A-genome Species Research Articles

A de novo genome assembly of Solanum bulbocastanum Dun., a Mexican diploid species reproductively isolated from the A-genome species, including cultivated potatoes.

Potato and its wild relatives are distributed mainly in the Mexican highlands and central Andes of South America. The South American A-genome species, including cultivated potatoes, are reproductively isolated from Mexican diploid species. Whole-genome sequencing has disclosed genome structure and similarity, mostly in cultivated potatoes and their closely related species. In this study, we generated a chromosome-scale assembly of the genome of a Mexican diploid species, Solanum bulbocastanum Dun., using PacBio long-read sequencing, optical mapping, and Hi-C scaffolding technologies. The final sequence assembly consisted of 737.9 Mb, among which 647.0 Mb were anchored to the 12 chromosomes. Compared with chromosome-scale assemblies of S. lycopersicum (tomato), S. etuberosum (non-tuber-bearing species with E-genome), S. verrucosum, S. chacoense, S. multidissectum, and S. phureja (all four are A-genome species), the S. bulbocastnum genome was the shortest. It contained fewer transposable elements (56.2%) than A-genome species. A cluster analysis was performed based on pairwise ratios of syntenic regions among the seven chromosome-scale assemblies, showing that the A-genome species were first clustered as a distinct group. Then, this group was clustered with S. bulbocastanum. Sequence similarity in 1,624 single-copy orthologous gene groups among 36 Solanum species and clones separated S. bulbocastanum as a specific group, including other Mexican diploid species, from the A-genome species. Therefore, the S. bulbocastanum genome differs in genome structure and gene sequences from the A-genome species. These findings provide important insights into understanding and utilizing the genetic diversity of S. bulbocastanum and the other Mexican diploid species in potato breeding.

Autotetraploid Induction of Three A-Genome Wild Peanut Species, Arachis cardenasii, A. correntina, and A. diogoi.

A-genome Arachis species (AA; 2n = 2x = 20) are commonly used as secondary germplasm sources in cultivated peanut breeding, Arachis hypogaea L. (AABB; 2n = 4x = 40), for the introgression of various biotic and abiotic stress resistance genes. Genome doubling is critical to overcoming the hybridization barrier of infertility that arises from ploidy-level differences between wild germplasm and cultivated peanuts. To develop improved genome doubling methods, four trials of various concentrations of the mitotic inhibitor treatments colchicine, oryzalin, and trifluralin were tested on the seedlings and seeds of three A-genome species, A. cardenasii, A. correntina, and A. diogoi. A total of 494 seeds/seedlings were treated in the present four trials, with trials 1 to 3 including different concentrations of the three chemical treatments on seedlings, and trial 4 focusing on the treatment period of 5 mM colchicine solution treatment of seeds. A small number of tetraploids were produced from the colchicine and oryzalin gel treatments of seedlings, but all these tetraploid seedlings reverted to diploid or mixoploid states within six months of treatment. In contrast, the 6-h colchicine solution treatment of seeds showed the highest tetraploid conversion rate (6-13% of total treated seeds or 25-40% of surviving seedlings), and the tetraploid plants were repeatedly tested as stable tetraploids. In addition, visibly and statistically larger leaves and flowers were produced by the tetraploid versions of these three species compared to their diploid versions. As a result, stable tetraploid plants of each A-genome species were produced, and a 5 mM colchicine seed treatment is recommended for A-genome and related wild Arachis species genome doubling.

