 50 InDel-based barcode system for genetic identification of tartary buckwheat resources.

Tartary buckwheat (Fagopyrum tataricum Gartn.) is a highly functional crop that is poised to be the target of many future breeding efforts. The reliable ex situ conservation of various genetic resources is essential for the modern breeding of tartary buckwheat varieties. We developed PCR-based co-dominant insertion/deletion (InDel) markers to discriminate tartary buckwheat genetic resources. First, we obtained the whole genome from 26 accessions across a superscaffold-scale reference genome of 569.37 Mb for tartary buckwheat cv. "Daegwan 3-7." Next, 171,926 homogeneous and 53,755 heterogeneous InDels were detected by comparing 26 accessions with the "Daegwan 3-7" reference sequence. Of these, 100 candidate InDels ranging from 5-20 bp in length were chosen for validation, and 50 of them revealed polymorphisms between the 26 accessions and "Daegwan 3-7." The validated InDels were further tested through the assessment of their likelihood to give rise to a single or a few PCR products in 50 other accessions, covering most tartary buckwheat genome types. The major allele frequencies ranged from 0.5616 at the TB42 locus to 0.9863 at the TB48 locus, with the average PIC value of 0.1532 with a range of 0.0267-0.3712. To create a user-friendly system, the homology of the genotypes between and among the accessions were visualized in both one- (1D) and two-dimensional (2D) barcode types by comparing amplicon polymorphisms with the reference variety, "Daegwan 3-7." A phylogenetic tree and population structure of the 76 accessions according to amplicon polymorphisms for the 50 InDel markers corresponded to those using non-synonymous single nucleotide polymorphism variants, indicating that the barcode system based on the 50 InDels was a useful tool to improve the reliability of identification of tartary buckwheat accessions in the germplasm stocks.

Comparative chloroplast genomes: insights into the evolution of the chloroplast genome of Camellia sinensis and the phylogeny of Camellia.

BACKGROUND: Chloroplast genome resources can provide useful information for the evolution of plant species. Tea plant (Camellia sinensis) is among the most economically valuable member of Camellia. Here, we determined the chloroplast genome of the first natural triploid Chinary type tea ('Wuyi narcissus' cultivar of Camellia sinensis var. sinensis, CWN) and conducted the genome comparison with the diploid Chinary type tea (Camellia sinensis var. sinensis, CSS) and two types of diploid Assamica type teas (Camellia sinensis var. assamica: Chinese Assamica type tea, CSA and Indian Assamica type tea, CIA). Further, the evolutionary mechanism of the chloroplast genome of Camellia sinensis and the relationships of Camellia species based on chloroplast genome were discussed. RESULTS: Comparative analysis showed the evolutionary dynamics of chloroplast genome of Camellia sinensis were the repeats and insertion-deletions (indels), and distribution of the repeats, indels and substitutions were significantly correlated. Chinese tea and Indian tea had significant differences in the structural characteristic and the codon usage of the chloroplast genome. Analysis of sequence characterized amplified region (SCAR) using sequences of the intergenic spacers (trnE/trnT) showed none of 292 different Camellia sinensis cultivars had similar sequence characteristic to triploid CWN, but the other four Camellia species did. Estimations of the divergence time showed that CIA diverged from the common ancestor of two Assamica type teas about 6.2 Mya (CI: 4.4-8.1 Mya). CSS and CSA diverged to each other about 0.8 Mya (CI: 0.4-1.5 Mya). Moreover, phylogenetic clustering was not exactly consistent with the current taxonomy of Camellia. CONCLUSIONS: The repeat-induced and indel-induced mutations were two important dynamics contributed to the diversification of the chloroplast genome in Camellia sinensis, which were not mutually exclusive. Chinese tea and Indian tea might have undergone different selection pressures. Chloroplast transfer occurred during the polyploid evolution in Camellia sinensis. In addition, our results supported the three different domestication origins of Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea. And, the current classification of some Camellia species might need to be further discussed.

INDEL detection, the 'Achilles heel' of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels.

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

Complete plastome assemblies from a panel of 13 diverse potato taxa.

