Expression of a phenylcoumaran benzylic ether reductase-like protein in the ovules of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to identify differentially expressed proteins in wild-type (DP 5690) and fiberless (SL 1-7-1) cotton ovules. One protein, designated V2 was unique to ovules of the fiber producing DP 5690 line. The protein was purified from 2D-PAGE of 4 d post anthesis DP 5690 ovules and partially sequenced. The short amino acid sequence was nearly identical to the deduced amino acid sequence for cotton phenylcoumaran benzylic ether reductase (PCBER) protein. A consensus sequence was assembled from ESTs encoding cotton PCBER genes, primers were designed, and a full length gene was amplified from plasmid DNA from a 72 h etiolated cotton cotyledon library. The polymerase chain reaction generated a 950 bp product with unique EcoRI (5′) and (3′) KpnI restriction sites for directional insertion into the expression vector pPICZA. Nucleotide sequencing was performed, and the full length coding region was 924 bp encoding a protein of 308 amino acids. The molecular mass and pI measured (2D PAGE) were similar to the theoretical protein.     

Next generation genetic mapping of the Ligon-lintless-2 (<Emphasis Type="Italic">Li</Emphasis><Subscript>2</Subscript>) locus in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Key message

Mapping-by-sequencing and novel subgenome-specific SNP markers were used to fine map the Ligon-lintless 2 ( Li ₂ ) short-fiber gene in tetraploid cotton. These methodologies will accelerate gene identification in polyploid species.

Abstract

Next generation sequencing offers new ways to identify the genetic mechanisms that underlie mutant phenotypes. The release of a reference diploid Gossypium raimondii (D₅) genome and bioinformatics tools to sort tetraploid reads into subgenomes has brought cotton genetic mapping into the genomics era. We used multiple high-throughput sequencing approaches to identify the relevant region of reference sequence and identify single nucleotide polymorphisms (SNPs) near the short-fiber mutant Ligon-lintless 2 (Li ₂) gene locus. First, we performed RNAseq on 8-day post-anthesis (DPA) fiber cells from the Li ₂ mutant and its wild type near isogenic line (NIL) Gossypium hirsutum cv. DP5690. We aligned sequence reads to the D₅ genome, sorted the reads into A and D subgenomes with PolyCat and called SNPs with InterSNP. We then identified SNPs that would result in non-synonymous substitutions to amino acid sequences of annotated genes. This step allowed us to identify a 1-Mb region with 24 non-synonymous SNPs, representing the introgressed region that differentiates Li ₂ from its NIL. Next, we sequenced total DNA from pools of F₂ plants, using a super bulked segregant analysis sequencing (sBSAseq) approach. The sBSAseq predicted 82 non-synonymous SNPs among 3,494 SNPs in a 3-Mb region that includes the region identified by RNAseq. We designed subgenome-specific SNP markers and tested them in an F₂ population of 1,733 individuals to construct a genetic map. Our resulting genetic interval contains only one gene, an aquaporin, which is highly expressed in wild-type fibers and is significantly under-expressed in elongating Li ₂ fiber cells.

     

A CMS-Related Gene, Ψatp6-2, Causes Increased ATP Hydrolysis Activity of the Mitochondrial F1Fo-ATP Synthase and Induces Male Sterility in Pepper (Capsicum annuum L.)

Pollen formation is a complex process that is strictly controlled by genetic factors. Although many novel mitochondrial genes have been implicated in the dysfunction of mitochondrial enzymes and the cytoplasmic male sterility (CMS), there is little empirical evidence to show that CMS-related genes actually result in the dysfunction of enzyme and little is known about the regulatory mechanisms of the aberrant mitochondrial enzymes in male sterility in CMS lines. Here, we report the characterization of a novel mitochondrial gene, Ψatp6-2, which is hypothesized to play a role in male sterility in pepper. Using virus-induced gene silencing (VIGS), we observed that silencing the atp6-2 gene in the maintainer line resulted in an increase in ATP hydrolysis activity of the mitochondrial F₁F_o-ATP synthase along with pollen abortion, while silencing the truncated Ψatp6-2 gene in the CMS line resulted in an inhibition of ATP hydrolysis activity and restoration of fertility. Altered ATP hydrolysis also affected the tolerance of the gene-silenced plants to abiotic stresses. Localization experiments showed that premature ATP hydrolysis occurred at the tetrad stage of pollen development in the CMS line, but no ATPase activity was observed in the microspores at the later stage. These results suggest that the Ψatp6-2 gene causes the alteration in ATP hydrolysis activity of the mitochondrial F₁F_o-ATP synthase during pollen development, which eventually leads to male sterility in pepper.     

Developing Fiber Specific Promoter-Reporter Transgenic Lines to Study the Effect of Abiotic Stresses on Fiber Development in Cotton

Cotton is one of the most important cash crops in US agricultural industry. Environmental stresses, such as drought, high temperature and combination of both, not only reduce the overall growth of cotton plants, but also greatly decrease cotton lint yield and fiber quality. The impact of environmental stresses on fiber development is poorly understood due to technical difficulties associated with the study of developing fiber tissues and lack of genetic materials to study fiber development. To address this important question and provide the need for scientific community, we have generated transgenic cotton lines harboring cotton fiber specific promoter (CFSP)-reporter constructs from six cotton fiber specific genes (Expansin, E6, Rac13, CelA1, LTP, and Fb late), representing genes that are expressed at different stages of fiber development. Individual CFSP::GUS or CFSP::GFP construct was introduced into Coker 312 via Agrobacterium mediated transformation. Transgenic cotton lines were evaluated phenotypically and screened for the presence of selectable marker, reporter gene expression, and insertion numbers. Quantitative analysis showed that the patterns of GUS reporter gene activity during fiber development in transgenic cotton lines were similar to those of the native genes. Greenhouse drought and heat stress study showed a correlation between the decrease in promoter activities and decrease in fiber length, increase in micronaire and changes in other fiber quality traits in transgenic lines grown under stressed condition. These newly developed materials provide new molecular tools for studying the effects of abiotic stresses on fiber development and may be used in study of cotton fiber development genes and eventually in the genetic manipulation of fiber quality.     

Discovery and identification of a novel Ligon lintless-like mutant (Lix) similar to the Ligon lintless (Li1) in allotetraploid cotton

Mutants are a powerful resource for studying gene structure, function, and evolution. In this present study, a novel Ligon lintless-like mutant (Lix), that has short fibers and deformed leaves and stems, was isolated from the progeny of transgenic cottons. The Lix mutant is similar in morphology to the Ligon lintless (Li₁) mutant. Genetic analysis and molecular mapping were performed for the Lix and Li₁ mutants. These two mutants are monogenic dominant mutants with distorted growth of vegetative and reproductive structures. Seedlings of the dominant homozygote Li ₁ Li ₁ genotype are lethal, while LixLix plants are viable but show no reproductive growth. Molecular tagging showed that the Lix gene is located on Chr. 04 in a 30.9-cM region spanned by NAU8376 and NAU3469. In a previous study, the Li ₁ gene was mapped to Chr. 22, and Chr. 04 and Chr. 22 are homoelogous chromosomes in tetraploid cotton. So, we propose that Lix and Li₁ mutants have similar mutated morphology, and Lix is mapped to a homoelogous chromosome carrying Li ₁ . The identification and genetic mapping of Lix/Li ₁ genes using mutants provides a foundation for isolating these genes. In turn, this will permit studies to elucidate the functional and evolutionary roles for these genes in cotton growth and development.