Inter-Specific Hybridization in Cotton (Gossypium hirsutum) for Crop Improvement

Interspecific hybridization has contributed significantly to land diversity, species evolution, and crops’ domestication, including upland cotton, the cultivated form of Gossypium hirsutum. Being the world’s most important fiber crop species, Gossypium hirsutum belongs to the allotetraploid Gossypium consisting of six additional tetraploid species. The lint fiber evolved once in diploid parent A-genome species in the Gossypium’s history and passed on during hybridization of the A-genome with the D-genome and was maintained in subsequent evolution. The domestication history of G. hirsutum involved the collection and use of lint fibers by indigenous people for the purpose of making strings and other textile products; hence, spinnable lint fibers were likely to have evolved under domestication. Crossing with G. barbadense has resulted in the development of multiple genetic lines in contemporary upland cotton. However, in later-generation hybrids between G. hirsutum and other polyploid species, reproductive barriers such as reduced fertility, segregation distortion, and hybrid breakdown are frequently observed, complicating the task of introgressing new, stably inherited allelic variation from inter-specific hybridization. Recent efforts in molecular genetics research have provided insights into the location and effects of QTLs from wild species that are associated with traits important to cotton production. These and future research efforts will undoubtedly provide the tools that can be utilized by plant breeders to access novel genes from wild and domesticated allotetraploid Gossypium for upland cotton improvement.

A de novo genome assembly of Solanum verrucosum Schlechtendal, a Mexican diploid species geographically isolated from other diploid A-genome species of potato relatives.

There are over 100 known species of cultivated potatoes and their wild relatives. Many of these species, including cultivated potatoes, share the A genome; these species are mainly distributed in South America and are reproductively isolated from Mexican diploid species. The only diploid A-genome species distributed in Mexico is Solanum verrucosum Schlechtendal, which is also a maternal progenitor of Mexican polyploid species. In this study, we constructed a high-quality de novo assembly of the S. verrucosum genome using PacBio long-read sequencing and Hi-C scaffolding technologies. A monohaploid clone (2n = x = 12) of S. verrucosum was used to reduce assembly difficulty due to the heterozygous nature of the species. The final sequence assembly consisted of 780.2 Mb of sequence, 684.0 Mb of which were anchored to the 12 chromosomes, with a scaffold N50 of 55.2 Mb. Putative centromeres were identified using publicly available data obtained via chromatin immunoprecipitation sequencing against a centromere-specific histone 3 protein. Transposable elements accounted for approximately 61.8% (482.1 Mb) of the genome, and 46,904 genes were functionally annotated. High gene synteny and similarity were revealed among the genomes of S. verrucosum, Solanum commersonii, Solanum chacoense, Solanum phureja, Solanum tuberosum, and Solanum lycopersicum. The reference-quality S. verrucosum genome will provide new insights into the evolution of Mexican polyploid species and contribute to potato breeding programs.

New Insights into the Genomic Structure of Avena L.: Comparison of the Divergence of A-Genome and One C-Genome Oat Species.

We used next-generation sequencing analysis of the 3′-part of 18S rDNA, ITS1, and a 5′-part of the 5.8S rDNA region to understand genetic variation among seven diploid A-genome Avena species. We used 4–49 accessions per species that represented the As genome (A. atlantica, A. hirtula, and wiestii), Ac genome (A. canariensis), Ad genome (A. damascena), Al genome (A. longiglumis), and Ap genome (A. prostrata). We also took into our analysis one C-genome species, A. clauda, which previously was found to be related to A-genome species. The sequences of 169 accessions revealed 156 haplotypes of which seven haplotypes were shared by two to five species. We found 16 ribotypes that consisted of a unique sequence with a characteristic pattern of single nucleotide polymorphisms and deletions. The number of ribotypes per species varied from one in A. longiglumis to four in A. wiestii. Although most ribotypes were species-specific, we found two ribotypes shared by three species (one for A. damascena, A. hirtula, and A. wiestii, and the second for A. longiglumis, A. atlantica, and A. wiestii), and a third ribotype shared between A. atlantica and A. wiestii. A characteristic feature of the A. clauda ribotype, a diploid C-genome species, is that two different families of ribotypes have been found in this species. Some of these ribotypes are characteristic of Cc-genome species, whereas others are closely related to As-genome ribotypes. This means that A. clauda can be a hybrid between As- and C-genome oats.

Genetic characterization and curation of diploid A-genome wheat species.

