Unilateral cross-incompatibility between Camellia oleifera and C. yuhsienensis provides new insights for hybridization in Camellia spp.

Camellia yuhsienensis was used to cross with Camellia oleifera to improve the resistance of oil camellia anthracnose. However, unilateral cross-incompatibility (UCI) between C. oleifera and C. yuhsienensis was found during the breeding process. Five C.oleifera cultivars and four C. uhsienensis materials were tested to confirm the UCI between C. oleifera and C. yuhsienensis. 'Huashuo' (HS) and 'Youza 2' (YZ2) were used to represent these two species to characterize the UCI, including pollen tube growth, fertilization and fruit development. The results demonstrated that UCI was prevalent between C. oleifera and C. yuhsienensis. The asynchronous flowering period was a pre-pollination barrier that limited mating between these two species under natural conditions. Interspecific pollen tubes were observed through the styles of these two plants, though the growth rates differed considerably. At 96 hours after pollination, the pollen tube of YZ2 barely entered the ovule, but remained at the base of the style and became swollen. However, the HS pollen tube entered the ovule 48 hours after pollination, double fertilization was observed, and the fruit and seeds developed commonly. Relative to compatible combinations, most unfertilized ovules in incompatible combinations failed to grow, turned brown 150 days after pollination, and the fruits were smaller than expected with uneven enlargement. Investigations on both semi-in vivo and in vitro pollen tubes gave us new idea for thought: the HS style has a stronger inhibitory effect on the interspecific pollen tubes, while calcium alleviates the inhibitory of styles but failed to prevent the appearance of abnormal pollen tube morphology. This study provides useful information on interspecific hybridization between C. oleifera and C. yuhsienensis for understanding reproductive isolation mechanisms and breeding programs in genus Camellia.

Transcriptome and Anatomical Comparisons Reveal the Effects of Methyl Jasmonate on the Seed Development of Camellia oleifera.

Seed is a major storage organ that determines the yield and quality of Camellia oleifera (C. oleifera). Methyl jasmonate (MeJA) is a signaling molecule involved in plant growth and development. However, the role of MeJA in the development of C. oleifera seeds remains a mystery. This study demonstrated that the larger seeds induced by MeJA resulted from more cell numbers and a larger cell area in the outer seed coat and embryo at the cellular level. At the molecular level, MeJA could regulate the expression of factors in the known signaling pathways of seed size control as well as cell proliferation and expansion, resulting in larger seeds. Furthermore, the accumulation of oil and unsaturated fatty acids due to MeJA-inducement was attributed to the increased expression of fatty acid biosynthesis-related genes but reduced expression of fatty acid degradation-related genes. CoMYC2, a key regulator in jasmonate signaling, was considered a potential hub regulator which directly interacted with three hub genes (CoCDKB2-3, CoCYCB2-3, and CoXTH9) related to the seed size and two hub genes (CoACC1 and CoFAD2-3) related to oil accumulation and fatty acid biosynthesis by binding to their promoters. These findings provide an excellent target for the improvement of the yield and quality in C. oleifera.

Integration of semi-in vivo assays and multi-omics data reveals the effect of galloylated catechins on self-pollen tube inhibition in Camellia oleifera.

Camellia oil extracted from the seeds of Camellia oleifera Abel. is a popular and high-quality edible oil, but its yield is limited by seed setting, which is mainly caused by self-incompatibility (SI). One of the obvious biological features of SI plants is the inhibition of self-pollen tubes; however, the underlying mechanism of this inhibition in C. oleifera is poorly understood. In this study, we constructed a semi-in vivo pollen tube growth test (SIV-PGT) system that can screen for substances that inhibit self-pollen tubes without interference from the genetic background. Combined with multi-omics analysis, the results revealed the important role of galloylated catechins in self-pollen tube inhibition, and a possible molecular regulatory network mediated by UDP-glycosyltransferase (UGT) and serine carboxypeptidase-like (SCPL) was proposed. In summary, galloylation of catechins and high levels of galloylated catechins are specifically involved in pollen tube inhibition under self-pollination rather than cross-pollination, which provides a new understanding of SI in C. oleifera. These results will contribute to sexual reproduction research on C. oleifera and provide theoretical support for improving Camellia oil yield in production.

