Comprehensive analysis of the complete mitochondrial genome of Lilium tsingtauense reveals a novel multichromosome structure.

KEY MESSAGE: Lilium tsingtauense mitogenome comprises 27 independent chromosome molecules, it undergoes frequent genomic recombination, and the rate of recombination and mutation between different repetitive sequences affects the formation of multichromosomal structures. Given the extremely large genome of Lily, which likely harbors additional genetic resources, it serves as an ideal material for studying the phylogenetic evolution of organisms. Although the Lilium chloroplast genome has been documented, the sequence of its mitochondrial genome (mitogenome) remains uncharted. Using BGI short reads and Nanopore long reads, we sequenced, assembled, and annotated the mitogenome of Lilium tsingtauense. This effort culminated in the characterization of Lilium's first complete mitogenome. Comparative analysis with other angiosperms revealed the unique multichromosomal structure of the L. tsingtauense mitogenome, spanning 1,125,108 bp and comprising 27 independent circular chromosomes. It contains 36 protein-coding genes, 12 tRNA genes, and 3 rRNA genes, with a GC content of 44.90%. Notably, three chromosomes in the L. tsingtauense mitogenome lack identifiable genes, hinting at the potential existence of novel genes and noncoding elements. The high degree of observed genome fragmentation implies frequent reorganization, with recombination and mutation rates among diverse repetitive sequences likely driving the formation of multichromosomal structures. Our comprehensive analysis, covering genome size, coding genes, structure, RNA editing, repetitive sequences, and sequence migration, sheds light on the evolutionary and molecular biology of multichromosomal mitochondria in Lilium. This high-quality mitogenome of L. tsingtauense not only enriches our understanding of multichromosomal mitogenomes but also establishes a solid foundation for future genome breeding and germplasm innovation in Lilium.

Genomic divergence and demographic history of Quercus aliena populations.

BACKGROUND: Quercus aliena is a major montane tree species of subtropical and temperate forests in China, with important ecological and economic value. In order to reveal the species' population dynamics, genetic diversity, genetic structure, and association with mountain habitats during the evolutionary process, we re-sequenced the genomes of 72 Q. aliena individuals. RESULTS: The whole chloroplast and nuclear genomes were used for this study. Phylogenetic analysis using the chloroplast genome dataset supported four clades of Q. aliena, while the nuclear dataset supported three major clades. Sex-biased dispersal had a critical role in causing discordance between the chloroplast and nuclear genomes. Population structure analysis showed two groups in Q. aliena. The effective population size sharply declined 1 Mya, coinciding with the Poyang Glaciation in Eastern China. Using genotype-climate association analyses, we found a positive correlation between allele frequency variation in SNPs and temperature, suggesting the species has the capacity to adapt to changing temperatures. CONCLUSION: Overall, this study illustrates the genetic divergence, genomic variation, and evolutionary processes behind the demographic history of Q. aliena.

Genome-wide analysis of the heat shock transcription factor family reveals saline-alkali stress responses in Xanthoceras sorbifolium.

