Development of SSR markers via de novo transcriptome assembly in Akebia trifoliata (Thunb.) Koidz.

Owing to its high nutritive, economic, and medicinal values, Akebia trifoliata has received increased attention, making worthy of being used as a new fruit crop for further domestication and commercialization in China. However, molecular research of A. trifoliata has lagged as investigations of its genomic resources and molecular markers are rare. In this study, a cDNA library of A. trifoliata leaves was sequenced using the Illumina NovaSeq. 6000 sequencing system. In total, 101 417 transcripts, 63 757 unigenes, and 9494 simple sequence repeats were assembled and identified from the transcriptome datasets. The majority of the SSRs were di- and trinucleotide repeats. Length and number of SSR motifs ranged from 15 to 66, and 5 to 48 bp, respectively. Of which, the A/T mononucleotide motif and AG/TC and CT/GA dinucleotide motifs were the most abundant. Furthermore, 100 SSR primers were randomly selected to validate amplification and polymorphism, and 88 A. trifoliata accessions were definitively distinguished by 49 primers. With the Qinling mountains and Huaihe River line as the boundaries, the northern and southern accessions were clustered into different groups, but no clear geographical patterns (city or origin) were observed in the southern accessions. These newly identified molecular markers may provide a foundation for the genetic identification and diversity analysis and marker-assisted selection breeding in species of Akebia.

Genome-wide association study discovered favorable single nucleotide polymorphisms and candidate genes associated with ramet number in ramie (Boehmeria nivea L.).

BACKGROUND: Ramie (Boehmeria nivea L.) is one of the most important natural fiber crops and an important forage grass in south China. Ramet number, which is a quantitative trait controlled by multigenes, is one of the most important agronomic traits in plants because the ramet number per plant is a key component of grain yield and biomass. However, the genetic variation and genetic architecture of ramie ramet number are rarely known. RESULTS: A genome-wide association study was performed using a panel of 112 core germplasms and 108,888 single nucleotide polymorphisms (SNPs) detected using specific-locus amplified fragment sequencing technology. Trait-SNP association analysis detected 44 significant SNPs that were associated with ramet number at P < 0.01. The favorable SNP Marker20170-64 emerged at least twice in the three detected stages and was validated to be associated with the ramie ramet number using genomic DNA polymerase chain reaction with an F1 hybrid progeny population. Comparative genome analysis predicted nine candidate genes for ramet number based on Marker20170-64. Real-time quantitative polymerase chain reaction analysis indicated that six of the genes were specific to upregulation in the ramie variety with high ramet number. These results suggest that these genes could be considered as ramet number-associated candidates in ramie. CONCLUSIONS: The identified loci or genes may be promising targets for genetic engineering and selection for modulating the ramet number in ramie. Our work improves understanding of the genetics of ramet number in ramie core germplasms and provides tools for marker-assisted selection for improvement of agricultural traits.

Genetic Diversity of a Natural Population of Akebia trifoliata (Thunb.) Koidz and Extraction of a Core Collection Using Simple Sequence Repeat Markers.

Understand genetic diversity and genetic structure of germplasm is premise of germplasm conservation and utilization. And core collection can reduce the cost and difficulty of germplasm conservation. Akebia trifoliata (Thunb.) Koidz is an important medicinal, fruit and oil crop, particularly in China. In this study, 28 simple sequence repeat (SSR) markers were used to assess the genetic diversity and genetic structure of 955 A. trifoliata germplasms, determine their molecular identity and extract a core collection. The genetic diversity of the 955 germplasms was moderately polymorphic. The average number of alleles (Na), observed heterozygosity (H O ), expected heterozygosity (H E ), Shannon's information index (I∗), and polymorphic information content (PIC) were 3.71, 0.24, 0.46, 0.81, and 0.41, respectively. Four subpopulations were identified, indicating a weak genetic structure. A 955 germplasms could be completely distinguished by the characters of s28, s25, s74, s89, s68, s30, s13, s100, s72, s77, and s3. And each germplasm's molecular identity was made up of eleven characters. The core collection was composed of 164 germplasms (17.2% of 955 total germplasms in the population) and diversity parameters differed significantly from those of a random core collection. These results have implications for germplasm conservation. At the same time, based on the results, the 955 germplasm could be better used and managed.

Comparative Analysis of Akebia trifoliata Fruit Softening at Different Flesh Ripening Stages Using Tandem Mass Tag Technology.

Owing to its medicinal and high nutritional values, Akebia trifoliata can be considered as a new type of medicinal and edible homologous resources, and it has begun to be widely cultivated in many areas of China. Over-softening of fruit would affect the sensorial quality, utilization rate, and consumer acceptance of the fruit postharvest. However, fruit softening has not been characterized and the molecular mechanism underlying A. trifoliata fruit softening during ripening remains unclear. A comparative proteomic analysis was performed on the fruit at three developmental stages using tandem mass tag technology. In total, 2,839 proteins and 302 differentially abundant proteins (DAPs) were identified. Bioinformatics analysis indicated that most DAPs were implicated in oxidoreductase activity, protein domain-specific binding and pyruvate metabolism. Moreover, 29 DAPs associated with cell wall metabolism, plant hormone, and stress and defense response pathways were validated using quantitative PCR. Notably, pectinesterase, pectate lyase, and ß-galactosidase, which are involved in cell wall degradation, as well as gibberellin regulated protein, cysteine protease, thaumatin-like protein and heat shock proteins which is involved in plant hormone, and stress and defense response, were significantly up-regulated in softening fruit compared with the levels in non-softening fruit. This indicated that they might play key roles in A. trifoliata fruit softening. Our findings will provide new insights into potential genes influencing fruit softening traits of A. trifoliata, which will help to develop strategies to improve fruit quality and reduce softening-related losses.