The chloroplasts are a crucial part of photosynthesizing plant cells and are extensively utilized in phylogenetic studies mainly due to their maternal inheritance. Characterization and analysis of complete plastome sequences is necessary to understand their diversity and evolutionary relationships. Here, a panel of thirteen plastomes from various potato taxa are presented. Though they are highly similar with respect to gene order and content, there is also a great extent of SNPs and InDels between them, with one of the Solanum bukasovii plastomes (BUK2) having the highest number of SNPs and InDels. Five different potato plastome types (C, S, A, W, W2) are present in the panel. Interestingly, the S. tuberosum subsp. tuberosum (TBR) accession has a W-type plastome, which is not commonly found in this species. The S-type plastome has a conserved 48 bp deletion not found in other types, which is responsible for the divergence of the S-type from the C-type plastome. Finally, a phylogenetic analysis shows that these plastomes cluster according to their types. Congruence between the nuclear genome and the plastome phylogeny of these accessions was seen, however with considerable differences, supporting the hypothesis of introgression and hybridization between potato species.

Accurate quantification of copy-number aberrations and whole-genome duplications in multi-sample tumor sequencing data.

Copy-number aberrations (CNAs) and whole-genome duplications (WGDs) are frequent somatic mutations in cancer but their quantification from DNA sequencing of bulk tumor samples is challenging. Standard methods for CNA inference analyze tumor samples individually; however, DNA sequencing of multiple samples from a cancer patient has recently become more common. We introduce HATCHet (Holistic Allele-specific Tumor Copy-number Heterogeneity), an algorithm that infers allele- and clone-specific CNAs and WGDs jointly across multiple tumor samples from the same patient. We show that HATCHet outperforms current state-of-the-art methods on multi-sample DNA sequencing data that we simulate using MASCoTE (Multiple Allele-specific Simulation of Copy-number Tumor Evolution). Applying HATCHet to 84 tumor samples from 14 prostate and pancreas cancer patients, we identify subclonal CNAs and WGDs that are more plausible than previously published analyses and more consistent with somatic single-nucleotide variants (SNVs) and small indels in the same samples.

CRISPR-induced indels and base editing using the Staphylococcus aureus Cas9 in potato.

Genome editing is now widely used in plant science for both basic research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its precision, high efficiency and versatility, allows for editing of many sites in plant genomes. This system has been highly successful to produce knock-out mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on demand nucleotide conversion, allowing for fine-tuning of protein function and generating gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to mediate different simultaneous catalytic activities. In this study, we successfully used the CRISPR-Cas9 system from Staphylococcus aureus (SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed a S. aureus-cytosine base editor that mediate nucleotide conversions, allowing for precise modification of specific residues or regulatory elements in potato. Our proof-of-concept in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications.

[Development and application of chloroplast molecular markers in Panax notoginseng].

The molecular markers(cpSSR, cpSNP and cpIndel) were developed based on the whole genome sequence of Panax notoginseng chloroplast genome, which provide a powerful tool for the evaluation and analysis of the future P. notoginseng germplasm resources. The 89 P. notoginseng samples from 9 groups were used for the experiment, and the data for the study were derived from NCBI and the GenBank numbers were: KJ566590, KP036468, KR021381 and KT001509. Through sequence alignment, 30 polymorphic sites(SNP and Indel) were identified, including 16 cpSNP and 14 cpIndel; cpSNP and cpIndel accounted for far more than the gene region in the intergenic region. The developed cpSSR reached 87-89, the repeat unit was mainly composed of trinucleotide, accounting for 70%-71%, and the dinucleotide was the least, accounting for 7%. Eighteen cpDNA molecular markers were developed, including 7 cpSSR primers, 6 cpIndel primers, and 5 cpSNP primers. The MatK gene and ycf1 primers were chosen as control. According to the results of DNA gel electrophoresis, cpSSR-5, pgcpir019 and pncp08 can be used to distinguish different cultivated populations of P. notoginseng. Among them, cpSSR-5 and pgcpir019 can also be used to distinguish the inter-species resources of ginseng by comprehensive sequence length, population π value and average nucleotide difference. However, pncp08 can only be used to distinguish different populations of P. notoginseng. In addition, the effect of distinguishing the groups of P. notoginseng, which the primer pncp-M(based on the MatK gene) is weaker than the cpSSR-5, pgcpir019 and pncp08.

Phylogenetic relationships of Atractylodes lancea, A. chinensis and A. macrocephala, revealed by complete plastome and nuclear gene sequences.

