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Naturally coloured cotton (NCC) fibres need little or no dyeing process in textile industry to low-carbon emission and are environment-friendly. Proanthocyanidins (PAs) and their derivatives were considered as the main components causing fibre coloration and made NCCs very popular and healthy, but the monotonous fibre colours greatly limit the wide application of NCCs. Here a G. hirsutum empurpled mutant (HS2) caused by T-DNA insertion is found to enhance the anthocyanidins biosynthesis and accumulate anthocyanidins in the whole plant. HPLC and LC/MS-ESI analysis confirmed the anthocyanidins methylation and peonidin, petunidin and malvidin formation are blocked. The deficiency of GhOMT1 in HS2 was associated with the activation of the anthocyanidin biosynthesis and the altered components of anthocyanidins. The transcripts of key genes in anthocyanidin biosynthesis pathway are significantly up-regulated in HS2, while transcripts of the genes for transport and decoration were at similar levels as in WT. To investigate the potential mechanism of GhOMT1 deficiency in cotton fibre coloration, HS2 mutant was crossed with NCCs. Surprisingly, offsprings of HS2 and NCCs enhanced PAs biosynthesis and increased PAs levels in their fibres from the accumulated anthocyanidins through up-regulated GhANR and GhLAR. As expected, multiple novel lines with improved fibre colours including orange red and navy blue were produced in their generations. Based on this work, a new strategy for breeding diversified NCCs was brought out by promoting PA biosynthesis. This work will help shed light on mechanisms of PA biosynthesis and bring out potential molecular breeding strategy to increase PA levels in NCCs.

Leucoanthocyanidin reductase (LAR) is the critical enzyme in the synthesis pathway of proanthocyanidins, which are the primary pigments in brown cotton fibers. Our previous study has revealed significant differences in the expression levels of GhLAR1 between white and brown cotton fibers at 10 DPA. In this work, the expression pattern of the GhLAR1 gene was further studied, and the promoter of GhLAR1 (1780 bp) was isolated and characterized. Bioinformatic analysis indicated that GhLAR1 promoter contained many known light response elements and several defenses related to transcriptional factor-binding boxes, which may partially explain the response of the GhLAR1 to temperature, NaCl, and PEG treatments. Furthermore, GhLAR1 was preferentially and strongly expressed in fibers and flowers of cotton, and the expression levels in all tested tissues (especially fibers) of brown cotton were significantly higher than those in white cotton. Consistent with the expression analysis, the GhLAR1 promoter mainly drove GUS expression in epidermal trichomes and floral organs.

Cotton male fertility-associated gene GhGLP4, encoding a germin-like protein, is essential for anthers development by keeping ROS homeostasis through reducing H2O2 level. Utilization of heterosis is an important way to increase cotton yield and improve fiber quality in hybrid cotton development programs. Male sterility is used in the development of cotton hybrids to reduce the cost of hybrid seed production by eliminating the process of emasculation. From the transcriptome analysis of genic male sterile mutant (ms1) and its background C312 of G. hirsutum, a gene encoding germin-like protein (GhGLP4) was found significantly down-regulated in different developmental stages of ms1 anthers. To explore the gene function in cotton fertility, GhGLP4 was further studied and interfered by virus-induced gene silencing. In the GhGLP4 interfered cotton lines, the expression level of GhGLP4 was significantly decreased in the stamens, and the down-regulation of GhGLP4 resulted in pollen sac closure, stigma exertion, filament shortening, decrease in the number of anthers and complete male sterility. The expression levels of respiratory burst oxidase homologs (Rboh, NADPH oxidase) were significantly altered. Further investigation showed that the SOD activity decreased while the H2O2 content increased in the atypical stamens. These results indicated that GhGLP4 gene affected the cotton anther development through maintenance of ROS homeostasis by H2O2 reduction.

