Functional Genomics

Cotton （Gossypium spp.）is the world’s most important fiber crop plant. Cotton fiber has been used in the textile and many other industies diffusely. Cotton fiber qualities including length, strength and fineness are known to be controlled by genes affecting cell elongation and secondary cell wall (SCW) biosynthesis. MicroRNAs are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of microRNAs are critical steps in finding their target genes and elucidating their functions. Here, we elucidated microRNAs from two developmental time points during elite upland cotton Ji228 fiber development by high-throughput sequencing. 41 conserved microRNAs belonging to 19 microRNAs families and 148 novel microRNAs were discovered, and 725 potential targets genes were predicted. On the other hand, 43,809 unigenes and 3,463 novel genes were mined from 7 development time points during elite upland cotton Ji228 fiber development by RNA-seq and DEG. 3 categories and 28 classes were found according to the expression profile of sequencing and gene expression of dynamic change in fiber development. MicroRNA-mRNA interactions potentially relevant for fiber development were explored using anti-correlation expression analysis and microRNAs target prediction algorithms. 9 microRNAs and their target genes were get from the relationship between the differences of microRNAs and differences mRNA. The data suggest critical roles of transcription relation and microRNA-mediated pathways in fiber development.

RNA interference to generate resistance against begomovirus/satellites in cotton U. Hameed,a M. Zia-Ur-Rehmana , M.J.Iqbala, M.S. Haidera and J.K. Brownb a Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan. ,b University of Arizona, Plant sciences, Tucson, USA In Pakistan major constrain in cotton production is Cotton leaf curl disease (CLCuD) caused by begomovirus (family Geminiviridae) and associated satellite that are transmitted by whitefly. CLCuD caused 5 billon US dollar losses in mid to late 1990s and it became the major limitation in increasing cotton production in Pakistan. The conventional techniques used for the control of viral diseases includes the application of insecticides to destroy putative insect vectors which transmitting viruses from infected to healthy plants and by breeding for resistance against viruses. Another approach that now used widely is production of transgenic plants using RNAi technique to induce resistance to viruses. RNAi is a specific mechanism that involves gene silencing by degradation of target transcripts. Hairpin (hp) RNAi construct containing viral sequence in sense and anti-sense direction separated by intron is used for plant transformation. We have used this strategy to control CLCuD by targeting the vital and conserved regions of begomovirus/satellite genome responsible for the disease. To generate broad spectrum resistance in cotton three RNAi constructs against begomoviruses/satellites are developed and the co-inoculation of these RNAi constructs along with cotton leaf curl virus-complex in model plant Nicotiana benthamiana, revealed that the technique significantly decreased the virus/satellite accumulation.These constructs are under process of transformation in cotton to generate broad spectrum resistance against viruses/satellites.

Cotton fiber quality traits are controlled by multiple genes of minor effect. Identification of significant and stable quantitative trait loci (QTLs) across environments and populations lays foundation for marker-assisted selection (MAS) for fiber quality improvement and studies of its molecular regulation. Here, a detailed genetic map is constructed and QTLs are detected based on an intraspecific recombinant inbred line population (RIL) derived from a cross between upland cotton ‘Yumian 1’ and ‘7235’. A total of 25,313 SSR primer pairs, including 5,000 developed from G. raimondii BAC-end sequences, were used to construct the genetic map which finally contained 1540 loci, spanning 2812.6 cM, with an average of 1.83 cM between adjacent markers. The population showed 662 distorted loci with more distorted loci located on the Dt subgenome (582) and favored Yumian 1 alleles (629). Fiber quality data were collected across four years. Variance analysis showed that fiber quality traits were significantly affected by both genetic and environmental factors. Significant correlations were also detected among fiber quality traits. A total of 62 QTLs， including 18 significant QTL were identified for five fiber quality traits with combined analysis and single environment analysis. Among these QTL, 12 were detected for fiber length, 14 for fiber uniformity, 13 for fiber micronaire, 11 for fiber elongation, and 11 for fiber strength. These QTLs explain phenotypic variation from 5.0% to 28.1%. For each trait, favorable alleles were conferred by both parents. Seventeen QTL were detected in multiple environments including four, three and ten QTL detected in four, three and two years respectively. QTL for different traits co-locate on Chr. 5, 8, 10, 18, and 24. The genetic map and stable QTLs are valuable for upland cotton genome research and breeding projects to improve fiber quality.