The heat shock transcription factor (HSF) family is involved in regulating growth, development, and abiotic stress. The characteristics and biological functions of HSF family member in X. sorbifolium, an important oil and ornamental plant, have never been reported. In this study, 21 XsHSF genes were identified from the genome of X. sorbifolium and named XsHSF1-XsHSF21 based on their chromosomal positions. Those genes were divided into three groups, A, B, and C, containing 12, one, and eight genes, respectively. Among them, 20 XsHSF genes are located on 11 chromosomes. Protein structure analysis suggested that XsHSF proteins were conserved, displaying typical DNA binding domains (DBD) and oligomerization domains (OD). Moreover, HSF proteins within the same group contain specific motifs, such as motif 5 in the HSFC group. All XsHSF genes have one intron in the CDS region, except XsHSF1 which has two introns. Promoter analysis revealed that in addition to defense and stress responsiveness elements, some promoters also contained a MYB binding site and elements involved in multiple hormones responsiveness and anaerobic induction. Duplication analysis revealed that XsHSF1 and XsHSF4 genes were segmentally duplicated while XsHSF2, XsHSF9, and XsHSF13 genes might have arisen from transposition. Expression pattern analysis of leaves and roots following salt-alkali treatment using qRT-PCR indicated that five XsHSF genes were upregulated and one XsHSF gene was downregulated in leaves upon NaCl treatment suggesting these genes may play important roles in salt response. Additionally, the expression levels of most XsHSFs were decreased in leaves and roots following alkali-induced stress, indicating that those XsHSFs may function as negative regulators in alkali tolerance. MicroRNA target site prediction indicated that 16 of the XsHSF genes may be regulated by multiple microRNAs, for example XsHSF2 might be regulated by miR156, miR394, miR395, miR408, miR7129, and miR854. And miR164 may effect the mRNA levels of XsHSF3 and XsHSF17, XsHSF9 gene may be regulated by miR172. The expression trends of miR172 and miR164 in leaves and roots on salt treatments were opposite to the expression trend of XsHSF9 and XsHSF3 genes, respectively. Promoter analysis showed that XsHSFs might be involved in light and hormone responses, plant development, as well as abiotic stress responses. Our results thus provide an overview of the HSF family in X. sorbifolium and lay a foundation for future functional studies to reveal its roles in saline-alkali response.

Resequencing of Rosa rugosa accessions revealed the history of population dynamics, breed origin, and domestication pathways.

BACKGROUND: Rosa rugosa is a shrub that originated in China and has economic and ecological value. However, during the development of R. rugosa, the genetic background was chaotic, and the genetic structure among different wild populations was unclear, as well as wild and cultivated accessions. Here, we report whole-genome resequencing of wild and cultivated R. rugosa accessions. RESULTS: A total of 19,041,284 SNPs were identified in 188 R. rugosa accessions and 3 R. chinensis accessions by resequencing. Population genetic analysis revealed that cultivated and wild groups were separated very early. All R. rugosa accessions were divided into 8 categories based on genetic structure: (1) Weihai, Yantai, and Liaoning category, (2) Jilin category, and (3) Hammonasset category (above three are wild); (4) traditional varieties, (5) hybrids between R. rugosa and R. chinensis, (6) Zizhi Rose, (7) Kushui Rose, (8) hybrids between R. rugosa and R. multiflora. We found that the heterozygosity and genetic diversity of wild accessions were generally lower than those of cultivated individuals. The genes that were selected during cultivation were identified, and it was found that these genes were mainly related to environmental adaptation and growth. CONCLUSIONS: The Jilin population was the oldest population and later migrated to Liaoning and then migrated to Yantai and Weihai by sea regression in the Bohai Basin. The Hammonasset naturalized population probably originated from the Jilin population and then experienced separate differentiation. The long-term asexual reproduction pattern of R. rugosa decreased genetic diversity in the wild population. During R. rugosa cultivation, the ancestors of the Jilin population were involved in breeding traditional varieties, after which almost no wild individuals were engaged in breeding. However, in recent decades, cross breeding of R. rugosa started the utilization of wild germplasms. In comparison, some other species play important roles in variety formation. Few genes related to economic traits were selected, suggesting no directional domestication in the R. rugosa cultivation process.

Sporogenesis, gametophyte development and embryogenesis in Glehnia littoralis.

BACKGROUND: Glehnia littoralis is an economic herb with both medicinal and edible uses. It also has important ecological value and special phylogenetic status as it is a monotypic genus species distributing around beach. Little information on its reproductive biology has been reported so far, which has hindered conservation and application of this species. In this study, we observed morphological changes from buds emergence to seeds formation and internal changes during sporogenesis, gametophyte development and embryo and endosperm development of G. littoralis using paraffin-embedded-sectioning and stereo microscope. RESULTS: The results showed that the stages of internal development events of G. littoralis corresponded to obvious external morphological changes, most of developmental features were consistent with other Apiaceae species. The development of male and female gametophytes was not synchronized in the same flower, however, exhibited temporal overlap. From mid-late April to mid-May, the anther primordial and ovule primordial developed into the trinucleate pollen grain and eight-nuclear embryo sac, respectively. From late-May to mid-July, the zygote developed into mature embryo. In addition, some defects in gynoecium or ovule development and abnormal embryo and endosperm development were found. We induced that the possible causes of abortion in G. littoralis were as follows: nutrient limitation, poor pollination and fertilization, and bad weather. CONCLUSIONS: This study revealed the whole process and morphological characteristics of the development of reproductive organ in G. littoralis, which not only provided important data for the study of systematic and conservation biology, but also provided a theoretical basis for cross breeding.

