Zabardast T. Buriev

Small RNA regulation of ovule development in the cotton plant, G. hirsutum L

BackgroundThe involvement of small RNAs in cotton fiber development is under explored. The objective of this work was to directly clone, annotate, and analyze small RNAs of developing ovules to reveal the candidate small interfering RNA/microRNAs involved in cotton ovule and fiber development.ResultsWe cloned small RNA sequences from 0–10 days post anthesis (DPA) developing cotton ovules. A total of 6691 individual colonies were sequenced from 11 ovule small RNA libraries that yielded 2482 candidate small RNAs with a total of 583 unique sequence signatures. The majority (362, 62.1%) of these 583 sequences were 24 nt long with an additional 145 sequences (24.9%) in the 21 nt to 23 nt size range. Among all small RNA sequence signatures only three mirBase-confirmed plant microRNAs (miR172, miR390 and ath-miR853-like) were identified and only two miRNA-containing clones were recovered beyond 4 DPA. Further, among all of the small RNA sequences obtained from the small RNA pools in developing ovules, only 15 groups of sequences were observed in more than one DPA period. Of these, only five were present in more than two DPA periods. Two of these were miR-172 and miR-390 and a third was identified as 5.8S rRNA sequence. Thus, the vast majority of sequence signatures were expressed in only one DPA period and this included nearly all of the 24 nt sequences. Finally, we observed a distinct DPA-specific expression pattern among our clones based upon sequence abundance. Sequences occurring only once were far more likely to be seen in the 0 to 2 DPA periods while those occurring five or more times were the majority in later periods.ConclusionThis initial survey of small RNA sequences present in developing ovules in cotton indicates that fiber development is under complex small RNA regulation. Taken together, the results of this initial small RNA screen of developing cotton ovules is most consistent with a model, proposed by Baulcombe, that there are networks of small RNAs that are induced in a cascade fashion by the action of miRNAs and that the nature of these cascades can change from tissue to tissue and developmental stage to developmental stage.

Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L

Simultaneous improvement of fibre quality, early-flowering, early-maturity and productivity in Upland cotton (G. hirsutum) is a challenging task for conventional breeding. The influence of red/far-red light ratio on the fibre length prompted us to examine the phenotypic effects of RNA interference (RNAi) of the cotton PHYA1 gene. Here we show a suppression of up to ~70% for the PHYA1 transcript, and compensatory overexpression of up to ~20-fold in the remaining phytochromes in somatically regenerated PHYA1 RNAi cotton plants. Two independent transformants of three generations exhibited vigorous root and vegetative growth, early-flowering, significantly improved upper half mean fibre length and an improvement in other major fibre characteristics. Small decreases in lint traits were observed but seed cotton yield was increased an average 10-17% compared with controls. RNAi-associated phenotypes were heritable and transferable via sexual hybridization. These results should aid in the development of early-maturing and productive Upland cultivars with superior fibre quality.

RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.)

RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization.

Genetic Diversity in Gossypium genus

Cotton (Gossypium spp.) is the unique, most important natural fiber crop in the world that brings significant economic income, with an annual average ranging from

Clustering, haplotype diversity and locations of MIC-3: a unique root-specific defense-related gene family in Upland cotton (Gossypium hirsutum L.).

27 – 29 billion worldwide from lint fiber production (Campbell et al., 2010). The worldwide economic impact of the cotton industry is estimated at ~

Genome Editing in Plants: An Overview of Tools and Applications

500 billion/yr with an annual utilization of ~115-million bales or ~27-million metric tons (MT) of cotton fiber (Chen et al., 2007). In 2011 and 2012, global cotton production is projected to increase 8% (to 26.9 million MT). This will be the largest crop since 2004 and 2005 (International Cotton Advisory Committee [ICAC], 2011).

Genome-wide identification and characterization of microRNAs differentially expressed in fibers in a cotton phytochrome A1 RNAi line

MIC-3 is a recently identified gene family shown to exhibit increased root-specific expression following nematode infection of cotton plants that are resistant to root-knot nematode. Here, we cloned and sequenced MIC-3 genes from selected diploid and tetraploid cotton species to reveal sequence differences at the molecular level and identify chromosomal locations of MIC-3 genes in Gossypium species. Detailed sequence analysis and phylogenetic clustering of MIC-3 genes indicated the presence of multiple MIC-3 gene members in Gossypium species. Haplotypes of a MIC-3 gene family member were discovered by comparative analysis among consensus sequences across genotypes within an individual clade in the phylogram to overcome the problem of duplicated loci in the tetraploid cotton. Deficiency tests of the SNPs delimited six At-genome members of the MIC-3 family clustered to chromosome arm 4sh, and one Dt-genome member to chromosome 19. Clustering was confirmed by long-PCR amplification of the intergenic regions using At-genome-specific MIC-3 primer pairs. The clustered distribution may have been favored by selection for responsiveness to evolving disease and/or pest pressures, because large variants of the MIC-3 gene family may have been recovered from small physical areas by recombination. This could give a buffer against selection pressure from a broad range of pest and pathogens in the future. To our knowledge, these are the first results on the evolution of clustering and genome-specific haplotype members of a unique cotton gene family associated with resistant response against a major pathogen.