Atractylodes lancea, A. chinensis, and A. macrocephala are the three most widely used medicinal species of the Atractylodes genus. Their similar morphological features cause disagreement as whether they are three unique species, leading to their frequent misuses in medical products. Our study aimed to understand their relationships through both the complete plastome sequences and nuclear sequences, to identify molecular markers for their differentiation and explore the evolutionary relationships among three species. We sequenced, annotated, and analyzed the plastomes of these three species. The plastomes are 153,201, 153,258, and 153,265 bps in length for A. lancea, A. chinensis, and A. macrocephaly, respectively. Similar to other Asteraceae species, their plastomes exhibit typical quadripartite structures. Each plastome consists of 119 distinct genes, including 78 protein-coding, 37 tRNA, and 4 rRNA genes. Analyses of indels, single-nucleotide polymorphisms and simple sequence repeats, and comparison of plastomes showed high degree of conservation, leading to difficulty in the discovery of differentiating markers. We identified eleven potential molecular markers using an algorithm based on interspecific and intraspecific nucleotide diversity gaps. Validation experiments with fifty-five individuals from the three species collected from the botanical garden and fields confirmed that the marker cz11 could effectively distinguish samples from the three different species. Analysis of the several nuclear sequences suggests that the species of A. macrocephala may be a hybrid of A. lancea and A. chinensis. In summary, the results from this study highlight the complex relationships among of these three medicinal plants.

Transcriptome analysis of Snow Mountain Garlic for unraveling the organosulfur metabolic pathway.

Snow Mountain Garlic grows in the high altitudes of the Himalayas under low temperature conditions. It contains various bioactive compounds whose metabolic pathways have not been worked out at genomic level. The present work is the first report on the transcriptome sequencing of this plant. >43 million paired-end reads (301 × 2) were generated using Illumina Miseq sequencing technology. Assembling of the sequencing data resulted in 326,785 transcripts. Differentially expressed genes between the clove and leaf tissues were identified and characterized. Besides, greater emphasis was laid on the genes, which were highly expressed in clove since the latter is assumed to contain high content of the bioactive compounds. Further analysis led to the identification of the genes plausibly involved in the organosulfur metabolism. We also identified several simple sequence repeats and single nucleotide polymorphism. These constitute valuable genetic resource for research and further genetic improvement of the plant.

Characterization of genome-wide genetic variations between two varieties of tea plant (Camellia sinensis) and development of InDel markers for genetic research.

BACKGROUND: Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) are the major genetic variations and are distributed extensively across the whole plant genome. However, few studies of these variations have been conducted in the long-lived perennial tea plant. RESULTS: In this study, we investigated the genome-wide genetic variations between Camellia sinensis var. sinensis 'Shuchazao' and Camellia sinensis var. assamica 'Yunkang 10', identified 7,511,731 SNPs and 255,218 InDels based on their whole genome sequences, and we subsequently analyzed their distinct types and distribution patterns. A total of 48 InDel markers that yielded polymorphic and unambiguous fragments were developed when screening six tea cultivars. These markers were further deployed on 46 tea cultivars for transferability and genetic diversity analysis, exhibiting information with an average 4.02 of the number of alleles (Na) and 0.457 of polymorphism information content (PIC). The dendrogram showed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or original places. Interestingly, we observed that the catechin/caffeine contents between 'Shuchazao' and 'Yunkang 10' were significantly different, and a large number of SNPs/InDels were identified within catechin/caffeine biosynthesis-related genes. CONCLUSION: The identified genome-wide genetic variations and newly-developed InDel markers will provide a valuable resource for tea plant genetic and genomic studies, especially the SNPs/InDels within catechin/caffeine biosynthesis-related genes, which may serve as pivotal candidates for elucidating the molecular mechanism governing catechin/caffeine biosynthesis.

Review: Molecular techniques to assess genetic variation within and between Panax ginseng and Panax quinquefolius.

A variety of methods have been used to examine genetic differences in P. ginseng and P. quinquefolius. They have shown genetic differences within populations of P. ginseng (within and between elite cultivars, landraces and wild accessions), within populations of P. quinquefolius (within and between wild and cultivated accessions) and between P. ginseng and P. quinquefolius as well as other Panax species. Some examples of their applications have been to show that some elite cultivars are not uniform, there are possible founder effects in certain populations, there has been the spread of cultivated types into wild populations, relative diversity differs between different populations and identification of the source and purity of commercial samples. More work in the use of molecular markers for ginseng are needed, however, particularly the use of Next Generation Sequencing. Potential applications are the use of sequence analysis for genetic selection, breeding to develop new cultivars and providing traceability from field to consumer. Research on molecular markers in ginseng has lagged compared to other crops probably because of less of an emphasis on breeding for cultivar development and relatively small areas of production. The many potential benefits for ginseng production have yet to be realized.

A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[Ser]Sec Gene.

The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3' UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism.

