Functional Genomics

Background: MicroRNAs, a kind of non-coding small RNAs, playing an improtant role in regulating gene expression during post-transcriptional stage in plant. To identify differentially expressed miRNAs related to differences in fiber length between two near-isogenic lines（NILs） that will facilitate the uncovering of the mechanism of fiber elongation, a high-throughput sequencing technology was applied. Result: Two libraries were constructed during elongation stage between the two NILs. A total of 35 known miRNAs families and 169 novel miRNAs were found by aligning to the cotton D genomic and the miRBase (version 18.0). A number of 16 known miRNAs and 60 novel miRNAs were found to have significant difference between the two NILs. Soon a bioinformatics and a degradome were taken to analyze the targets of miRNAs. A total of 236 targets were found, with obvious function related to plant hormone pathways and RIG-I-like receptor signaling pathways, such as auxin, brassinosteroid and so on. Then a real-time fluorescence quantitative PCR with some chosen miRNAs and targets were taken to measure the relationship among miRNAs, targets, and the length of fiber. Conclusion: This study shows the function of miRNAs during fiber elongation by regulating their targets. And some novel miRNAs in allopolyploid cotton were found. We also revealed a possible pathway of some miRNAs influencing the fiber length, which deserved further study. Key words: Allopolyploid cotton, fiber elongation, miRNA, high-throughput sequence

Plant microtubule cytoskeleton plays an important role in cell elongation and cell wall formation. The orientation of cortical microtubule array was consistent well with cellulose microfibril. At cell elongation period, transverse microtubule array is observed while longitudinal and oblique array present in cell stopped elongation. In cotton fiber cell, one of the longest cells ever characterized in plant kingdom, it is closely related between the arrangement of cortical microtubules and fiber cell elongation. The major component of microtubules is heterodimer of highly conserved α- and β-tubulin. There were 12 α-tubulin genes and 19 β-tubulin genes in cotton genome and some genes expressed preferentially in fiber cell or a certain developmental stage of cotton fiber. These indicated that tubulin gene had an important role in the growth and development of fibers. However, the function and mechanism of each tubulin gene in fiber development is largely unknown to date. To illuminate their function in fiber cell elongation, we have examined the expression difference of 31 microtubule genes between ligon lintless-1 (li-1) mutant and it’s isogenic wild-type TM-1 by quantitative real-time RT-PCR. The li-1 has extremely short fibers and was employed as an ideal material to study fiber elongation. The expression of GhTUB17, one of the β-tubulin genes, was drastically suppressed in 10 DPA fibers of li-1 mutant. The gene specifically expressed in fiber cell and the expression peak present in 6 and 8 DPA fibers. Moderate expression level was in 10 and 12 DPA fibers while the transcript was hardly detected in the fiber after 14 DPA. These results revealed that the GhTUB17 primarily related to fiber elongation. The length of cDNA of GhTUB17 was 1475bp in upland cotton (Gossypium hirsutum). It contains a 1335bp ORF encoding 444 amino acid residues with a molecular weight of 50kD. isoelectric point was 4.65. The amino acid sequence was high homology with the β-tubulin from Arabidopsis thaliana, tobacco, and cocoa. However, GhTUB17 was in a specific branch in phylogenetic tree. It was suggested that the gene might play a special role in fiber cell development. To further elucidate the mechanism of expression decrease in 1i-1 fiber, the cDNAs, genomic DNAs, and about 2.5 kb promoter sequences of GhTUB17 gene were cloned both from li-1 and TM-1. There was little diversity in the cDNA and genomic sequence while an 180bp deletion was in the promoter from li-1 mutant. Five brassinosteroid response elements were in the fragment. Correspondently, the expression of GhTUB17 was significantly induced by Brassinolide (BL) treatment in wild type fibers while less response for BL treatment in li-1 fibers. These results indicate that GhTUB17 play an important role in fiber growth and might involve in fiber elongation induced by brassinosteroids. Keywords: cytoskeleton; GhTUB17 gene; cotton fiber; li-1 mutant; brassinosteroid