Phytochromes are family of photoreceptors involved in regulation of complex developmental and molecular processes in plant cells. Recently, we characterized phytochrome gene family in cotton genome including PHYA, PHYB, PHYC and PHYE genes. Each phytochrome is responsible for many processes in cotton which directly related to light effects such as vegetative growth, plant architecture, photoperiodic flowering, maturity, fiber quality, tolerance to environmental stresses and productivity. It is known that the red light photoreceptor PHYB gene is responsible for inhibition of plant flowering. In order to study exact biological function of cotton PHYB gene(s), we designed synthetic short oligonucleotide duplex for cotton PHYB gene that introduced to cells, should specifically suppress the targeted gene expression. A binary pART27 vector construct bearing synthetic phyB cassette (SynB, driven by 35S promoter) and kanamycin resistance gene marker was developed and somatically transformed into Coker 312. RNAi plants were obtained using somatic embryogenesis. Candidate RNAi plants bearing the synthetic PHYB RNAi duplex were verified using PCR reaction that amplifies specifically phyB duplex insertions from the vector construct. Phenotypic observations of SynB RNAi T1-5 plants showed early flowering and fiber Micronaire (MIC) improvement compared to wild-type and null segregant controls. Our efforts helped to accurately regulate gene function and related agronomically important traits in complex plant genomes such as allopolyploid cotton. Details of phenotypic and molecular characterization of cotton PHYB gene RNAi plants will be discussed.

In this study, a RIL population (F6:8) with 196 lines was developed from an upland cotton cross of sGK9708, derived from an elite widely planted cultivar CRI 41, and 0-153, an excellent cultivar with superb fiber strength. Multi-environmental experiments in ten environments were conducted at six locations in four years and all five fiber quality traits were evaluated. A genetic linkage map harbouring 400 SSR loci and 54 linkage groups was constructed, which spanned 1260.68 cM, with an average marker interval of 3.15 cM between two markers and approximately covered 28.33% of the cotton genome. Among the total 48 QTL detected at least in 3 environments, 5 major QTL were detected at least in 8 environments: 2 for fiber length (qFL-1-1and qFL-9-2) which were mapped on Chr25 and Chr7 respectively and 3 for fiber strength (qFS-1-2, qFS-1-4 and qFS-9-5) which were mapped on Chr25 and Chr7 respectively. All the 5 QTL displayed stably in multiple environments and explained a larger phenotypic variance (with average PV>10%), among which qFS-1-4 might be the same as STR-25 in Lacape’s report (Lacape et al. 2005), qFS-1-2 and qFS-9-5 were new identified ones.