Cotton Verticillium wilt, caused by the notorious fungal phytopathogen Verticillium dahliae (V.dahliae), is a destructive soil-borne vascular disease and severely decreases cotton yield and quality worldwide. Transcriptional and post-transcriptional regulation of genes responsive to V.dahliae are crucial for V.dahliae tolerance in plants. However, the specific microRNAs (miRNAs) and the miRNA/target gene crosstalk involved in cotton resistance to Verticillium wilt remain largely limited. To investigate the roles of regulatory RNAs under V.dahliae induction in upland cotton, mRNA and small RNA libraries were constructed from mocked and infected roots of two upland cotton cultivars with the V.dahliae-sensitive cultivar Jimian 11 (J11) and the V.dahliae-tolerant cultivar Zhongzhimian 2 (Z2). A comparative transcriptome analysis revealed 8330 transcripts were differentially expressed under V.dahliae stress and associated with several specific biological processes. Moreover, small RNA sequencing identified a total of 383 miRNAs, including 330 unique conserved miRNAs and 53 novel miRNAs. Analysis of the regulatory network involved in the response to V.dahliae stress revealed 31 differentially expressed miRNA–mRNA pairs, and the up-regulation of GhmiR395 and down-regulation of GhmiR165 were possibly involved in the response to V. dahliae by regulating sulfur assimilation through the GhmiR395-APS1/3 module and the establishment of the vascular pattern and secondary cell wall formation through GhmiR165-REV module, respectively. The integrative analysis of mRNA and miRNA expression profiles from upland cotton lays the foundation for further investigation of regulatory mechanisms of resistance to Verticillium wilt in cotton and other crops.

The wild cotton species Gossypium stocksii produces a brown fiber that provides a valuable resource for the color improvement of naturally colored cotton (NCC) fiber. However, the biochemical basis and molecular mechanism of its fiber pigmentation remain unclear. Herein, we analyzed the dynamics of proanthocyanidins (PAs) accumulation in developing the fiber of G. stocksii, which suggested a similar role of PAs and/or their derivatives in the fiber coloration of G. stocksii. In addition, comparative transcriptomics analyses revealed that the PA biosynthetic genes were expressed at higher levels and for a longer period in developing fibers of G. stocksii than G. arboreum (white fiber), and the transcription factors, such as TT8, possibly played crucial regulatory roles in regulating the PA branch genes. Moreover, we found that the anthocyanidin reductase (ANR) was expressed at a higher level than the leucoanthocyanidin reductases (LARs) and significantly upregulated during fiber elongation, suggesting a major role of ANR in PA synthesis in G. stocksii fiber. In summary, this work revealed the accumulation of PAs and the expression enhancement of PA biosynthetic genes in developing fibers of G. stocksii. We believe this work will help our understanding of the molecular mechanisms of cotton fiber coloration and further promote the future breeding of novel NCCs.

Cotton (Gossypium spp.) is one of the most important fiber crops globally, with the main cultivated species Gossypium hirsutum L. and G. barbadence L. contributing more than 90% of the total fiber production (Wang et al., 2019). However, low genetic diversity in germplasm has largely impeded the continuous improvement of cultivated cotton. Wild cotton species have desirable characters such as resistance to various biotic and abiotic stresses and high-quality fiber products, which are therefore natural reservoirs for cotton genetic improvement (Wendel and Cronn, 2003).

The cultivated diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function compared to the allotetraploid cotton species through genetic transformation, the reliable and efficient method for high frequency somatic embryogenesis and plant regeneration in G. arboreum is urgent need to be established. Callus was induced from hypocotyl, root and cotyledon of G. arboreum seedlings on MSB (MS salts and B5 vitamins) medium with 0.09 µM 2,4-D and 2.32 µM KT. The embryogenic callus was induced on MS5 medium from the suspended cultures of several cycles of alternate liquid-solid culture, which was critical step for somatic embryogenesis. The liquid medium of MS4 was supplemented with 0.1g/L NaCl, 0.1g/L KCl and 0.1g/L CuSO4. The solid medium of MS5 for embryogenic callus effective induction was supplemented with 37.59 mM KNO3 + 62.47 µM NH4NO3 and 2.46 µM IBA + 0.93 µM KT or 0.045 µM 2,4-D + 2.46 µM IBA + 0.465 µM KT. During callus growing on different media, callus was effectively selected for subculture or treatment according to cell morphology to induce embryogenic callus and somatic embryos. Somatic embryo maturation and germination were better on MS5 medium with maltose or glucose + maltose than the single glucose. The regenerated plantlets with well-developed roots were directly transferred to soil or grafted onto the germinated cotton plantlets. The feasible process of plant regeneration via somatic embryogenesis in diploid cultivated species was established and needed to be improved and optimized for the gene functional analysis and gene editing in the diploid cotton species.