Sequencing and Comparative Analysis of the Chloroplast Genome of Angelica polymorpha and the Development of a Novel Indel Marker for Species Identification.

The genus Angelica (Apiaceae) comprises valuable herbal medicines. In this study, we determined the complete chloroplast (CP) genome sequence of A. polymorpha and compared it with that of Ligusticum officinale (GenBank accession no. NC039760). The CP genomes of A. polymorpha and L. officinale were 148,430 and 147,127 bp in length, respectively, with 37.6% GC content. Both CP genomes harbored 113 unique functional genes, including 79 protein-coding, four rRNA, and 30 tRNA genes. Comparative analysis of the two CP genomes revealed conserved genome structure, gene content, and gene order. However, highly variable regions, sufficient to distinguish between A. polymorpha and L. officinale, were identified in hypothetical chloroplast open reading frame1 (ycf1) and ycf2 genic regions. Nucleotide diversity (Pi) analysis indicated that ycf4⁻chloroplast envelope membrane protein (cemA) intergenic region was highly variable between the two species. Phylogenetic analysis revealed that A. polymorpha and L. officinale were well clustered at family Apiaceae. The ycf4-cemA intergenic region in A. polymorpha carried a 418 bp deletion compared with L. officinale. This region was used for the development of a novel indel marker, LYCE, which successfully discriminated between A. polymorpha and L. officinale accessions. Our results provide important taxonomic and phylogenetic information on herbal medicines and facilitate their authentication using the indel marker.

Transcriptome analysis identifies strong candidate genes for ginsenoside biosynthesis and reveals its underlying molecular mechanism in Panax ginseng C.A. Meyer.

Ginseng, Panax ginseng C.A. Meyer, is one of the most important medicinal herbs for human health and medicine in which ginsenosides are known to play critical roles. The genes from the cytochrome P450 (CYP) gene superfamily have been shown to play important roles in ginsenoside biosynthesis. Here we report genome-wide identification of the candidate PgCYP genes for ginsenoside biosynthesis, development of functional SNP markers for its manipulation and systems analysis of its underlying molecular mechanism. Correlation analysis identified 100 PgCYP genes, including all three published ginsenoside biosynthesis PgCYP genes, whose expressions were significantly correlated with the ginsenoside contents. Mutation association analysis identified that six of these 100 PgCYP genes contained SNPs/InDels that were significantly associated with ginsenosides biosynthesis (P ≤ 1.0e-04). These six PgCYP genes, along with all ten published ginsenoside biosynthesis genes from the PgCYP and other gene families, formed a strong co-expression network, even though they varied greatly in spatio-temporal expressions. Therefore, this study has identified six new ginsenoside biosynthesis candidate genes, provided a genome-wide insight into how they are involved in ginsenoside biosynthesis and developed a set of functional SNP markers useful for enhanced ginsenoside biosynthesis research and breeding in ginseng and related species.

Bromein, a Bromelain Inhibitor from Pineapple Stem: Structural and Functional Characteristics.

Bromelain inhibitor, "bromein", is a proteinase-inhibitor specific to the cysteine proteinase bromelain from pineapple stem. In the stem, eight bromein isoforms are known to exist, and each isoform has a short peptide (light chain) and a long one (heavy chain) with five disulfide bonds. The three-dimensional structure of the sixth isoform (bromein-6) is composed of inhibitory and stabilizing domains, and each domain contains a three-stranded antiparallel ß-sheet. The genomic sequence of a bromein precursor encodes three homologous bromein isoform domains, and each isoform domain has a signal peptide, three interchain peptides between the light chain and heavy chain, two interdomain peptides and a propeptide. Interestingly, at the protein level, bromein- 6 appears to share a similar folding and disulfide-bonding connectivity with Bowman-Birk serine proteinase inhibitors and shows weak inhibition toward chymotrypsin and trypsin. However, no significant similarity was found between them at the genomic level. This indicates that they have evolved convergently to possess such a structural similarity. To identify the essential reactive site(s) with bromelain, we investigated the inhibitory activity of 44 kinds of the single/double and insertion/ deletion mutants of bromein-6 towards stem bromelain. As a result, it was shown that both the appropriate positioning and the complete side-chain structure of Leu10 in the light chain are absolutely crucial for the inhibition, with an additional measure of importance for the preceding Pro9. Bromein and stem bromelain coexist in the acidic vacuoles of the stem tissue, and one of the key role of bromein appears to be the regulation of the bromelain activity.