Hormone analysis and candidate genes identification associated with seed size in Camellia oleifera.

Camellia oleifera is an important woody oil species in China. Its seed oil has been widely used as a cooking oil. Seed size is a crucial factor influencing the yield of seed oil. In this study, the horizontal diameter, vertical diameter and volume of C. oleifera seeds showed a rapid growth tendency from 235 days after pollination (DAP) to 258 DAP but had a slight increase at seed maturity. During seed development, the expression of genes related to cell proliferation and expansion differ greatly. Auxin plays an important role in C. oleifera seeds; YUC4 and IAA17 were significantly downregulated. Weighted gene co-expression network analysis screened 21 hub transcription factors for C. oleifera seed horizontal diameter, vertical diameter and volume. Among them, SPL4 was significantly decreased and associated with all these three traits, while ABI4 and YAB1 were significantly increased and associated with horizontal diameter of C. oleifera seeds. Additionally, KLU significantly decreased (2040-fold). Collectively, our data advances the knowledge of factors related to seed size and provides a theoretical basis for improving the yield of C. oleifera seeds.

Chromosome-level genome of Camellia lanceoleosa provides a valuable resource for understanding genome evolution and self-incompatibility.

The section Oleifera (Theaceae) has attracted attention for the high levels of unsaturated fatty acids found in its seeds. Here, we report the chromosome-scale genome of the sect. Oleifera using diploid wild Camellia lanceoleosa with a final size of 3.00 Gb and an N50 scaffold size of 186.43 Mb. Repetitive sequences accounted for 80.63% and were distributed unevenly across the genome. Camellia lanceoleosa underwent a whole-genome duplication event approximately 65 million years ago (65 Mya), prior to the divergence of C. lanceoleosa and Camellia sinensis (approx. 6-7 Mya). Syntenic comparisons of these two species elucidated the genomic rearrangement, appearing to be driven in part by the activity of transposable elements. The expanded and positively selected genes in C. lanceoleosa were significantly enriched in oil biosynthesis, and the expansion of homomeric acetyl-coenzyme A carboxylase (ACCase) genes and the seed-biased expression of genes encoding heteromeric ACCase, diacylglycerol acyltransferase, glyceraldehyde-3-phosphate dehydrogenase and stearoyl-ACP desaturase could be of primary importance for the high oil and oleic acid content found in C. lanceoleosa. Theanine and catechins were present in the leaves of C. lanceoleosa. However, caffeine can not be dectected in the leaves but was abundant in the seeds and roots. The functional and transcriptional divergence of genes encoding SAM-dependent N-methyltransferases may be associated with caffeine accumulation and distribution. Gene expression profiles, structural composition and chromosomal location suggest that the late-acting self-incompatibility of C. lanceoleosa is likely to have favoured a novel mechanism co-occurring with gametophytic self-incompatibility. This study provides valuable resources for quantitative and qualitative improvements and genome assembly of polyploid plants in sect. Oleifera.

Favorable pleiotropic loci for fiber yield and quality in upland cotton (Gossypium hirsutum).

Upland cotton (Gossypium hirsutum L.) is an important economic crop for renewable textile fibers. However, the simultaneous improvement of yield and fiber quality in cotton is difficult as the linkage drag. Compared with breaking the linkage drag, identification of the favorable pleiotropic loci on the genome level by genome-wide association study (GWAS) provides a new way to improve the yield and fiber quality simultaneously. In our study restriction-site-associated DNA sequencing (RAD-seq) was used to genotype 316 cotton accessions. Eight major traits in three categories including yield, fiber quality and maturation were investigated in nine environments (3 sites × 3 years). 231 SNPs associated with these eight traits (- log10(P) > 5.27) were identified, located in 27 genomic regions respectively by linkage disequilibrium analysis. Further analysis showed that four genomic regions (the region 1, 6, 8 and 23) held favorable pleiotropic loci and 6 candidate genes were identified. Through genotyping, 14 elite accessions carrying the favorable loci on four pleiotropic regions were identified. These favorable pleiotropic loci and elite genotypes identified in this study will be utilized to improve the yield and fiber quality simultaneously in future cotton breeding.