A-genome diploid wheats represent the earliest domesticated and cultivated wheat species in the Fertile Crescent and include the donor of the wheat A sub-genome. The A-genome species encompass the cultivated einkorn (Triticum monococcum L. subsp. monococcum), wild einkorn (T. monococcum L. subsp. aegilopoides (Link) Thell.), and Triticum urartu. We evaluated the collection of 930 accessions in the Wheat Genetics Resource Center (WGRC) using genotyping by sequencing and identified 13,860 curated single-nucleotide polymorphisms. Genomic analysis detected misclassified and genetically identical (>99%) accessions, with most of the identical accessions originating from the same or nearby locations. About 56% (n = 520) of the WGRC A-genome species collections were genetically identical, supporting the need for genomic characterization for effective curation and maintenance of these collections. Population structure analysis confirmed the morphology-based classifications of the accessions and reflected the species geographic distributions. We also showed that T. urartu is the closest A-genome diploid to the A-subgenome in common wheat (Triticum aestivum L.) through phylogenetic analysis. Population analysis within the wild einkorn group showed three genetically distinct clusters, which corresponded with wild einkorn races α, β, and γ described previously. The T. monococcum genome-wide FST scan identified candidate genomic regions harboring a domestication selection signature at the Non-brittle rachis 1 (Btr1) locus on the short arm of chromosome 3Am at ∼70 Mb. We established an A-genome core set (79 accessions) based on allelic diversity, geographical distribution, and available phenotypic data. The individual species core set maintained at least 79% of allelic variants in the A-genome collection and constituted a valuable genetic resource to improve wheat and domesticated einkorn in breeding programs.

Comparative Functional Studies on Two Diploid Cotton Genomes Reveals Functional Differences of Basic Helix-loop-helix Proteins in Arabidopsis Trichome Initiation

Background: The cultivated tetraploid cotton species (AD genomes) was originated from two ancestral diploid species (A and D genomes). While the ancestral A-genome species produce spinnable fibers, the D- genome species do not. Cotton fibers are unicellular trichomes originating from seed coat epidermal cells, and currently there is an immense interest in understanding the process of fiber initiation and development. Current knowledge demonstrates that there is a great of deal of resemblance in initiation mechanism between by Arabidopsis trichome and cotton fiber.
 Methodology: In this study, we performed comparative functional studies between A genome and D-genome species in cotton by using Arabidopsis trichome initiation as a model. Four cotton genes TTG3, MYB2, DEL61 and DEL65 were amplified from A-genome and D-genome species, and transformed into their homolog trichomeless mutants Arabidopsis ttg1, gl1, and gl3egl3, respectively.
 Results: Our data indicated that the transgenic plants expressing TTG3 and MYB2 genes from A-genome and D-genome species complement the ttg1 and gl1 mutants, respectively. We also discovered complete absences of two functional basic helix loop helix (bHLH) proteins (DEL65/DEL61) in D- diploid species and one (DEL65) that is functional in A-genome species, but not from D-genome species. This observation is consistent with the natural phenomenon of spinnable fiber production in A- genome species and absence in D-genome species.

Comparative studies on seed protein characteristics in eight lines of two Gossypium species

BackgroundIn order to achieve the targets aiming at the improvement of protein quality, knowledge regarding seed protein fractions and polypeptides constituting them in different crops is essential. Besides having high nutritional value as animal feed and human food, the protein isolates from cottonseed meal have also been proven promising as industrial raw materials for a number of applications. As far as Indian work on the characterization of cotton seed proteins is concerned, relatively meagre reports are available. Keeping in mind the importance of cotton seed proteins, lines belonging to Gossypium arboreum L. (Indian cotton) and G. hirsutum L. (American cotton) which are grown in all the major cotton growing states in India were selected for analysing their seed protein characteristics.ResultsWhereas G. arboreum (A-genome) lines revealed a lower range of seed protein content i.e. 19.5~24.3%, an upper range (21.8~29.5%) could be observed in lines of G. hirsutum (AD-genome). Globulins represented dominating fraction in both species followed by albumins, glutelins and prolamins. A significant positive correlation between albumins/globulins and seed protein content in G. arboreum /G. hirsutum, respectively, was observed. Intraspecific electrophoretic variation in seed protein extracts was observed in the region of molecular weight 22 kDa - 27 kDa in lines of both the species; however some lines with A-genome showed similarity in banding pattern with AD-genome. Four polypeptides with disulphide-linkages were also reported for the first time. Albumins were observed to reveal more variations in their electrophoretic pattern between the lines of two species followed by globulins.ConclusionOn the basis of present and previous studies, screening the lines with low or high protein content will lead the selection of lines with superior polypeptide fraction important for nutritional and industrial purposes. On comparing the composition and behaviour of four 2-S linked polypeptides with other plant groups, these were suggested to be legumin-like in nature. The similarity in banding patterns between the lines of A-genome and AD-genome species marked towards the close evolutionary relationship between these two. Albumin fractions on the basis of our results could be taken for cultivar differentiation in cotton crop.