As water-soluble, natural pigments, anthocyanins are responsible for the red, purple and blue colors of many flowers, which attract pollinators to spread pollen. The colors of flowers are also essential for plants to survive in the nature and become one of the most significant characteristics of ornamental plants. In the booming floriculture industry, to produce various flower colors could increase the richness of natural colors, but it is still difficult to breed flowers with coveted blue color. The diversity of flower color is mainly determined by the types and contents of anthocyanins and their derivatives. The synthesis of delphinidin pigments is the key factor for breeding blue flowers. However, there are no structural genes in many plants to biosynthesize delphinidin pigments. Blue flowers are successfully created by genetic engineering in recent years. In this paper, using common ornamental plants as examples, we review the mechanism of plant flower coloration from the aspects of the key factors affecting the synthesis of delphinidin pigment and the production strategies of blue flowers based on the regulation of anthocyanin metabolism. Different strategies of molecular breeding could provide opportunities to improve colors of other floriculture plants and to develop anthocyanin-rich economic crops, such as colored cotton with blue fibers.花色是植物吸引昆虫传播花粉的主要因素，对于植物在自然界的生存必不可少，也是观赏植物最重要的性状之一。在蓬勃发展的花卉产业中，色彩各异花卉的培育，可以弥补自然花色的匮乏，但是令人垂涎的蓝色花比较难培育。花色的多样性主要是由花青素及其衍生物的种类和含量等因素决定的，飞燕草色素的合成是形成蓝色花的关键因素，许多植物体内缺少合成飞燕草色素的结构基因。近年来，利用基因工程技术培育蓝色花的研究也时有报道。文中以常见的观赏植物为例，基于花青素代谢调控，从影响飞燕草色素合成的关键因素和不同分子改良途径培育蓝色花等几个方面对植物花朵呈色的机制进行了综述，并展示不同分子育种策略可能在其他领域的应用，为其他植物或经济作物的色泽改良如彩色棉蓝色纤维的培育等提供参考和技术支持。.

Background The formation of natural colored fibers mainly results from the accumulation of different anthocyanidins and their derivatives in the fibers of Gossypium hirsutum L. Chalcone synthase (CHS) is the first committed enzyme of flavonoid biosynthesis, and anthocyanidins are transported into fiber cells after biosynthesis mainly by Anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR) to present diverse colors with distinct stability. The biochemical and molecular mechanism of pigment formation in natural colored cotton fiber is not clear. Results The three key genes of GhCHS, GhANR and GhLAR were predominantly expressed in the developing fibers of colored cotton. In the GhCHSi, GhANRi and GhLARi transgenic cottons, the expression levels of GhCHS, GhANR and GhLAR significantly decreased in the developing cotton fiber, negatively correlated with the content of anthocyanidins and the color depth of cotton fiber. In colored cotton Zongxu1 (ZX1) and the GhCHSi, GhANRi and GhLARi transgenic lines of ZX1, HZ and ZH, the anthocyanidin contents of the leaves, cotton kernels, the mixture of fiber and seedcoat were all changed and positively correlated with the fiber color. Conclusion The three genes of GhCHS, GhANR and GhLAR were predominantly expressed early in developing colored cotton fibers and identified to be a key genes of cotton fiber color formation. The expression levels of the three genes affected the anthocyanidin contents and fiber color depth. So the three genes played a crucial part in cotton fiber color formation and has important significant to improve natural colored cotton quality and create new colored cotton germplasm resources by genetic engineering.

Additional file 1: Table S1. Statistics of RNA-seq and short reads mapping. Table S2. Significantly varied GO terms between 158A and SGDH284 for line specific differentially expressed genes. Table S3. Statistics of differentially expressed transcription factors in 158A and SGDH284. Table S4. Enrichment analysis of cold responsive transcription factors in 158A and SGDH284. Table S5. Primer pairs used to detect the expression of selected transcription factors.

Additional file 1: Table S1. Primers used in the experiments. Figure S1. Relative expression levels of GhCHS, GhANR, GhANR in the developing fiber of 5 DPA, 10 DPA and 15 DPA in GhCHSi, GhANRi, GhANRi, GhPDSi transgenic cotton lines and wild cotton ZX1.