The genomic basis of geographic differentiation and fiber improvement in cultivated cotton.

Large-scale genomic surveys of crop germplasm are important for understanding the genetic architecture of favorable traits. The genomic basis of geographic differentiation and fiber improvement in cultivated cotton is poorly understood. Here, we analyzed 3,248 tetraploid cotton genomes and confirmed that the extensive chromosome inversions on chromosomes A06 and A08 underlies the geographic differentiation in cultivated Gossypium hirsutum. We further revealed that the haplotypic diversity originated from landraces, which might be essential for understanding adaptative evolution in cultivated cotton. Introgression and association analyses identified new fiber quality-related loci and demonstrated that the introgressed alleles from two diploid cottons had a large effect on fiber quality improvement. These loci provided the potential power to overcome the bottleneck in fiber quality improvement. Our study uncovered several critical genomic signatures generated by historical breeding effects in cotton and a wealth of data that enrich genomic resources for the research community.

Identification, characterization and expression profiling of circular RNAs in the early cotton fiber developmental stages.

Circular RNA is one of the endogenous non-coding RNAs with a covalently closed loop structure and largely involved in regulating gene expression. However, the abundance of circular RNAs and their regulatory functions during the early stages of fiber development are still not known. In this work, we conducted high-throughput sequencing of the Ligonlintless-1 and its wild-type at 0 DPA, 8 DPA and stem. A total of 2811 circular RNAs were identified and unevenly distributed across cotton chromosomes. We found 34, 142 and 27 circular RNAs were differentially expressed between Ligonlintless-1 and wild-type at 0 DPA, 8 DPA and stem, respectively. Both circular RNA-microRNA-mRNA network and MeJA treatment results in Ligonlintless-1 and wild-type might provide a strong indication of four circular RNAs and ghr_miR169b being important biological molecular associating with fiber development. The results provide new insight into the putative molecular function of circular RNAs in the regulation of fiber development.

Large-fragment insertion activates gene GaFZ (Ga08G0121) and is associated with the fuzz and trichome reduction in cotton (Gossypium arboreum).

Cotton seeds are typically covered by lint and fuzz fibres. Natural 'fuzzless' mutants are an ideal model system for identifying genes that regulate cell initiation and elongation. Here, using a genome-wide association study (GWAS), we identified a ~ 6.2 kb insertion, larINDELFZ , located at the end of chromosome 8, composed of a ~ 5.0 kb repetitive sequence and a ~ 1.2 kb fragment translocated from chromosome 12 in fuzzless Gossypium arboreum. The presence of larINDELFZ was associated with a fuzzless seed and reduced trichome phenotypes in G. arboreum. This distant insertion was predicted to be an enhancer, located ~ 18 kb upstream of the dominant-repressor GaFZ (Ga08G0121). Ectopic overexpression of GaFZ in Arabidopsis thaliana and G. hirsutum suggested that GaFZ negatively modulates fuzz and trichome development. Co-expression and interaction analyses demonstrated that GaFZ might impact fuzz fibre/trichome development by repressing the expression of genes in the very-long-chain fatty acid elongation pathway. Thus, we identified a novel regulator of fibre/trichome development while providing insights into the importance of noncoding sequences in cotton.

A copy number variant at the HPDA-D12 locus confers compact plant architecture in cotton.

