A2 Genomes Research Articles

Gossypium Genomics: Trends, Scope, and Utilization for Cotton Improvement.

Cotton (Gossypium spp.) is the most important natural fiber crop worldwide. The diversity of Gossypium species also provides an ideal model for investigating evolution and domestication of polyploids. However, the huge and complex cotton genome hinders genomic research. Technical advances in high-throughput sequencing and bioinformatics analysis have now largely overcome these obstacles, bringing about a new era of cotton genomics. Here, we review recent progress in Gossypium genomics based on whole genome sequencing, resequencing, and comparative genomics, which have provided insights about the genomic basis of fiber biogenesis and the landscape of cotton functional genomics. We address current challenges and present multidisciplinary genomics-enabled breeding strategies covering the breadth of high fiber yield, quality, and environmental resilience for future cotton breeding programs.

Analysis of Genome Grimelysin-Containing Locus in the Genome of Serratia grimesii A2

Serratia grimesii strain A2 was isolated from buffered solution of actin by workers of Institute of Marine Biology, Vladivostok city [1]. This bacterium attracts special interest because of its ability to produce intracellular proteinase grimelysin which hydrolyzes actin in the single site and thus abolishes actin polymerization [1]. In addition to this well-characterized function, grimelysin is also required for invasion of bacteria into eukaryotic cells [1]. Here, we present the analysis of genome sequence of S. grimesii strain A2. The 5133.068 bp draft genome consists of 120 contigs with 4999 coding sequences, including 82 RNA genes. Genome sequencing and annotation revealed the presence of grimelysin gene in the operon with the gene of hypothetical protein of unknown function and the presence of regulatory gene upstream of it. In silico analysis of promoter sequences allowed the identification of putative binding sites for regulatory proteins. We anticipate that the S. grimesii strain A2 genome analysis will advance the current knowledge about grimelysin functions and regulation of its expression.

English

Genus cotton has total 50 species, diploid species fall into 8 genomic groups (A–G, and K). To the extent of our knowledge, frequency of single nucleotide polymorphisms (SNPs) has not been calculated in genes of multiple genomes of the genus Gossypium. Here we present the frequency of SNPs in the transcribed regions of the multiple genes, and their utility in resolving phylogenies among 11 diploid species representing five of the eight diploid genomes of the genus Gossypium. We explored the expressed sequence tags (ESTs) data set of G. arboreum showing homology with genes encoding for mitochondrial small heat shock protein (MT-sHSP), histone H2B1, S-adenosyl methionine synthetase, cytochrome p450, actin-depolymerizing factor 2, C-terminal domain of helicases and histone H2B3 for designing primers. The resultant PCR products amplifying partial gene sequences were sequenced. In total, 61 SNPs were detected in cotton genomes which include 53 substitutions and 8 Indels in the total 1920 bp genome length. Phylogenetic analysis using this data revealed grouping of genomes comparable with previous studies. A1 and A2 genomes were most similar (98.0%) while D9 and Oryza genomes were least similar (31.2%). Oryza was most distantly related with other genomes. In conclusion SNPs are potent markers to delineate cotton genomes according to their evolutionary positions.

Quantitative proteomic analysis of Giardia duodenalis assemblage A: A baseline for host, assemblage, and isolate variation.

Giardia duodenalis is a gastrointestinal protozoan parasite of vertebrates and is a species complex comprised of eight assemblages, with the zoonotic assemblage A one of two subtypes infective for humans. With increasing genomic and transcriptomic data publicly available through the centralized giardiaDB.org, we have quantitatively analyzed the proteomes of eight G. duodenalis assemblage A strains (seven A1 and one A2) to provide a proteomic baseline to complement the available data. A nonredundant total of 1197 subassemblage A1 proteins and 719 subassemblage A2 proteins were identified with an average of 770 proteins in each strain. The eight strains were also searched against both assemblage A genome sequences (subassemblage A1 and A2 genomes) and demonstrated subassemblage specific differences in protein identifications, especially for variable gene families. Substantial differences were observed in the numbers and abundance in the variable surface protein family, and two different variable surface protein expression profiles that were independent of host origin, subassemblage, or geographic origin. We hypothesize that this variation in surface antigen switching events may be related to karotype and chromosomal variation, which would indicate an assemblage-independent mechanism of diversity generation in G. duodenalis. All MS data have been deposited in the ProteomeXchange with identifier PXD001272 (http://proteomecentral.proteomexchange.org/dataset/PXD001272).