Integrative transcriptome and proteome analyses provide new insights into different stages of Akebia trifoliata fruit cracking during ripening.

BACKGROUND: Akebia trifoliata (Thunb.) Koidz may have applications as a new potential source of biofuels owing to its high seed count, seed oil content, and in-field yields. However, the pericarp of A. trifoliata cracks longitudinally during fruit ripening, which increases the incidence of pests and diseases and can lead to fruit decay and deterioration, resulting in significant losses in yield. Few studies have evaluated the mechanisms underlying A. trifoliata fruit cracking. RESULTS: In this study, by observing the cell wall structure of the pericarp, we found that the cell wall became thinner and looser and showed substantial breakdown in the pericarp of cracking fruit compared with that in non-cracking fruit. Moreover, integrative analyses of transcriptome and proteome profiles at different stages of fruit ripening demonstrated changes in the expression of various genes and proteins after cracking. Furthermore, the mRNA levels of 20 differentially expressed genes were analyzed, and parallel reaction monitoring analysis of 20 differentially expressed proteins involved in cell wall metabolism was conducted. Among the molecular targets, pectate lyases and pectinesterase, which are involved in pentose and glucuronate interconversion, and ß-galactosidase 2, which is involved in galactose metabolism, were significantly upregulated in cracking fruits than in non-cracking fruits. This suggested that they might play crucial roles in A. trifoliata fruit cracking. CONCLUSIONS: Our findings provided new insights into potential genes influencing the fruit cracking trait in A. trifoliata and established a basis for further research on the breeding of cracking-resistant varieties to increase seed yields for biorefineries.

Comparative transcriptomics reveals the selection patterns of domesticated ramie.

Although domestication has dramatically altered the phenotype, physiology, and life history of ramie (Boehmeria nivea) plants, few studies have investigated the effects of domestication on the structure and expression pattern of genes in this fiber crop. To investigate the selective pattern and genetic relationships among a cultivated variety of ramie (BNZ: B. nivea, ZZ1) and four wild species, BNT (B. nivea var. tenacissima), BNN (B. nivea var. nipononivea), BNW (B. nivea var. nivea), and BAN (B. nivea var. viridula), in the section Tilocnide, we performed an RNA sequencing analysis of these ramie species. The de novo assembly of the "all-ramie" transcriptome yielded 119,114 unigenes with an average length of 633 bp, and a total of 7,084 orthologous gene pairs were identified. The phylogenetic tree showed that the cultivar BNZ clustered with BAN in one group, BNW was closely related to BNT, and BNN formed a separate group. Introgression analysis indicated that gene flow occurred from BNZ to BNN and BAN, and between BAN and BNN. Among these orthologs, 2,425 and 269 genes underwent significant purifying and positive selection, respectively. For these positively selected genes, oxidation-reduction process (GO:0055114) and stress response pathways (GO:0006950) were enriched, indicating that modulation of the cellular redox status was important during both ramie fiber evolution and improvement. Two genes related to the suppression of flowering and one gene annotated as a flowering-promoting factor were subjected to positive selection, probably caused by human manipulation. Additionally, five genes were homologs of those involved in abiotic stress tolerance and disease resistance, with higher expression levels in the cultivar BNZ than in the wild species. Collectively, the results of this study indicated that domestication has resulted in the upregulation of many genes involved in the abiotic and biotic stress responses, fiber yield, and plant growth of ramie.

Transcriptional Profiling and Identification of Heat-Responsive Genes in Perennial Ryegrass by RNA-Sequencing.

Perennial ryegrass (Lolium perenne) is one of the most widely used forage and turf grasses in the world due to its desirable agronomic qualities. However, as a cool-season perennial grass species, high temperature is a major factor limiting its performance in warmer and transition regions. In this study, a de novo transcriptome was generated using a cDNA library constructed from perennial ryegrass leaves subjected to short-term heat stress treatment. Then the expression profiling and identification of perennial ryegrass heat response genes by digital gene expression analyses was performed. The goal of this work was to produce expression profiles of high temperature stress responsive genes in perennial ryegrass leaves and further identify the potentially important candidate genes with altered levels of transcript, such as those genes involved in transcriptional regulation, antioxidant responses, plant hormones and signal transduction, and cellular metabolism. The de novo assembly of perennial ryegrass transcriptome in this study obtained more total and annotated unigenes compared to previously published ones. Many DEGs identified were genes that are known to respond to heat stress in plants, including HSFs, HSPs, and antioxidant related genes. In the meanwhile, we also identified four gene candidates mainly involved in C4 carbon fixation, and one TOR gene. Their exact roles in plant heat stress response need to dissect further. This study would be important by providing the gene resources for improving heat stress tolerance in both perennial ryegrass and other cool-season perennial grass plants.