Along with many other biological and environmental threats, one of the main biological threats in cotton production is a devastating and aggressive wilting disease known as Fusarium wilt caused by the fungus Fusarium oxysporum f.sp. vasinfectum (FOV). Molecular mechanisms associated with Fusarium wilt resistance of Upland cotton (Gossypium hirsutum L.) are largely unknown. Because small RNAs and microRNAs play an important role in plant defense we characterized small RNA profiles during FOV race 3 pathogenesis in cotton. Four separate small RNA libraries (sRNAs) were prepared from cotton (G. hirsutum) roots (infected and uninfected), including FOV resistant Mebane B-1 line and FOV susceptible № 11970 line. We sequenced 4179 clones and detected 4116 small RNAs sequences of 16-30nt. Although the total number of unique tags were approximately the same among libraries (except № 11970 FOV susceptible infected line), these tags were substantially different based on the length distribution. However, the highest percentage of these unique sequence signatures was 21nt in length in all of the libraries. The Mebane B-1 line had the highest number of unique 21nt sequence signatures and the uninfected library had a greater number than its infected counterpart library suggesting the distribution of 21nt size sRNAs is different between these two lines depending upon FOV infection. BLAST results show that there are several kinds of RNA fragments such as mRNAs, rRNAs and other unknown short fragments. Greater than 73% of unique sRNAs from four libraries matched to Gossypium L. (G. arboreum, G. hirsutum, and G. barbadense) expressed sequence tags (ESTs). A small percentage of unique sRNAs matched to A.thaliana (1.68%), T. cacao (1.26%), Fungal (2%), and other organism (21.33%) ESTs. MirBase database search detected 4% of unique sRNAs to be homologous to some plant microRNAs described in the literature such as miR156i-3p, miR160a, miR169n-3p, miR171d-5p, miR172a, miR390a-3p, miR398b-5p, miR395l-5p, miR1069-3p, miR2949a-5p, miR2911, miR2916, miR166m-5p, and miR3476-5p. Our results showed only two sequence tags aligning with known plant miRNAs not registered in the cotton database yet. Mir-160, which was sequenced from FOV uninfected Mabane-1 young root tissues, is highly conserved in plants and mir-2911, which was seen in all four small RNA libraries, is known from Nicotiana tabacum and Populus euthratica databases. Analysis of target proteins from small RNA signatures detected important cellular components helping to explain the FOV pathogenesis regulation in cotton. Small RNA and microRNA sequence signatures characterized in this work will be helpful for developing innovative biotechnology tools to improve FOV resistance of cotton cultivars.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules found in all eukaryotes, and they play significant roles in developmental and environmental signal transduction. In this study, a MAPK gene (GbMPK3), which showed homologous to AtMPK3 and NtWIPK, was isolated from sea-island cotton (Gossypium barbadense) and induced during multiple abiotic stress treatments including salt, cold, heat, dehydration and oxidative stress. Transgenic tobacco (Nicotiana benthamiana) with constitutively higher expression of GbMPK3 was conferred with enhanced drought tolerance, reduced water loss during drought treatment and improved plant height and survival rates after re-watering. Additionally, the gene expression levels and enzymatic activity of antioxidant enzymes were more strongly induced with depressed hydrogen peroxide accumulation in GbMPK3-overexpressing tobacco compared with wild-type under drought condition. Furthermore, observation of seed germination and leaf morphology showed that tolerance of transgenic plants to methyl viologen (MV) was improved due to increased antioxidant enzyme expression, suggesting that GbMPK3 may positively regulate drought tolerance through enhanced reactive oxygen species (ROS) scavenging ability.

Discovery and development of intra-specific single nucleotide polymorphism markers in Upland cotton (G. hirsutum L.) Qian-Hao Zhu, Andrew Spriggs, Jen Taylor, Danny Llewellyn, Iain Wilson CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia Single nucleotide polymorphisms (SNPs) are the most abundant type of molecular markers in plants. Only varietal SNPs that are different between varieties and not sub-genome SNPs that are just differences between the two sub-genomes (At and Dt) of tetraploid cotton are useful as markers in breeding. Varietal SNPs have not yet been practically used in cotton breeding because they are difficult to discover due to low intra-specific polymorphism and very high sequence identity between homoeologous genes in cotton. Next-generation sequencing is now facilitating genome-wide SNP discovery in many crops, including cotton; however, identification of reliable varietal SNPs is still a challenge in polyploids. We have used transcriptome sequencing (RNA-seq), restriction-site associated DNA (RAD) sequencing, and novel bioinformatic strategies to identify varietal SNPs among 18 commercial Upland cotton varieties. Using the RNA-seq data, we identified 37413 varietal SNPs based on the rationale that they can be more confidently called when flanked by genome-specific SNPs that assign reads to their respective sub-genomes. Of these SNPs, 22121 did not have an additional varietal SNP within their 20-bp flanking regions so can be used in most common SNP genotyping assays. Based on the gene annotations of G. raimondii, 40.52% and 25.39% of these SNPs had non-synonymous and synonymous effects, respectively. Approximately 2.49% of these SNPs affected translation start, stop or splice sites, while the remaining (~31.60%) were located in non-coding regions. From the RAD data, we identified an additional 3090 varietal SNPs between two of the varieties. Verification rates of 72.6-91.7% were achieved for subsets of these varietal SNPs using different genotyping platforms. Depending on the assay platform, however, many of the SNPs behave as dominant markers because of amplification from both homoeologous loci, but the number of SNPs acting as co-dominant markers increases when one or more sub-genome-specific SNP(s) are incorporated in their assay primers, giving them greater utility for breeding applications. A G. hirsutum genetic map with 1,244 SNP markers and covering 5557.42 cM was constructed and used to map quantitative trait loci for leaf shape, leaf trichome and pollen colour. Our collection of G. hirsutum varietal SNPs provides the cotton community with a valuable marker resource applicable to genetic analyses and breeding programs.