Enrichment of an intraspecific genetic map of upland cotton by developing markers using parental RAD sequencing.

RAD sequencing was performed using DH962 and Jimian5 as upland cotton mapping parents. Sequencing data for DH962 and Jimian5 were assembled into the genome sequences of ≈55.27 and ≈57.06 Mb, respectively. Analysing genome sequences of the two parents, 1,323 SSR, 3,838 insertion/deletion (InDel), and 9,366 single-nucleotide polymorphism (SNP) primer pairs were developed. All of the SSRs, 121 InDels, 441 SNPs, and other 6,747 primer pairs were screened in the two parents, and a total of 535 new polymorphic loci were identified. A genetic map including 1,013 loci was constructed using these results and 506 loci previously published for this population. Twenty-seven new QTLs for yield and fibre quality were identified, indicating that the efficiency of QTL detection was greatly improved by the increase in map density. Comparative genomics showed there to be considerable homology and collinearity between the AT and A2 genomes and between the DT and D5 genomes, although there were a few exchanges and introgressions among the chromosomes of the A2 genome. Here, the development of markers using parental RAD sequencing was effective, and a high-density intraspecific genetic map was constructed. This map can be used for molecular marker-assisted selection in cotton.

RNA-Seq transcriptome profiling of upland cotton (Gossypium hirsutum L.) root tissue under water-deficit stress.

An RNA-Seq experiment was performed using field grown well-watered and naturally rain fed cotton plants to identify differentially expressed transcripts under water-deficit stress. Our work constitutes the first application of the newly published diploid D5 Gossypium raimondii sequence in the study of tetraploid AD1 upland cotton RNA-seq transcriptome analysis. A total of 1,530 transcripts were differentially expressed between well-watered and water-deficit stressed root tissues, in patterns that confirm the accuracy of this technique for future studies in cotton genomics. Additionally, putative sequence based genome localization of differentially expressed transcripts detected A2 genome specific gene expression under water-deficit stress. These data will facilitate efforts to understand the complex responses governing transcriptomic regulatory mechanisms and to identify candidate genes that may benefit applied plant breeding programs.

Introgression of Root‐Knot Nematode Resistance into Tetraploid Cottons

Root‐knot nematode (RKN) resistance introgression into tetraploid cotton (Gossypium spp.) and its ancestral genome origin were examined. Resistance sources (‘Acala NemX’, ‘Clevewilt 6’, Auburn 623 RNR) were compared with diverse germplasm using simple sequence repeat (SSR) markers from chromosomes 7, 11, and 14 and DNA sequence information. Differences (P < 0.05) were observed for mean root galling index (GI; scale 0–10) between the 56 resistant and susceptible entries. In resistance sources, GI ranged from 0.3 to 2.9, but no consistent differences were observed at allele‐marker or DNA sequence level. Except for CIR316 allele (206–207 bp) on chromosome 11, no alleles from other SSRs were observed on resistant entries (GI < 3). Allotetraploid G. hirsutum L. (AD1) and G. barbadense L. (AD2) showing the same SSR marker alleles as G. arboreum L. (A2) might suggest resistance introduction from the diploid cotton A2 genome (genetic distance 0.19 to 0.27). However, percent sequence identity from MUCS088 and CIR316 revealed G. barbadense and resistant G. hirsutum (206–207 bp) also were close to diploids G. herbaceum L. (A1), G. thurberi Tod. (D1), and G. trilobum (DC.) Skovst. (D8). Other G. hirsutum DNA sequences were closer to G. raimondii Ulbr. (D5). These analyses indicated resistance introgression into G. hirsutum allotetraploid cottons occurred by artificial hybridization with ancestral genome origin from G. arboreum as well as G. thurberi and not during cotton genome evolution.