Comparative Chloroplast Genome Analyses of Species in Gentiana section Cruciata (Gentianaceae) and the Development of Authentication Markers.

Gentiana section Cruciata is widely distributed across Eurasia at high altitudes, and some species in this section are used as traditional Chinese medicine. Accurate identification of these species is important for their utilization and conservation. Due to similar morphological and chemical characteristics, correct discrimination of these species still remains problematic. Here, we sequenced three complete chloroplast (cp) genomes (G. dahurica, G. siphonantha and G. officinalis). We further compared them with the previously published plastomes from sect. Cruciata and developed highly polymorphic molecular markers for species authentication. The eight cp genomes shared the highly conserved structure and contained 112 unique genes arranged in the same order, including 78 protein-coding genes, 30 tRNAs, and 4 rRNAs. We analyzed the repeats and nucleotide substitutions in these plastomes and detected several highly variable regions. We found that four genes (accD, clpP, matK and ycf1) were subject to positive selection, and sixteen InDel-variable loci with high discriminatory powers were selected as candidate barcodes. Our phylogenetic analyses based on plastomes further confirmed the monophyly of sect. Cruciata and primarily elucidated the phylogeny of Gentianales. This study indicated that cp genomes can provide more integrated information for better elucidating the phylogenetic pattern and improving discriminatory power during species authentication.

Cytological and transcriptome analyses reveal abrupt gene expression for meiosis and saccharide metabolisms that associated with pollen abortion in autotetraploid rice.

Autotetraploid rice is a useful germplasm that has four chromosome sets and strong biological advantages; however, low fertility limits its commercial utilization. Little information is available about the DNA variation and differential gene expressions associated with low fertility in autotetraploid rice. In the present study, 81 SNPs and 182 InDels were identified in T449 (an autotetraploid rice line with low fertility) compared to E249 (diploid counterpart) by whole-genome re-sequencing. We detected only three non-synonymous SNPs and six large-effect InDels, which were associated with three and six genes, respectively. A total of 75 meiosis-related differentially expressed genes were detected during the meiosis stage by transcriptome analysis, including OsMTOPVIB, which is essential for meiotic DSB formation, and OsMOF, which takes part in homologous chromosome pairing and synapsis. Approximately 20.69% lagging chromosome at metaphase I and 4.65% abnormal tetrad were observed in T449. Moreover, transcriptome analysis revealed down-regulation of a sucrose transporter (OsSUT5) and two monosaccharide transporters (OsMST1 and OsMST8) in T449 at the single microspore stage, and their expression levels were verified by qRT-PCR. Cytological observation of saccharide distribution showed abnormal accumulation of saccharides in T449 and the contents of fructose and glucose were markedly higher in T449 than E249 at the single microspore stage. Our results suggested that polyploidy not only induces abrupt expression changes in the meiosis-related genes that lead to abnormal chromosome behavior, but also causes changes in the saccharide distribution and expression patterns of saccharide-related genes, which jointly causes sterility in the autotetraploid rice.

Molecular Discrimination of Cynanchum wilfordii and Cynanchum auriculatum by InDel Markers of Chloroplast DNA.

The tuber of Cynanchum wilfordii (Baekshuoh Radix in Korean) is an important medicinal herb in Korea and China; however, it is difficult to differentiate C. wilfordii from a related medicinal herb, C. auriculatum (Baishouwu Radix in Chinese). We sought to develop a molecular method that could be used to distinguish between the tubers of C. wilfordii and C. auriculatum. We aligned the chloroplast genome sequences (available in the NCBI database) of the two species and identified three species-specific insertion and deletion (InDel) sites in the trnQ-psbK, rps2-rpoC2, and psaJ-rpl33 intergenic spacer (IGS) regions. To confirm the presence of these three InDels and validate their use as markers, we designed three primer pairs to amplify the trnQ-psbK, rps2-rpoC2, and psaJ-rpl33 IGS regions. Polymerase chain reaction (PCR) amplification of the trnQ-psbK IGS region yielded a 249 bp fragment for C. wilfordii, and 419 bp fragment for C. auriculatum, whereas the rps2-rpoC2 IGS primers produced a 629 bp fragment from C. wilfordii and a 282 bp fragment from C. auriculatum. In the psaJ-rpl33 IGS region, allele fragments of 342 and 360 bp in length were amplified from C. wilfordii, whereas 249 and 250 bp fragment were amplified from C. auriculatum. We propose these three InDel markers as a valuable, simple, and efficient tool for identifying these medicinal herbs and will thus reduce adulteration of these herbal materials in commercial markets.