Fiber and leaf trichome are extremely-elongated single cells that initiate and develop on epidermal cells, and are likely to have common regulatory mechanisms. In our previous study, a major QTL affecting multiple fiber quality traits at T1 locus (trichome ) on chromosome 6 has been identified in a RIL population from a cross between high quality cultivar Yumian 1 and multiple dominant marker line T586. To fine-map the QTL at t1 locus, a 1440-individuals F2 population was developed from a cross between Yumian 1 and recombinant line RIL118 whose QTL region derived from T586 in 2010. Firstly, 104 markers on chromosome 6 were used to genotype 360 plants randomly selected from F2 population and a genetic map covering 146.3 cM was constructed, and QTL affecting fiber length, fiber uniformity, fiber micronaire and fiber strength was confirmed. T1 co-segregated with 10 markers, QTL affecting fiber quality traits covered 0.8 cM. Secondly, to fine map T1 and QTL affecting fiber quality traits, 380 new SSR primer pairs designed from the QTL corresponding region on chromosome 10 in G. raimondii genome, and 13 markers were obtained. Twenty-tree markers within the QTL region were used to genotype the 1440 plants and the saturated genetic map spanned 0.28 cM and T1 was flanked by SWU2518 and SWU2302. Based on fiber quality trait from the 1440 plants in 2010, the QTL identified explains 54.7%, 40.5%, 30.1% and 50.0% of the fiber length, micronaire, uniformity and strength phenotypic variation respectively. In 2011 and 2013, another 5535 F2-plants developed from (Yumian 1 × RIL118) were used to screen new recombinants in the QTL region, and thirty-five recombinants were obtained. However, T1 and QTL affecting fiber quality traits were still mapped in the same interval between SWU2518 and SWU2302. In G. raimondii genome, the QTL corresponding region covers about 2.7-Mb physical distance and contains 100 genes. The present study proves that QTL affecting fiber quality traits at T1 is stable and pleiotropic. However, the candidates for T1 and QTL affecting fiber quality traits need to study further.

Upland cotton is the chief source of white fiber around the world. Unfortunately its vulnerability to various biological and a biological stresses arrest its yield and production for the last several years especially in Pakistan. Besides the improvement of this vital crop regarding the fiber quality traits and productivity has always been hard to be manipulated either by using the classical breeding approaches or by advanced molecular genetic approaches. The recent progress in the area of plant molecular biology and plant genomics has the potential to initiate a new Green Revolution. However, these discoveries are to be implemented in the development of new cultivars to realize that potential. Genetic mutation is a powerful tool to create the genetic variability. TILLING (Target Induced Local Lesions In Genomes) is a reverse genetic approach for mutation based crop improvement that combines conventional mutagenesis with DNA level mutation identification. It has been used successfully by researchers as a functional genomic discovery platform in model organisms. But now it has proven effectively as a crop improvement tool. Besides genetic engineering approaches are hindered mainly by non-availability of tissue culture in elite cotton cultivars, cost of producing the transgenic plants, behavior of the transgene and the ethical questions associated with GMO’s. In contrast, TILLING provides a non-transgenic genetic therapy for cotton genome to deal with the prevailing problems. Cotton TILLING project at the Department of Plant Breeding and Genetics, University of Agriculture Faisalabad, Pakistan is the initiative towards the creation and functionally characterization of the cotton TILLING populations. The major objective of this project is the development and testing of cotton TILLING populations for disease and fiber related genes with theme to circumvent the inferior fiber quality and unrestrained epidemic Cotton Leaf Curl Disease (CLCuD) in subcontinent. We targeted fiber related gene families like pectin methyl esterase, actin, sucrose synthase, and defense related gene families including resistance gene analogues (RGAs) and defense gene analogues (DGAs). Two cotton cultivars “PB-899 and PB-900” were mutagenized on the calculated optimum doses of EMS and raised up to their M2 populations. A total of 8000 to 10,000 M2 plants were screened for mutations in targeted genes. The phenotyping presented a number of mutants including branching pattern, leaf morphology, disease resistance, photosynthetic lesions and flower sterility. The molecular screening of point mutations was performed by TILLING PCR aided with CEL1 mismatch cleavage assay that also showed significant proportion of induced mutations in TILLING populations that verifies it an efficient tool in improvement of cotton.