Velvet bentgrass (Agrostis canina L.) is the likely ancestral diploid maternal parent of allotetraploid creeping bentgrass (Agrostis stolonifera L.)

Understanding genetic relationships among the three most important Agrostis species will be important in breeding and genomic studies aimed at cultivar improvement. Creeping, colonial, and velvet bentgrasses (Agrostis stolonifera L., A. capillaris L., and A. canina L., respectively) are commercially important turfgrass species often used on golf courses. Velvet bentgrass is a diploid and creeping and colonial bentgrasses are both allotetraploids. A model for the genomic relationships among these species was previously developed from cytological evidence. The genome designations were A1A1 for velvet bentgrass, A1A1A2A2 for colonial bentgrass, and A2A2A3A3 for creeping bentgrass. Here we used phylogenetic analysis based on DNA sequences of nuclear ITS and protein coding genes and the plastid trnK intron and matK gene to reexamine these relationships. In contrast to the previous model, the DNA sequence analysis suggested that velvet bentgrass was closely related to creeping bentgrass and it is likely the maternal parent of creeping bentgrass. Phylogenetic analysis of some conserved nuclear genes revealed a close relationship of the velvet bentgrass sequences with the A2 subgenome sequences of creeping bentgrass. We therefore propose that velvet bentgrass be designated as having the A2 genome, rather than the A1 genome as in the previous model.

Dynamics of the Evolution of the Genus of Agrostis Revealed by GISH/FISH

ABSTRACTGenomic changes in related species undergoing human‐driven and natural selection are important and biologically interesting. The genus Agrostis is an ideal model for studying the evolution of plants since it has not undergone artificial selection for the past thousands of years. Fluorescence in situ hybridization (FISH) or genomic in situ hybridization (GISH) technology is an effective method for analyzing genome homology and organization, detecting genomic rearrangements, and karyotyping based on visual features. In this study, GISH was performed on two tetraploid species, A. capillaris (2n = 4x = 28, A1A1 A2A2) and A. stolonifera (2n = 4x = 28, A2A2 A3A3), using genomic DNA from a putative ancestral diploid species A. canina (2n = 2x = 14, A1A1) as a probe. Genomic in situ hybridization results revealed that the two tetraploid species underwent intergenomic translocations and genomic rearrangements and demonstrated that the A1 genome is highly homologous to the A2 genome. We discovered the phenomenon of concentric parental genome separation in the nucleus of A. stolonifera in which the two parental genomes (A2 and A3) were spatially separated with limited intermixing and were of concentric arrangements. The FISH results showed that the distribution of 45S rDNA loci varied among the three species. The results of our study suggest that substantial genomic alterations occurred during the evolution of the genus of Agrostis

Analysis of EST sequences suggests recent origin of allotetraploid colonial and creeping bentgrasses

Advances in plant genomics have permitted the analysis of several members of the grass family, including the major domesticated species, and provided new insights into the evolution of the major crops on earth. Two members, colonial bentgrass (Agrostis capillaris L.) and creeping bentgrass (A. stolonifera L.) have only recently been domesticated and provide an interesting case of polyploidy and comparison to crops that have undergone human selection for thousands of years. As an initial step of characterizing these genomes, we have sampled roughly 10% of their gene content, thereby also serving as a starting point for the construction of their physical and genetic maps. Sampling mRNA from plants subjected to environmental stress showed a remarkable increase in transcription of transposable elements. Both colonial and creeping bentgrass are allotetraploids and are considered to have one genome in common, designated the A2 genome. Analysis of conserved genes present among the ESTs suggests the colonial and creeping bentgrass A2 genomes diverged from a common ancestor approximately 2.2 million years ago (MYA), thereby providing an enhanced evolutionary zoom in respect to the origin of maize, which formed 4.8 MYA, and tetraploid wheat, which formed only 0.5 MYA and is the progenitor of domesticated hexaploid wheat.