Improving yield is a primary mission for cotton (Gossypium hirsutum) breeders; development of cultivars with suitable architecture for high planting density (HPDA) can increase yield per unit area. We characterized a natural cotton mutant, AiSheng98 (AS98), which exhibits shorter height, shorter branch length, and more acute branch angle than wild-type. A copy number variant at the HPDA locus on Chromosome D12 (HPDA-D12), encoding a dehydration-responsive element-binding (DREB) transcription factor, GhDREB1B, strongly affects plant architecture in the AS98 mutant. We found an association between a tandem duplication of a c. 13.5 kb segment in HPDA-D12 and elevated GhDREB1B expression resulting in the AS98 mutant phenotype. GhDREB1B overexpression confers a significant decrease in plant height and branch length, and reduced branch angle. Our results suggest that fine-tuning GhDREB1B expression may be a viable engineering strategy for modification of plant architecture favorable to high planting density in cotton.

Full-Length Transcriptome from Camellia oleifera Seed Provides Insight into the Transcript Variants Involved in Oil Biosynthesis.

Camellia oleifera Abel., belonging to the genus Camellia of Theaceae, has been widely used as a cooking oil, lubricant, and in cosmetics. Because of complicated polyploidization and large genomes, reference genome information is still lacking. Systematic characterization of gene models based on transcriptome data is a fast and economical approach for C. oleifera. Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) and Illumina RNA-Seq combined with gas chromatography were performed for exploration of oil biosynthesis, accumulation, and comprehensive transcriptome analysis in C. oleifera seeds at five different developmental stages. We report the first full-length transcriptome data set of C. oleifera seeds comprising 40,143 deredundant high-quality isoforms. Among these isoforms, 37,982 were functionally annotated, and 271 (2.43%) belonged to fatty acid metabolism. A total of 8,344 full-length unique transcript models were obtained, and 8,151 (97.69%) of them produced more than two isoforms, suggesting a high degree of transcriptome complexity in C. oleifera seeds. A total of 783 alternative splicing (AS) events were identified, among which the retained intron was the most abundant. We also obtained 1,910 long noncoding RNAs (lncRNAs) and found that AS events occurred in these lncRNAs. Potential transcript variants of genes involved in oil biosynthesis were also investigated. After performing weighted correlation network analysis, we found seven "gene modules" and hub genes for each module showing a significant association with oil content. The series test of clusters classified these modules into four significant profiles based on gene expression patterns. Protein-protein interaction network analysis showed that upregulated WRI1 interacted with 17 genes encoding the enzymes playing key roles in oil synthesis. MYB and ZIP transcriptional factors also showed significant interactions with key genes involved in oil synthesis. Collectively, our data advance the knowledge of RNA isoform diversity in seeds at different developmental stages and provide a rich resource for functional studies on oil synthesis in C. oleifera.

Casparian strip membrane domain proteins in Gossypium arboreum: genome-wide identification and negative regulation of lateral root growth.

BACKGROUND: Root systems are critical for plant growth and development. The Casparian strip in root systems is involved in stress resistance and maintaining homeostasis. Casparian strip membrane domain proteins (CASPs) are responsible for the formation of Casparian strips. RESULTS: To investigate the function of CASPs in cotton, we identified and characterized 48, 54, 91 and 94 CASPs from Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively, at the genome-wide level. However, only 29 common homologous CASP genes were detected in the four Gossypium species. A collinearity analysis revealed that whole genome duplication (WGD) was the primary reason for the expansion of the genes of the CASP family in the four cotton species. However, dispersed duplication could also contribute to the expansion of the GaCASPs gene family in the ancestors of G. arboreum. Phylogenetic analysis was used to cluster a total of 85 CASP genes from G. arboreum and Arabidopsis into six distinct groups, while the genetic structure and motifs of CASPs were conserved in the same group. Most GaCASPs were expressed in diverse tissues, with the exception of that five GaCASPs (Ga08G0113, Ga08G0114, Ga08G0116, Ga08G0117 and Ga08G0118) that were highly expressed in root tissues. Analyses of the tissue and subcellular localization suggested that GaCASP27 genes (Ga08G0117) are membrane protein genes located in the root. In the GaCASP27 silenced plants and the Arabidopsis mutants, the lateral root number significantly increased. Furthermore, GaMYB36, which is related to root development was found to regulate lateral root growth by targeting GaCASP27. CONCLUSIONS: This study provides a fundamental understanding of the CASP gene family in cotton and demonstrates the regulatory role of GaCASP27 on lateral root growth and development.