Genome-Wide Identification and Expression Profiling of Heat Shock Protein 20 Gene Family in Sorbus pohuashanensis (Hance) Hedl under Abiotic Stress.

Small heat shock proteins (HSP20s) are a significant factor in plant growth and development in response to abiotic stress. In this study, we investigated the role of HSP20s' response to the heat stress of Sorbus pohuashanensis introduced into low-altitude areas. The HSP20 gene family was identified based on the genome-wide data of S. pohuashanensis, and the expression patterns of tissue specificity and the response to abiotic stresses were evaluated. Finally, we identified 38 HSP20 genes that were distributed on 16 chromosomes. Phylogenetic analysis of HSP20s showed that the closest genetic relationship to S. pohuashanensis (SpHSP20s) is Malus domestica, followed by Populus trichocarpa and Arabidopsis thaliana. According to phylogenetic analysis and subcellular localization prediction, the 38 SpHSP20s belonged to 10 subfamilies. Analysis of the gene structure and conserved motifs indicated that HSP20 gene family members are relatively conserved. Synteny analysis showed that the expansion of the SpHSP20 gene family was mainly caused by segmental duplication. In addition, many cis-acting elements connected with growth and development, hormones, and stress responsiveness were found in the SpHSP20 promoter region. Analysis of expression patterns showed that these genes were closely related to high temperature, drought, salt, growth, and developmental processes. These results provide information and a theoretical basis for the exploration of HSP20 gene family resources, as well as the domestication and genetic improvement of S. pohuashanensis.

A high-quality genome assembly and annotation of Quercus acutissima Carruth.

Introduction: Quercus acutissima is an economic and ecological tree species often used for afforestation of arid and semi-arid lands and is considered as an excellent tree for soil and water conservation. Methods: Here, we combined PacBio long reads, Hi-C, and Illumina short reads to assemble Q. acutissima genome. Results: We generated a 957.1 Mb genome with a contig N50 of 1.2 Mb and scaffold N50 of 77.0 Mb. The repetitive sequences constituted 55.63% of the genome, among which long terminal repeats were the majority and accounted for 23.07% of the genome. Ab initio, homology-based and RNA sequence-based gene prediction identified 29,889 protein-coding genes, of which 82.6% could be functionally annotated. Phylogenetic analysis showed that Q. acutissima and Q. variabilis were differentiated around 3.6 million years ago, and showed no evidence of species-specific whole genome duplication. Conclusion: The assembled and annotated high-quality Q. acutissima genome not only promises to accelerate the species molecular biology studies and breeding, but also promotes genome level evolutionary studies.

Complete mitochondrial genome sequence of Acer miaotaiense (Aceraceae).

Acer miaotaiense P. C. Tsoong is a rare and endangered tree endemic to the Qinling Mountains of China and is listed as a national third-class protected plant. In this study, we sequenced the complete mitochondrial genome of Acer miaotaiense using the Illumina Novaseq 6000 and Nanopore platforms. The total mitochondrial genome length is 819,227 bp and has 69 genes, including 41 protein-coding, 25 tRNA, and 3 rRNA genes. The genome nucleotide composition was asymmetric, with an overall G + C content of 45.7%. Phylogenetic analysis indicated that Acer miaotaiense is closely related to the congeneric Acer yangbiense.

Complex Physical Structure of Complete Mitochondrial Genome of Quercus acutissima (Fagaceae): A Significant Energy Plant.