Nitrate Transporter 1.1 (NRT1.1) is a nitrate transporter and sensor that modulates plant metabolism and growth. It has previously been shown that NRT1.1 is involved in the regulation of flowering time in Arabidopsis thaliana. In this study, we mainly used genetic and molecular methods to reveal the key flowering pathway that NRT1.1 may be involved in. Mutant alleles of CO and FLC, two crucial components in the flowering pathway, were introduced into the NRT1.1 defective mutant background by crossing. When the CO mutation was introduced into chl1-5 plants, the double mutant had delayed flowering time, and the CO transcription levels did not change in the chl1-5 plants. These results indicate that the CO-dependent photoperiod may be not associated with the delayed flowering shown by chl1-5. However, FLC loss of function could rescue the late flowering phenotype of the chl1-5 mutant, and FLC expression levels significantly increased in the NRT1.1 defective mutant plants. The FT expression levels in the chl1-5flc-3 double mutant plants recovered when the FLC mutation was introduced into chl1-5 plants and the up-regulation of FLC transcripts in the chl1-5 mutant plants was not related to nitrate availability. Our findings suggest that NRT1.1 affects flowering time via interaction with the FLC-dependent flowering pathway to influence its target gene FT, and that NRT1.1 may be included in an additional signaling pathway that represses the expression of FLC in a nitrate-independent manner.

Cotton (Gossypium spp.) is an important economic crop and there is obvious heterosis in cotton, fertility has played an important role in this heterosis. However, the genes that exhibit critical roles in anther development and fertility are not well understood. Here, we report an acyl-CoA N-acyltransferase (EC2.3; GhACNAT) that plays a key role in anther development and fertility. Suppression of GhACNAT by virus-induced gene silencing in transgenic cotton (G. hirsutum L. cv. C312) resulted in indehiscent anthers that were full of pollen, diminished filaments and stamens and plant sterility. We found GhACNAT was involved in lipid metabolism and jasmonic acid (JA) biosynthesis. The genes differentially expressed in GhACNAT-silenced plants and C312 were mainly involved in catalytic activity and transcription regulator activity in lipid metabolism. In GhACNAT-silenced plants, the expression levels of genes involved in lipid metabolism and jasmonic acid biosynthesis were significantly changed, the amount of JA in leaves and reproductive organs was significantly decreased compared with the amounts in C312. Treatments with exogenous methyl jasmonate rescued anther dehiscence and pollen release in GhACNAT-silenced plants and caused self-fertility. The GhACNAT gene may play an important role in controlling cotton fertility by regulating the pathways of lipid synthesis and JA biogenesis.

The commercial utilization of heterosis in seed yield by means of hybrid varieties is of great importance for increasing oilseed rape production in China. This requires a functional system for the production of hybrid seed. The Brassica napus oilseed rape line 9012AB is a recessive epistatic genic male sterility (GMS) two-type line, in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms1 and ms2) interacting with one pair of a recessive epistatic inhibitor gene (rf). Homozygosity at the rf locus (rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms1ms1ms2ms2 plants. This study was conducted to identify molecular markers for one of the male fertility/sterility loci in the B. napus male sterility line 9012AB. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from male sterile and male fertile plants of the homozygous two-type line 9012AB were subjected to amplified fragment length polymorphic (AFLP) analysis. A total of 256 primer combinations were used and seven markers tightly linked to one recessive genic male sterile gene (ms) were identified. Among them, six fragments co-segregated with the target gene in the tested population, and the other one had a genetic distance of 4.3 cM. The markers identified in this study will greatly enhance the utilization of recessive GMS for the production of hybrid seed in B. napus oilseed rape in China.

117AB is a recessive genic male sterility (RGMS) line in which the sterility is controlled by a duplicate recessive gene named ms, located at two separate loci. In the RGMS line, the genotype of the sterile plant (117A) is msmsmsms, and that of the fertile plant (117B) is Msmsmsms. The present study was aimed to identify DNA markers linked to the ms locus by amplified fragment length polymorphism (AFLP). From the survey of 512 AFLP primer combinations, 6 AFLP fragments (y1, k1, k2, k3, k4, k5) were identified as being tightly linked to the Ms locus. The genetic distances between the markers and the Ms locus were all less than 8 cM, among which two fragments, designated as k2 and k3, co-segregated with the target gene in the tested population. Fragment k2 was successfully converted into a sequence characterized amplified region (SCAR) marker. The markers detected could be valuable in marker-assisted breeding of RGMS in Brassica napus.

Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel). In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L) and bentazon (4.2 µmol). A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon) tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops.

Growth habit and developmental phase transitions in plants are acutely responsive to the quantity and quality of incident light. Information about neighbouring plants is primarily perceived by changes in the light environment. Arabidopsis is able to distinguish these changes through the phytochrome (PHYA-E) red light receptors that perceive changes in the red to far red (R:FR) ratio, and the cryptochrome and phototropin blue light receptors. Phytochrome, in its biologically active far-red light-absorbing form (Pfr), mediates its effects by directly binding with the basic helix-loop-helix (bHLH) phytochrome-interacting factors (PIFs). High R:FR ratios initiate phytochrome (Pfr)-PIF binding and PIF protein degradation. Low R:FR ratios photo-convert Pfr into the inactive form Pr allowing the gene expression that leads to a range of adaptations, including the shade avoidance responses. PIF1 regulates the expression of a number of genes by directly binding to a G-box element in their promoter. T-DNA insertional mutagenesis of a number of these genes results in pleiotropic effects associated with an altered capacity to respond appropriately to environmental cues, including those signalling changes in soil water status and the ambient light environment. The data show evidence of a perturbation of light regulated response processes, including germination, the shade avoidance responses, stomatal conductance, flowering and the circadian clock perturbation in the PIF-regulated gene mutants. The pif1,3,4,5 quadruple and one of the PIF-regulated gene mutants fail to show shade avoidance responses in older plants grown under shade inducing conditions. This suggests that the encoded proteins mediate a parallel pathway in the induction of the SAS. The implications of our findings for an understanding of how plants appropriately respond to changes in their environment will be discussed. Figure 1

PREMISE OF THE STUDY Microsatellite markers were developed in Myrica rubra to investigate potential hybridization events within or between M. rubra and its closely related species. METHODS AND RESULTS Using an ISSR-suppression PCR method, 12 primer pairs were temporarily developed with GSG(GT)(6) as the primer for enriching microsatellite sequences and the genomic DNA of M. rubra cv. 'Heijing' as template. The average allele number per locus was 4.9 within 26 individuals including two species, M. rubra and M. nana. Three pairs of primers produced species-specific alleles, and the other nine showed polymorphisms among 26 accessions. CONCLUSIONS Results indicated that the ISSR-suppression PCR method is suitable for developing microsatellite markers, especially for this species with little understanding of genomic information. The developed microsatellite markers provide a useful tool for further studies of population structure within or between M. rubra and M. nana or other closely related species.

9012AB, a recessive genic male sterility (RGMS) line developed from spontaneous mutation in Brassica napus (Chen et al. in Acta Agron Sin 24:431-438, 1998), has been playing an increasing role in hybrid cultivar development in China. The male sterility of 9012AB is controlled by two recessive genes (designated Bnms3 and Bnms4) interacting with one recessive epistatic suppressor gene (esp). Previous study has identified seven AFLP markers, six of which were co-segregated with the Bnms3 gene in a small population (Ke et al. in Plant Breed 124:367-370, 2005). By cloning these AFLP markers and their flanking sequences, five of the six co-segregated markers were successfully converted into sequence characterized amplified region (SCAR) markers. For fine mapping of the Bnms3 gene, these SCAR markers were analyzed in a NIL population of 4,136 individuals. The Bnms3 gene was then genetically mapped to a region of 0.56 cM, with 0.15 cM from marker SEP8 and 0.41 from marker SEP4, respectively. BLAST analysis with these SCAR marker sequences identified a collinear genomic region in Arabidopsis chromosome 5, from which two specific PCR markers further narrowed the Bnms3 locus from an interval of 0.56 to 0.14 cM. These results provide additional information for map-based cloning of the Bnms3 gene and will be helpful for marker-assisted selection (MAS) of elite RGMS lines and maintainers.