Development, genetic mapping and QTL association of cotton PHYA , PHYB , and HY5 -specific CAPS and dCAPS markers

The emergence of genome manipulation methods promises a real revolution in biotechnology and genetic engineering. Targeted editing of the genomes of living organisms not only permits investigations into the understanding of the fundamental basis of biological systems but also allows addressing a wide range of goals towards improving productivity and quality of crops. This includes the creation of plants with valuable compositional properties and with traits that confer resistance to various biotic and abiotic stresses. During the past few years, several novel genome editing systems have been developed; these include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These exciting new methods, briefly reviewed herein, have proved themselves as effective and reliable tools for the genetic improvement of plants.

Molecular evolution of the clustered MIC-3 multigene family of Gossypium species

Cotton fiber is an important commodity throughout the world. Fiber property determines fiber quality and commercial values. Previous studies showed that silencing phytochrome A1 gene (PHYA1) by RNA interference in Upland cotton (Gossypium hirsutum L. cv. Coker 312) had generated PHYA1 RNAi lines with simultaneous improvements in fiber quality (longer, stronger and finer fiber) and other key agronomic traits. Characterization of the altered molecular processes in these RNAi genotypes and its wild-type controls is a great interest to better understand the PHYA1 RNAi phenotypes. In this study, a total of 77 conserved miRNAs belonging to 61 families were examined in a PHYA1 RNAi line and its parental Coker 312 genotype by using multiplex sequencing. Of these miRNAs, seven (miR7503, miR7514, miR399c, miR399d, miR160, miR169b, and miR2950) were found to be differentially expressed in PHYA1 RNAi cotton. The target genes of these differentially expressed miRNAs were involved in the metabolism and signaling pathways of phytohormones, which included Gibberellin, Auxin and Abscisic Acid. The expression of several MYB transcription factors was also affected by miRNAs in RNAi cotton. In addition, 35 novel miRNAs (novel miR1-novel miR35) were identified in fibers for the first time in this study. Target genes of vast majority of these novel miRNAs were also predicted. Of these, nine novel miRNAs (novel-miR1, 2, 16, 19, 26, 27, 28, 31 and 32) were targeted to cytochrome P450-like TATA box binding protein (TBP). The qRT-PCR confirmed expression levels of several differentially regulated miRNAs. Expression patterns of four miRNAs-targets pairs were also examined via RNA deep sequencing. Together, the results imply that the regulation of miRNA expression might confer to the phenotype of the PHYA1 RNAi line(s) with improved fiber quality.

QTL mapping for flowering-time and photoperiod insensitivity of cotton Gossypium darwinii Watt

BackgroundAmong SNP markers that become increasingly valuable in molecular breeding of crop plants are the CAPS and dCAPS markers derived from the genes of interest. To date, the number of such gene-based markers is small in polyploid crop plants such as allotetraploid cotton that has A- and D-sub-genomes. The objective of this study was to develop and map new CAPS and dCAPS markers for cotton developmental-regulatory genes that are important in plant breeding programs.ResultsGossypium hirsutum and G. barbadense, are the two cultivated allotetraploid cotton species. These have distinct fiber quality and other agronomic traits. Using comparative sequence analysis of characterized GSTs of the PHYA1, PHYB, and HY5 genes of G. hirsutum and G. barbadense one PHYA1-specific Mbo I/Dpn II CAPS, one PHYB-specific Alu I dCAPS, and one HY5-specific Hinf I dCAPS cotton markers were developed. These markers have successfully differentiated the two allotetraploid genomes (AD1 and AD2) when tested in parental genotypes of ‘Texas Marker-1’ (‘TM-1’), ‘Pima 3–79’ and their F1 hybrids. The genetic mapping and chromosome substitution line-based deletion analyses revealed that PHYA1 gene is located in A-sub-genome chromosome 11, PHYB gene is in A-sub-genome chromosome 10, and HY5 gene is in D-sub-genome chromosome 24, on the reference ‘TM-1’ x ‘Pima 3–79’ RIL genetic map. Further, it was found that genetic linkage map regions containing phytochrome and HY5-specific markers were associated with major fiber quality and flowering time traits in previously published QTL mapping studies.ConclusionThis study detailed the genome mapping of three cotton phytochrome genes with newly developed CAPS and dCAPS markers. The proximity of these loci to fiber quality and other cotton QTL was demonstrated in two A-subgenome and one D-subgenome chromosomes. These candidate gene markers will be valuable for marker-assisted selection (MAS) programs to rapidly introgress G. barbadense phytochromes and/or HY5 gene (s) into G. hirsutum cotton genotypes or vice versa.