Quercus acutissima Carruth. is a Chinese important energy plant with high ecological and economic values. While the species chloroplast genome has been reported, its mitochondrial genome (mitogenome) is still unexplored. Here, we assembled and annotated the Q. acutissima mitogenome, and we compared its characteristic differences with several closely related species. The Q. acutissima mitogenome's main structure is branched with three distinguished contigs (linear molecule 1, circular molecule 2, and circular molecule 3) with 448,982 bp total length and 45.72% GC content. The mitogenome contained 51 genes, including 32 protein-coding, 16 tRNA and 3 rRNA genes. We examined codon usage, repeated sequences, genome recombination, chloroplast to mitochondrion DNA transformation, RNA editing, and synteny in the Q. acutissima mitogenome. Phylogenetic trees based on 29 species mitogenomes clarified the species classification. Our results provided comprehensive information of Q. acutissima mitogenome, and they are expected to provide valuable information for Fagaceae evolutionary biology and to promote the species germplasm utilization.

Characterization of the complete plastome of Thalictrum aquilegiifolium var. sibiricum (Ranunculaceae), an annual herb.

Thalictrum aquilegiifolium var. sibiricum, is an annual herb that grows on slopes or in mountain gullies in areas of damp forest. In this study, we report for the first time the complete plastome sequence of T. aquilegiifolium var. sibiricum. The plastome sequence is 156,244 bp in length and comprises a large single-copy region (85,447 bp), a small single-copy region (17,599 bp), and a pair of inverted repeat regions (26,480 bp). The genome encodes 112 unique genes, including 78 protein-coding genes, 30 tRNAs, and four rRNAs, and has total GC content of 38.4%. Phylogenomic analysis based on the plastome sequences of 20 species in the family Ranunculaceae indicated that T. aquilegiifolium var. sibiricum is clustered with Thalictrum minus.

Characterization of the complete chloroplast genome of Achnatherum pekinense (Poaceae), a widespread weed.

Achnatherum pekinense belongs to Poaceae. The complete chloroplast genome of A. pekinense was reported in this study. The chloroplast genome was 137,837 bp in size with a canonical quadripartite structure, including two inverted repeat regions (IR) of 21,635 bp for each, a large single-copy (LSC) region of 81,787 bp in length, and a small single-copy (SSC) region of 12,780 bp in length. The overall guanine-cytosine (GC) content of this chloroplast genome was 38.8%, and the corresponding values of the LSC, SSC, and IR regions were 36.9%, 33.1%, and 44.1%, respectively. A total of 113 unique genes were annotated in this chloroplast genome, including four rRNA genes, 31 tRNA genes, and 78 protein-coding genes. The phylogenetic analysis showed that A. pekinense was clustered with A. inebrians.

Genome-wide analysis of BURP genes and identification of a BURP-V gene RcBURP4 in Rosa chinensis.

KEY MESSAGE: Nine RcBURPs have been identified in Rosa chinensis, and overexpression of RcBURP4 increased ABA, NaCl sensitivity, and drought tolerance in transgenic Arabidopsis. BURP proteins are unique to plants and may contribute greatly to growth, development, and stress responses of plants. Despite the vital role of BURP proteins, little is known about these proteins in rose (Rosa spp.). In the present study, nine genes belonging to the BURP family in R. chinensis were identified using multiple bioinformatic approaches against the rose genome database. The nine RcBURPs, with diverse structures, were located on all chromosomes of the rose genome, except for Chr2 and Chr3. Phylogenic analysis revealed that these RcBURPs can be classified into eight subfamilies, including BNM2-like, PG1ß-like, USP-like, RD22-like, BURP-V, BURP-VI, BURP-VII, and BURP-VIII. Conserved motif and exon-intron analyses indicated a conserved pattern within the same subfamily. The presumed cis-regulatory elements (CREs) within the promoter region of each RcBURP were analyzed and the results showed that all RcBURPs contained different types of CREs, including abiotic stress-, light response-, phytohormones response-, and plant growth and development-related CREs. Transcriptomic analysis revealed that a BURP-V member, RcBURP4, was induced in rose leaves and roots under mild and severe drought treatments. We then overexpressed RcBURP4 in Arabidopsis and examined its role under abscisic acid (ABA), NaCl, polyethylene glycol (PEG), and drought treatments. Nine stress-responsive genes expression were changed in RcBURP4-overexpressing leaves and roots. Furthermore, RcBURP4-silenced rose plants exhibited decreased tolerance to dehydration. The results obtained from this study provide the first comprehensive overview of RcBURPs and highlight the importance of RcBURP4 in rose plant.