Genotyping by Sequencing Revealed QTL Hotspots for Trichome-Based Plant Defense in Gossypium hirsutum.

Cotton possesses certain physical features, including leaf and stem trichomes that help plants deter damage caused by insect pests, and to some extent, from abiotic factors as well. Among those features, trichomes (pubescence) hold a special place as a first line of defense and a managemental tool against sucking insect pests of cotton. Different insect pests of cotton (whiteflies, aphids, jassids, and boll weevil) severely damage the yield and quality of the crop. Likewise, whiteflies, aphids, jassids, and other insect pests are considered as potential carriers for cotton leaf curl viruses and other diseases. Genotyping by sequencing (GBS) study was conducted to understand and explore the genomic regions governing hairy (Pubescence) leaves and stem phenotypes. A total of 224 individuals developed from an intraspecific cross (densely haired cotton (Liaoyang duomao mian) × hairless cotton (Zong 128)) and characterized phenotypically for leaf and stem pubescence in different environments. Here we identify and report significant QTLs (quantitative trait loci) associated with leaf and stem pubescence, and the response of plant under pest (aphid) infestation. Further, we identified putative genes colocalized on chromosome A06 governing mechanism for trichome development and host-pest interaction. Our study provides a comprehensive insight into genetic architecture that can be employed to improve molecular marker-assisted breeding programs aimed at developing biotic (insect pests) resilient cotton cultivars.

A Genome-Wide Association Study Revealed Key SNPs/Genes Associated With Salinity Stress Tolerance In Upland Cotton.

Millions of hectares of land are too saline to produce economically valuable crop yields. Salt tolerance in cotton is an imperative approach for improvement in response to ever-increasing soil salinization. Little is known about the genetic basis of salt tolerance in cotton at the seedling stage. To address this issue, a genome-wide association study (GWAS) was conducted on a core collection of a genetically diverse population of upland cotton (Gossypium hirsutum L.) comprising of 419 accessions, representing various geographic origins, including China, USA, Pakistan, the former Soviet Union, Chad, Australia, Brazil, Mexico, Sudan, and Uganda. Phenotypic evaluation of 7 traits under control (0 mM) and treatment (150 mM) NaCl conditions depicted the presence of broad natural variation in the studied population. The association study was carried out with the efficient mixed-model association eXpedited software package. A total of 17,264 single-nucleotide polymorphisms (SNPs) associated with different salinity stress tolerance related traits were found. Twenty-three candidate SNPs related to salinity stress-related traits were selected. Final key SNPs were selected based on the r2 value with nearby SNPs in a linkage disequilibrium (LD) block. Twenty putative candidate genes surrounding SNPs, A10_95330133 and D10_61258588, associated with leaf relative water content, RWC_150, and leaf fresh weight, FW_150, were identified, respectively. We further validated the expression patterns of twelve candidate genes with qRT-PCR, which revealed different expression levels in salt-tolerant and salt-sensitive genotypes. The results of our GWAS provide useful knowledge about the genetic control of salt tolerance at the seedling stage, which could assist in elucidating the genetic and molecular mechanisms of salinity stress tolerance in cotton plants.

Genome-wide analysis of cotton C2H2-zinc finger transcription factor family and their expression analysis during fiber development.