A segregating mutant population has been developed from the standard Bulgarian variety “Chirpan 603” by applying seed gamma irradiation. M4 and M5 generations were used for identification of quantitative trait loci (QTLs) related to fiber quality characteristics. SSR markers developed in interspecific crosses and further confirmed in intraspecific crosses, together with in-house developed ISSR markers were used for association mapping of QTLs for fiber strength, length, uniformity, micronaire and elongation. QTLs with major effects on studied traits were confirmed in M6 generation. Further ones were identified and used for map saturation and linkage group confirmation.

Plant steroid hormones, brassinosteroids (BRs) regulate plant growth and development and play an important role in the development of cotton fiber. However, the BR signaling pathway in cotton fiber cell is largely unknown. AtBIL4 (Brz-insensitive-long hypocotyls 4, NM_116196) is a critical component of plant cell elongation occurring upon BR signaling. Considering that cell elongation was a main process in fiber growth, a homologous gene of AtBIL4 was cloned from upland cotton (Gossypium hirsutum L.) and its expression pattern was analyzed in fiber growth. Four BIL4-like genes, designed GhBIL4-1, GhBIL4-2, GhBIL4-3 and GhBIL4-4 were isolated from 10-DPA fiber cells. The amino acids deduced from the ORF of the genes were 243, 248, 248 and 248, respectively. Four GhBIL4 proteins were membrane proteins and seven transmembrane domains were in each sequence. BLAST searches of GhBIL4s amino acid sequence identified similar genes in Arabidopsis, poplar, grape, bean, tomato, potato, and rice. Interestingly, the GhBIL4s have high homology with BI, a Bax Inhibitor involved in programmed cell death. Overexpressing BI in plant could improve the tolerance to cell death induced by biotic or abiotic stress. GhBIL4s expressed widely in all detected samples although different expression level present in various tissues, organs, and cells. The highest levels of GhBIL4-1 transcript were in fiber cells. Moderate and similar expression levels were observed in root, stem, leaf, hypocotyl, pollen, and ovule. The expression peak of GhBIL4-3 was in fiber cells while identical expression in the other samples. Either GhBIL4-2 or GhBIL4-4 expressed highly in stem, hypocotyl, fiber cell, and ovule. The lowest expression level was detected in pollen. Therefore, GhBIL4s expressed preferentially in fiber cells. In the various developmental stages of fibers, four genes expressed slightly in 0~4 DPA ovules (with fibers) and highly expressed in fiber cells. GhBIL4-1 and GhBIL4-3 shared a similar expression pattern in the growth of fiber cells. Identical and moderate levels present in the fibers of 6~10 DPA while the highest levels was displayed in the fibers of 14~20 DPA. The expression levels of GhBIL4-2 and GhBIL4-4 were elevated drastically in 6 DPA fiber cells compared to 4 DPA ovules. Consequently, the expression decreased slightly and sustained high expression level in 8~20 DPA fiber. These results suggested that the GhBIL4s might play important roles in the growth and development of fiber, especially in fiber elongation and secondary cell wall deposition. Furthermore, GhBIL4s might be involved in the resistance to biotic or abiotic stress induced by BRs. Using a vector provided kindly by professor Nakano Takeshi (RIKEN), we have obtained transgenic cotton lines overexpressed AtBIL4. The terminal bud development of transgenic plants was suppressed and the reproductive development was retarded. The flower bud abolished and the pollen was sterility in transgenic plants. So that cotton bolls were hardly harvested. These results further indicated that GhBIL4s play important roles in the growth and development of cotton plant. Keywords: upland cotton; brassinosteroids(BRs); GhBIL4;