Complete sequence and comparative analysis of the mitochondrial genome of the rare and endangered Clematis acerifolia, the first clematis mitogenome to provide new insights into the phylogenetic evolutionary status of the genus.

Clematis is one of the large worldwide genera of the Ranunculaceae Juss. Family, with high ornamental and medicinal value. China is the modern distribution centre of Clematis with abundant natural populations. Due to the complexity and high morphological diversity of Clematis, the genus is difficult to classify systematically, and in particular, the phylogenetic position of the endangered Clematis acerifolia is highly controversial. The use of the mitochondrial complete genome is a powerful molecular method that is frequently used for inferring plants phylogenies. However, studies on Clematis mitogenome are rare, thus limiting our full understanding of its phylogeny and genome evolution. Here, we sequenced and annotated the C. acerifolia mt genome using Illumina short- and Nanopore long-reads, characterized the species first complete mitogenome, and performed a comparative phylogenetic analysis with its close relatives. The total length of the C. acerifolia mitogenome is 698,247 bp and the main structure is multi-branched (linear molecule 1 and circular molecule 2). We annotated 55 genes, including 35 protein-coding, 17 tRNA, and 3 rRNA genes. The C. acerifolia mitogenome has extremely unconserved structurally, with extensive sequence transfer between the chloroplast and mitochondrial organelles, sequence repeats, and RNA editing. The phylogenetic position of C. acerifolia was determined by constructing the species mitogenome with 24 angiosperms. Further, our C. acerifolia mitogenome characteristics investigation included GC contents, codon usage, repeats and synteny analysis. Overall, our results are expected to provide fundamental information for C. acerifolia mitogenome evolution and confirm the validity of mitochondrial analysis in determining the phylogenetic positioning of Clematis plants.

The complete chloroplast genome of Waldheimia glabra.

Waldheimia glabra (Decne.) Rgl. 1879 (family Asteraceae) is a perennial herb with high economic and medicinal values. In this study, we sequenced the complete chloroplast (cp) genome of W. glabra by high-throughput Illumina sequencing. The size of the W. glabra cp genome is 151,499 bp, with overall GC content of 37.3%. It contains a large single copy and a small single copy region of 83,078 bp and 18,457 bp, respectively, separated by a pair of inverted repeats regions of 24,982 bp. We also discovered 131 genes, including 86 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes in the genome. The maximum-likelihood phylogenetic tree demonstrated that W. glabra is closely related to Leucanthemella linearis.

The first complete chloroplast genome of Hovenia dulcis Thunb. (Rhamnaceae).

The complete chloroplast genome of Hovenia dulcis was sequenced with Illumina HiSeq 2000 platform. It was a typical quadruple structure as other plants of Hovenia with 162,962 bp in length, including a large single-copy (LSC: 90,900 bp) region and a small single-copy (SSC: 18,920 bp) which were separated by a pair of inverted repeats (IRa, b: 26,571 bp) region. The overall GC content is 36.6%. A total of 130 genes was annotated which contained 85 protein-coding genes including the Trans splicing gene of rps12, 37 tRNA genes, and 8 rRNA genes. ML phylogenetic analysis compared with 6 expressed chloroplast genomes of Rhamnaceae revealed that H. dulcis was closely related to the species of Zizyphus, and which were clustered into a group with Z. jujuba, Z. mauritiana and Z. spina-christi. Hovenia dulcis was relatively distant to other species of Berchemiella and Rhamnus, which were clustered into another group.

Complete chloroplast genome of Prunus canescens: an endemic shrub in China.

Prunus canescens is an endemic cherry species in China, which is distributed in Shaanxi, Gansu, Hubei, and Sichuan provinces of China. The chloroplast (cp) genome of P. canescens is 157,890 bp in size containing 125 unique genes, including 8 rRNA genes, 37 tRNA genes, and 80 protein-coding genes (PCGs). Phylogenetic analysis exhibited that P. canescens is most related to P. pseudocerasus.