Overexpression of the M2-2 Protein of Respiratory Syncytial Virus Inhibits Viral Replication

The M2-2 protein of respiratory syncytial virus (RSV) is involved in regulation of viral RNA transcription and replication. Encoded by the next-to-last gene of RSV, the M2-2 open reading frame (ORF) overlaps with the upstream M2-1 ORF, suggesting that the production of the M2-2 protein might be tightly regulated during virus replication. To evaluate the effect of M2-2 overexpression on RSV replication, the M2-2 gene was separated from M2-1 by leaving it at the position prior to the M2-1 or moving it to the promoter proximal position as an independent transcriptional unit in the RSV A2 genome. Although recombinant viruses bearing the shuffled M2-2 gene were recovered and expressed higher levels of M2-2, most of these viruses grew poorly in HEp-2 cells. Sequence analysis revealed that various mutations (substitution, insertion, and deletion) occurred in the M2-2 gene, resulting in reduced M2-2 activity as measured by the RSV minigenome system. Further examination of the M2-2 sequence and its function showed that either one of the first two AUG codons located at the 5' end of M2-2 could be used to produce a functional M2-2 protein and that deletion of the first six amino acids from its N terminus or four amino acids from its C terminus greatly reduced its function. The effect of M2-2 protein on RSV replication was also studied by examining RSV replication in cells transiently expressing M2-2. The M2-2 protein expressed at a high level completely inhibited RSV replication. These results strongly suggested that the level of the M2-2 protein produced in the infected cells is critical to RSV replication.

Cloning and expressionanalysis of two cotton (Gossypium hirsutum L.) genes encodingcell wall proline-rich proteins.

Two cotton (Gossypium hirsutum L.) genes, ghprp1 and ghprp2, encoding cell wall proline-rich proteins (PRPs) have been cloned and characterized. The ghprpl gene has an open reading frame (ORF) that encodes a PRP of 299 amino acids (aa), whereas the ghprp2 gene contains an ORF that codes for a 310-aa PRP. The GhPRP1 has an 80% identity in aa sequence with that of GhPRP2. Like other plant cell wall PRPs, both cotton PRPs have a hydrophobic signal peptide at their N-termini, followed by repeating peptide units. Northern blot analyses showed that the ghprpl gene is predominantly expressed in the fiber during the elongation stage of fiber development. Reverse transcription (RT)-PCR analysis showed that ghprpl is expressed in both fiber and root tissues, whereas ghprp2 is in roots only. The ghprpl gene was shown to be present in the A1, A2, D1 and D5 genomes of Gossypium by PCR amplification, whereas the ghprp2 gene is only present in the A1 and A2 genomes. The ghprpl gene was over-expressed in the yeast Pichia pastoris, and the expressed GhPRP1 protein was used as an antigen to raise polyclonal antibodies (anti-GhPRP1). Western analysis using the anti-GhPRP1 probe detected a major protein band (50 kDa) in 5-31-day postanthesis (DPA) fibers. However, the 50 kDa protein was absent in other cotton tissues.

The Site-Specific Recombination System of theLactobacillusSpecies Bacteriophage A2 Integrates in Gram-Positive and Gram-Negative Bacteria

The region of the bacteriophage A2 genome involved in site-specific recombination with the DNA ofLactobacillusspp. has been identified. Twoorfs, transcribed from the same strand, have been found immediately upstream of the phage attachment site (attP). Theorfadjacent toattPpredicts a 385-amino-acid protein that presents significant similarity with site-specific recombinases of the integrase family. The otherorfencodes a basic polypeptide of 76 amino acid residues. The junctions of the prophage with the genomes of its hosts have been determined, allowing the identification of the host attachment site (attB), which has a common 19-nucleotide core region withattP. TheattBsite is located at the 3′ end of the transfer RNALeugene (anticodon CAA). Nonreplicative plasmids containing the A2-specific recombination cassette integrate into different lactobacilli but also into unrelated Gram-positive bacteria such asLactococcus lactisand even intoEscherichia coli. InLc. lactis, integration occurs in a previously unknown intergenic region, whereas inE. coli, it maps within therrnDoperon, 5′ ofrrsDgene. Comparison of the integration sites in the different hosts indicates that some flexibility is permitted in theattBsequence, sinceLc. lactisandE. colionly share 13 and 11 nucleotides, respectively, with the 19-nucleotide core sequence of the lactobacilli.