Cloning and Functional Analysis of Pectin methylesterases Gene GhPME6 in Upland Cotton Wang Lin，Shang Hai-Hong，Li Jun-Wen， Wang Shao-Gan，Liu Ai-Ying，Shi Yu-Zhen，Gong Ju-Wu，Gong Wan-Kui，Yuan You-Lu* (State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS, Anyang, 455000) Pectin Methylesterase (PME) is common in plants which has some connection with the change of the cell wall’s structures. Pectin methylesterases can catalyze the demethylesterification of pectin in cell wall releasing acidic pectins and methanol. The free carboxyl can interact with bivalent ions (Ca2+) , so ridigifying the cell wall. Based on the bioinformatic analysis of the ESTs from the cotton fiber SSH-cDNA library about the secondary wall thickening, we obtained a cDNA sequence, which has the homolog sequence of Pectin methylesterases, and it was designated as GhPME6. The full length of cDNA was amplified by RACE from upland cotton TM-1. And the characteristics of this sequence was analyzed by bioinformatic softwares. The analysis of gene structure indicate that GhPME6 has a ORF which length is 150bp and encodes a polypeptide of 519 amino acid. And GhPME6 were composed of two exons and one intron in comparison of their sequences of genomic DNA and cDNA by DNAMAN. The deduced amino acids showed high identity with the Pectin methylesterases by multiple alignments analysis. The deduced amino acid sequence contains typical structures of PMEI(pectin methylesterase inhibitor) and PME(pectin methylesterase) by using Hmmsearch. PME6 is highly conserved during evolution of the Gossypium’s genome through the analysis of comparing the PME6 in G. raimondii, G. arboreum, G. hirsutum and G. barbadense. The tissue expression analysis by using quantitative RT-PCR revealed that GhPME6 expressed predominantly during the stage of secondary cell wall thickening of fiber development. The result suggested GhPME6 might may play an important role in the cotton fiber strength forming. By using E. coli expression system and SDS-PAGE assay, the result show the molecular weight of the prokaryotic expressed protein is 57 KDa. It is same as expected and can lay the foundation for the deeper study of the PME6’s action during the stage of the cotton fiber strength forming.

Genes are crucial not only to the study of the mechanisms of traits or biological processes that they control, but also to the development of knowledge and tools enabling effectively enhanced plant breeding. However, few genes controlling fiber quality and yield have been cloned and characterized to date. In this study, a newly-developed high-throughput gene and QTL cloning system is used to isolate genes controlling traits important to fiber quality, such as length, strength, uniformity, elongation and micronaire, lint and seed yield, and lint percentage. A total of 474 genes controlling fiber length (GFL) have been cloned. Systems analysis of the genes showed that genes controlling fiber length are involved in a variety of biological processes and metabolic pathways, with each having an effect ranging 2.64% – 7.94%. Of the 474 GFL genes, 88.6% decreased fiber length when turned on or actively expressed, whereas only 11.4% increased fiber length when turned on or actively expressed. Using these newly cloned GFL genes, we have deciphered the molecular basis and regulation mechanisms of cotton fiber length. The cloned GFL genes and the findings resulting from their analysis have provided new knowledge and tools for development of a gene-based breeding system in cotton, which is far more powerful than the currently used marker-assisted selection in plant breeding.