Comparative effect of allopolyploidy on transposable element composition and gene expression between Gossypium hirsutum and its two diploid progenitors.

An allopolyploidization event formed allotetraploid Gossypium species from an A-genome diploid species and a D-genome diploid species. To explore the responses of transposable elements (TEs) to allopolyploidy, we assembled parallel TE datasets from G. hirsutum, G. arboreum and G. raimondii and analyzed the TE types and the effects of TEs on orthologous gene expression in the three Gossypium genomes. Gypsy was the most abundant TE type and most TEs were located ∼500 bp from genes in all three genomes. In G. hirsutum, 35.6% of genes harbored TE insertions, whereas insertions were more frequent in G. arboreum and G. raimondii. G. hirsutum had the highest proportion of uniquely matching 24-nt small interfering RNAs (siRNAs) that targeted TEs. TEs, particularly those targeted by 24-nt siRNAs, were associated with reduced gene expression, but the effect of TEs on orthologous gene expression varied substantially among species. Orthologous gene expression levels in G. hirsutum were intermediate between those of G. arboreum and G. raimondii, which did not experience TE expansion or reduction resulting from allopolyploidization. This study underscores the diversity of TEs co-opted by host genes and provides insights into the roles of TEs in regulating gene expression in Gossypium.

Rapid proliferation and nucleolar organizer targeting centromeric retrotransposons in cotton

Centromeric chromatin in most eukaryotes is composed of highly repetitive centromeric retrotransposons and satellite repeats that are highly variable even among closely related species. The evolutionary mechanisms that underlie the rapid evolution of centromeric repeats remain unknown. To obtain insight into the evolution of centromeric repeats following polyploidy, we studied a model diploid progenitor (Gossypium raimondii, D-genome) of the allopolyploid (AD-genome) cottons, G.hirsutum and G.barbadense. Sequence analysis of chromatin-immunoprecipitated DNA showed that the G.raimondii centromeric repeats originated from retrotransposon-related sequences. Comparative analysis showed that nine of the 10 analyzed centromeric repeats were absent from the centromeres in the A-genome and related diploid species (B-, F- and G-genomes), indicating that they colonized the centromeres of D-genome lineage after the divergence of the A- and D- ancestral species or that they were ancestrally retained prior to the origin of Gossypium. Notably, six of the nine repeats were present in both the A- and D-subgenomes in tetraploid G.hirsutum, and increased in abundance in both subgenomes. This finding suggests that centromeric repeats may spread and proliferate between genomes subsequent to polyploidization. Two repeats, Gr334 and Gr359 occurred in both the centromeres and nucleolar organizer regions (NORs) in D- and AD-genome species, yet localized to just the NORs in A-, B-, F-, and G-genome species. Contained within is a story of an established centromeric repeat that is eliminated and allopolyploidization provides an opportunity for reinvasion and reestablishment, which broadens our evolutionary understanding behind the cycles of centromeric repeat establishment and targeting.

The starch-bound alpha-amylase/trypsin-inhibitors in Avena.