BACKGROUND: C2H2-zinc finger protein family is commonly found in the plant, and it is known as the key actors in the regulation of transcription and vital component of chromatin structure. A large number of the C2H2-zinc finger gene members have not been well characterized based on their functions and structure in cotton. However, in other plants, only a few C2H2-zinc finger genes have been studied. RESULTS: In this work, we performed a comprehensive analysis and identified 386, 196 and 195 C2H2-zinc finger genes in Gossypium hirsutum (upland cotton), Gossypium arboreum and Gossypium raimondii, respectively. Phylogenetic tree analysis of the C2H2-zinc finger proteins encoding the C2H2-zinc finger genes were classified into seven (7) subgroups. Moreover, the C2H2-zinc finger gene members were distributed in all cotton chromosomes though with asymmetrical distribution patterns. All the orthologous genes were detected between tetraploid and the diploid cotton, with 154 orthologous genes pair detected between upland cotton and Gossypium arboreum while 165 orthologous genes were found between upland cotton and Gossypium raimondii. Synonymous (Ks) and non-synonymous (Ka) nucleotide substitution rates (Ka/Ks) analysis indicated that the cotton C2H2-zinc finger genes were highly influenced mainly by negative selection, which maintained their protein levels after the duplication events. RNA-seq data and RT-qPCR validation of the RNA seq result revealed differential expression pattern of some the C2H2-zinc finger genes at different stages of cotton fiber development, an indication that the C2H2-zinc finger genes play an important role in initiating and regulating fiber development in cotton. CONCLUSIONS: This study provides a strong foundation for future practical genome research on C2H2-zinc finger genes in upland cotton. The expression levels of C2H2-zinc finger genes family is a pointer of their involvement in various biochemical and physiological functions which are directly related to cotton fiber development during initiation and elongation stages. This work not only provides a basis for determining the nominal role of the C2H2-zinc finger genes in fiber development but also provide valuable information for characterization of potential candidate genes involved in regulation of cotton fiber development.

Long non-coding RNAs and their potential functions in Ligon-lintless-1 mutant cotton during fiber development.

BACKGROUND: Long non-coding RNAs (LncRNAs) are part of genes, which are not translated into proteins and play a vital role in plant growth and development. Nevertheless, the presence of LncRNAs and how they functions in Ligon-lintless-1 mutant during the early cessation of cotton fiber development are still not well understood. In order to investigate the function of LncRNAs in cotton fiber development, it is necessary and important to identify LncRNAs and their potential roles in fiber cell development. RESULTS: In this work, we identified 18,333 LncRNAs, with the proportion of long intergenic noncoding RNAs (LincRNAs) (91.5%) and anti-sense LncRNAs (8.5%), all transcribed from Ligon-lintless-1 (Li1) and wild-type (WT). Expression differences were detected between Ligon-lintless-1 and wild-type at 0 and 8 DPA (day post anthesis). Pathway analysis and Gene Ontology based on differentially expressed LncRNAs on target genes, indicated fatty acid biosynthesis and fatty acid elongation being integral to lack of fiber in mutant cotton. The result of RNA-seq and RT-qPCR clearly singles out two potential LncRNAs, LNC_001237 and LNC_017085, to be highly down-regulated in the mutant cotton. The two LncRNAs were found to be destabilized or repressed by ghr-miR2950. Both RNA-seq analysis and RT-qPCR results in Ligon-lintless-1 mutant and wild-type may provide strong evidence of LNC_001237, LNC_017085 and ghr-miR2950 being integral molecular elements participating in various pathways of cotton fiber development. CONCLUSION: The results of this study provide fundamental evidence for the better understanding of LncRNAs regulatory role in the molecular pathways governing cotton fiber development. Further research on designing and transforming LncRNAs will help not only in the understanding of their functions but will also in the improvement of fiber quality.

Association Analysis of Salt Tolerance in Asiatic cotton (Gossypium arboretum) with SNP Markers.