Cotton Leaf Curl Virus (CLCuV) disease is one of the major diseases playing havoc with the cotton yield globally and especially in Pakistan. The DNA virus causing this disease is transmitted by white fly which serves as a carrier and spreads the virus from one plant to another. The virus enters the cotton leaf vascular bundle by white fly bite and multiplies in the plant vascular sap resulting in the curling of leaves, stunted growth and ultimately low yield. Gossypium hirsutum which is largely cultivated in Pakistan is susceptible to CLCuV while the old world diploid cotton, G. arboreum is more resistant to this virus but unfortunately produces lower quality fibers. In CEMB, we have produced a mutant of G. arboreum which is susceptible to this virus. Moreover, we have access to CLCuV resistant, G. hirsutum Mac7 USA gerplasm and the susceptible cultivars of G. hirsutum. These resistant and susceptible cultivars of diploid and tetra-ploid cotton could be very useful in investigating the molecular basis of CLCuV resistance. We are using comparative global transcriptional profiling of resistant and sensitive cultivars to find the molecular basis of resistance. For this purpose inoculation of CLCuV resistant and sensitive cultivars of cotton would be done by traditional vector based method and RNA would be isolated after the symptoms appear. To avoid variability and for consistency of the results, at least 5 biological replicates of each cultivar would be used in this study. cDNA synthesis and labeling of samples with Cy3 and Cy5 florescent dyes will be done followed by hybridization of samples on custom designed microarray chips. Comparative transcriptional profiling of sensitive and resistant varieties and their response to inoculation with CLCuV would be very helpful in identification of key regulators of CLCuV resistance. Transformation of key regulators (genes) in Gossypium hirsutum to produce CLCuV resistant cotton will be the future outcome of this project.

Begomoviruses are known to be a major problem in the cotton and other crops in Pakistan particularly in the Punjab region. Crops infected with these viruses show a notable decrease in the yield which results in major losses for farmers in particular and for the nation’s economy in general. Genetically-engineered resistance against begomoviruses is at advanced stage of investigation and crops such as cotton are more likely to benefit of solving this problem. In current study a collaborative effort has been made by different institutes namely IAGS, NIBGE and CEMB with financial support of USDA. In this effort strategies employed includes nine construct namely C1 to C6 of which C1 was based on pathogen derived resistance against vector, three constructs from C2 to C4 were based upon pathogen derived resistance against Gemini viruses while remaining two constructs i.e C5 and C6 were developed on the principle of non-pathogen derived resistance by NIBGE i.e protein mediated resistance against CLCuV were handed over to CEMB for transformation Similarly One construct of pathogen derived resistance against Gemini viruses (RNAi based construct against sense and anti-sense region of replication of begomoviruses) namely C7, other RNAi based constructs against beta satellite of CLCuV namely C8 and one RNAi based construct against cathepsin gut region of whitefly namely C9 was developed by IAGS and provided to CEMB for transformation in local cotton varieties. A local modified gene transformation technology in local cotton cultivar namely MNH-786, VH-289 and CIM-496 has been applied with increased efficiency. Multiplication of putative transgenic cotton plants has been done in controlled condition of green house after one and half month screening on selection medium. Generation advancement and screening of best event from T0 to T1 and T2 of transgenic cotton plants was done and also in process. Confirmation of successful integration of transgene was done through amplification by using gene specific primers for C1, C2, C3, C4, C5, C6 and C7. Quantification of miRNA in transgenic cotton plants was done on relative basis through real time PCR in both To and T1 generation. Variable quantity of miRNA was found in different events in both To and T1 generation of transgenic cotton plants. Morphological characteristics of these transgenic plants in comparison of miRNA quantity have shown that CLCuV symptoms were less in plants having greater quantity of miRNA and greater in plants having less quantity of miRNA. Similarly absolute quantification of virus titer was done in nine C2 transgenic plants. Four transgenic plants showed least quantity of virus with no morphological symptoms as compared to other five which have shown high titer of virus with maximum viral symptoms. Further generation advancement and selfing of these transgenic cotton plants with determination of their copy no and virus titer will be helpful for the selection of best events which will be expected outcome of this study in near future.