Oat kernels exhibit an extra-soft texture, a trait recently demonstrated to be largely modulated by starch-bound tryptophan-rich 2S proteins, the vromindolines. In this study, fractionation by two-dimensional electrophoresis of starch-bound proteins in 25 oat (Avena sativa) cultivars and 11 diploid or tetraploid Avena species revealed novel 2S proteins called Avena α-amylase/trypsin-inhibitors (AATI) because of their sequence similarity with wheat α-amylase/trypsin inhibitors. Thirty-seven AATI polypeptides, about 14kDa in size, were split into three families named AATI-1, AATI-2, and AATI-3 with different primary structures and isoelectric points. AATI-1 and AATI-2 proteins showed 55.5-60.0% sequence similarity with wheat α-amylase inhibitors CM1, CM2, and CM16, which have been found to cause innate immunity responses in celiac disease and non-celiac gluten sensitivity. Diploid A-genome and tetraploid AC-genome oat species possess three and five genes encoding for the AATI proteins, respectively, whereas hexaploid A. sativa exhibits 12 genes dispersed over the A-, C-, and D-genomes. Some AATI proteins expressed in hexaploid oats were assigned to the A-genome based on similarity to their counterparts in diploid species, contributing to further clarify the genetic origin of hexaploid oats. Moreover, AATI may interact with starch-bound vromindolines in determining the extra-soft texture of oat kernels and, due to their balanced amino acid compositions, may contribute to the biological value of oat proteins in a positive manner.

Diversification of the Homoeologous Lr34 Sequences in Polyploid Wheat Species and Their Diploid Progenitors.

Allopolyploidization induces a multiple processes of genomic reorganization, including the structurally functional diversification of the homoeologous genes. An example of such diversification is the appearance of the Lr34 gene on chromosome 7D of bread wheat T. aestivum (BAD), the gene conferring durable, race non-specific protection against three fungal pathogens. In this study, we focused on the variability of a functionally critical region between exons 10-12 of Lr34 among diploid progenitors of wheat genomes and their respective polyploids. In the diploid A-genome species, two basic forms of the studied region have been revealed: (1) non-functional forms containing stop codons, or/and frameshifts (T. monococcum/T. urartu) and (2) forms with no such a mutations (T. boeoticum). The Lr34 sequence of T. urartu containing a TGA stop codon was inherited by the first tetraploid T. dicoccoides (BA), and then reorganized in some accessions of this species due to the insertion of an LTR retroelement in exon 10. Besides T. boeoticum, the second form of the Lr34 sequence is also characteristic of A. speltoides, which presumably donated this form to all polyploid descendants bearing B-genome. No differences were found between the D-genome-specific Lr34 sequences studied here and downloaded from databases, implying the highest level of conservation of the Lr34 predecessor throughout evolution. The sequence data were later used to construct phylograms, and apparent peculiarities in the evolution of the studied region of Lr34 genes discussed.

Successful crosses between fungal-resistant wild species of Arachis (section Arachis) and Arachis hypogaea.

Peanut (Arachis hypogaea) is the fifth most produced oil crop worldwide. Besides lack of water, fungal diseases are the most limiting factors for the crop. Several species of Arachis are resistant to certain pests and diseases. This study aimed to successfully cross the A-genome with B-K-A genome wild species previously selected for fungal disease resistance, but that are still untested. We also aimed to polyplodize the amphihaploid chromosomes; cross the synthetic amphidiploids and A. hypogaea to introgress disease resistance genes into the cultivated peanut; and analyze pollen viability and morphological descriptors for all progenies and their parents. We selected 12 A-genome accessions as male parents and three B-genome species, one K-genome species, and one A-genome species as female parents. Of the 26 distinct cross combinations, 13 different interspecific AB-genome and three AA-genome hybrids were obtained. These sterile hybrids were polyploidized and five combinations produced tetraploid flowers. Next, 16 combinations were crossed between A. hypogaea and the synthetic amphidiploids, resulting in 11 different hybrid combinations. Our results confirm that it is possible to introgress resistance genes from wild species into the peanut using artificial hybridization, and that more species than previously reported can be used, thus enhancing the genetic variability in peanut genetic improvement programs.