Recently microRNAs (miRNAs) have been found to be differentially expressed during cotton fiber development. However, what specific miRNAs and how they are involved in fiber development is unclear. Here, using deep sequencing, 65 conserved miRNA families were identified; 32 families were differentially expressed between leaf and ovule. A total of 128 pre-miRNAs, including 120 conserved and 8 novel pre-miRNAs were found in cotton. At least 40 miRNAs were either leaf or ovule-specific, whereas 62 miRNAs were shared in both leaf and ovule. A total of 863 genes were potentially targeted by 120 identified miRNAs, whose functions are involved in a series of biological processes including transcription factors, fiber development, metabolism, and signal transduction. GO and KEGG analyses showed that the identified miRNAs and their targets were classified to 1,119 GO terms including 563 biological processes, 416 molecular functions, and 140 cellular components, and were enriched to 74 KEGG pathways. At least 7 unique miRNAs participate in trichome regulatory interaction network. Eleven tasiRNA-derived candidate genes were identified in cotton. One has never been found in other plant species and two of them are MYB and ARF, both play important role in cotton fiber development. Sixteen genes were predicted to be tasiRNA targets, including sucrose synthase, polyphenol oxidase, MYB2, and glycosyl hydrolase. Together, this study discovered new miRNAs in cotton and offered evidences that miRNAs play important roles in cotton fiber development. The identification of tasiRNAs and their targets broadens our understanding of the complicated regulatory mechanism of miRNAs in cotton.

Due to its toxic nature, cadmium (Cd) causes ultramorphological, metabolic, and proteomic changes in plants. We designed the present study to study Cd stress (0, 500 µM) in upland cotton cultivar (ZMS-49) using physiological, metabolic and proteomic biomarkers. One way ANOVA was performed along with transmission electron microscopy, energy diffraction X-Ray analysis and 2-D gel electrophoresis to fulfill the objectives of the designed study. Data revealed that 500 µM Cd level altered physiology-based traits such as fresh and dry weights, tolerance index and chlorophyll-related parameters. It also caused lowering down of various essential plants nutrients as compared to control. The antioxidative metabolism was also significantly influenced by the higher level of Cd (500 µM). Oxidative stress biomarkers in all parts of the plant increased, while there was found either increase or decrease in the expression levels of various antioxidants. Ultramorphology of both roots and leaves was insignificantly changed. Cd was localized in cell wall, vacuoles and intracellular regions. Leaf proteomic data reveal differential expression of a number of proteins under Cd stressful conditions. Proteins such as methionine synthase, ribulose 1, 5-bisphosphate carboxylase, apoplastic anionic guiacol peroxidase etc. were upregulated. While proteins involved in energy pathway (ATP-dependent Clp protease ATP-binding subunit ClpC), chaperon and stress protein (HSP70 (AA6-651) etc. were supressed. Upregulation of antioxidant machinery, Cd-deposition in dead parts of the cell and active regulation of certain proteins show that ZMS-49 can be a potential candidate to be grown in Cd-contaminated areas.