Salinity is not only a major environmental factor which limits plant growth and productivity, but it has also become a worldwide problem. However, little is known about the genetic basis underlying salt tolerance in cotton. This study was carried out to identify marker-trait association signals of seven salt-tolerance-related traits and one salt tolerance index using association analysis for 215 accessions of Asiatic cotton. According to a comprehensive index of salt tolerance (CIST), 215 accessions were mainly categorized into four groups, and 11 accessions with high salinity tolerance were selected for breeding. Genome-wide association studies (GWAS) revealed nine SNP rich regions significantly associated with relative fresh weight (RFW), relative stem length (RSL), relative water content (RRWC) and CIST. The nine SNP rich regions analysis revealed 143 polymorphisms that distributed 40 candidate genes and significantly associated with salt tolerance. Notably, two SNP rich regions on chromosome 7 were found to be significantly associated with two salinity related traits, RFW and RSL, by the threshold of -log10P ≥ 6.0, and two candidate genes (Cotton_A_37775 and Cotton_A_35901) related to two key SNPs (Ca7_33607751 and Ca7_77004962) were possibly associated with salt tolerance in G. arboreum. These can provide fundamental information which will be useful for future molecular breeding of cotton, in order to release novel salt tolerant cultivars.

Biomimetic design and fabrication of scaffolds integrating oriented micro-pores with branched channel networks for myocardial tissue engineering.

The ability to fabricate three-dimensional (3D) thick vascularized myocardial tissue could enable scientific and technological advances in tissue engineering and drug screening, and may accelerate its application in myocardium repair. In this study, we developed a novel biomimetic scaffold integrating oriented micro-pores with branched channel networks to mimic the anisotropy and vasculature of native myocardium. The oriented micro-pores were fabricated using an 'Oriented Thermally Induced Phase Separation (OTIPS)' technique, and the channel network was produced by embedding and subsequently dissolving a 3D-printed carbohydrate template after crosslinking. Micro-holes were incorporated on the wall of channels, which greatly enhanced the permeability of channels. The effect of the sacrificial template on the formation of oriented micro- pores was assessed. The mechanical properties of the scaffold were tuned by varying the temperature gradient and chitosan/collagen ratio to match the specific stiffness of native heart tissue. The engineered cardiac tissue achieved synchronized beating with electrical stimulation. Calcium transient results suggested the formation of connection between cardiomyocytes within scaffold. All the results demonstrated that the reported scaffold has the potential to induce formation of a perfusable vascular network and to create thick vascularized cardiac tissue that may advance further clinical applications.

Comparative transcriptome analysis of TUCPs in Gossypium hirsutum Ligon-lintless-1 mutant and their proposed functions in cotton fiber development.

Transcripts of uncertain coding potential (TUCP) are part of the LncRNAs, which encode some polypeptides. However, the abundance of TUCP transcripts and their roles in Ligon-linless-1 (Li-1) cotton mutant during the early termination of fiber development are still not documented. Li-1 mutant is one of the excellent modules for investigating fiber elongation processes due to its unique fiber developmental stages. To examine the function of TUCP in cotton fiber development, it is important to identify TUCPs and their involvement in fiber development. In this study, we found that 11104 TUCP transcripts were removed by coding potential criteria of Pfam domain scan. Additionally, differential expression levels of TUCP transcripts were detected between Li-1 mutant and the wild-type (WT), which imply their possible functions in cotton fiber development. These results further revealed that a great number of differentially expressed TUCP transcripts in cotton were identified at 8 DPA, followed by 0 DPA and stem. However, these might explain an undesirable function in cotton fiber development. The gene ontology and pathway analysis, based on differential expression patterns of TUCP transcripts on targeted genes, identified the transport process, cytoskeleton structure, membrane permeability and fatty acids. These give new insight into significant involvement in early cessation of cotton fiber development and abnormal stem. The RNA-seq and qRT-PCR expression analyses of TUCP transcripts evidently singled out three possible genes, TUCP_010675, TUCP_001475, TUCP_009444 and other targeted mRNAs. The expression pattern of TUCP transcripts and their mRNA targets provided valuable evidence for further investigations on the biological functions of TUCP in cotton fiber development. The study findings may serve as a useful tool for comparative analysis of TUCP transcripts in cotton species and assist in selection of the applicable candidate genes for further functional analyses, genetic improvement and genetic engineering of cotton fiber development.