Cytoplasmic diversity of the cotton genus as revealed by chloroplast microsatellite markers

The diversity of chloroplast genomes has played an important role, as have those of nuclear and mitochondrial genomes, in the evolution of plants. The sequences of the chloroplast genome supply unsub- stituted information for genome analysis. In order to understand the genetic differentiation and relationship of cotton species, we investigated the cytoplasmic diversity of chloroplast genomes in 41 Gossypium accessions with 75 chloroplast simple sequence repeat (cpSSR) markers. The markers were developed from reference sequences of the chloroplast genomes of G. hirsutum and G. barbadense and covered approx- imately 12.6 kb. Among the 75 markers, 50 were polymorphic, with polymorphism information content values ranging from 0.11 to 0.88. Analyses of the dataset demonstrated that single copy regions were much more informative than inverted repeat regions. The non-coding sequences were well differentiated among these species. For some common cpDNA haplotypes, the E-genome species that may be the oldest of the extant cotton species was deduced. The differentiation of A-genome species lagged behind that of AD-genome species. Neither G. herbaceum nor G. arboreum was the cytoplasmic donor of tetraploid species, strongly suggesting that AD genomes origi- nated from an extinct ancestor of modern A-genome species. We speculate that the genetic differentiation of the chloroplast genome of each cotton species resulted from the dispersal of that species and its adaptations to local ecological conditions. These cpSSR markers provided valuable information to reveal the diversity and differentiation of cotton during evolution.

Single-copy gene fluorescence in situ hybridization and genome analysis: Acc-2 loci mark evolutionary chromosomal rearrangements in wheat

Fluorescence in situ hybridization (FISH) is a useful tool for physical mapping of chromosomes and studying evolutionary chromosome rearrangements. Here we report a robust method for single-copy gene FISH for wheat. FISH probes were developed from cDNA of cytosolic acetyl-CoA carboxylase (ACCase) gene (Acc-2) and mapped on chromosomes of bread wheat, Triticum aestivum L. (2n = 6x = 42, AABBDD), and related diploid and tetraploid species. Another nine full-length (FL) cDNA FISH probes were mapped and used to identify chromosomes of wheat species. The Acc-2 probe was detected on the long arms of each of the homoeologous group 3 chromosomes (3A, 3B, and 3D), on 5DL and 4AL of bread wheat, and on homoeologous and nonhomoeologous chromosomes of other species. In the species tested, FISH detected more Acc-2 gene or pseudogene sites than previously found by PCR and Southern hybridization analyses and showed presence/absence polymorphism of Acc-2 sequences. FISH with the Acc-2 probe revealed the 4A-5A translocation, shared by several related diploid and polyploid species and inherited from an ancestral A-genome species, and the T. timopheevii-specific 4A(t)-3A(t) translocation.

A high-density genetic map of Arachis duranensis, a diploid ancestor of cultivated peanut

BackgroundCultivated peanut (Arachis hypogaea) is an allotetraploid species whose ancestral genomes are most likely derived from the A-genome species, A. duranensis, and the B-genome species, A. ipaensis. The very recent (several millennia) evolutionary origin of A. hypogaea has imposed a bottleneck for allelic and phenotypic diversity within the cultigen. However, wild diploid relatives are a rich source of alleles that could be used for crop improvement and their simpler genomes can be more easily analyzed while providing insight into the structure of the allotetraploid peanut genome. The objective of this research was to establish a high-density genetic map of the diploid species A. duranensis based on de novo generated EST databases. Arachis duranensis was chosen for mapping because it is the A-genome progenitor of cultivated peanut and also in order to circumvent the confounding effects of gene duplication associated with allopolyploidy in A. hypogaea.ResultsMore than one million expressed sequence tag (EST) sequences generated from normalized cDNA libraries of A. duranensis were assembled into 81,116 unique transcripts. Mining this dataset, 1236 EST-SNP markers were developed between two A. duranensis accessions, PI 475887 and Grif 15036. An additional 300 SNP markers also were developed from genomic sequences representing conserved legume orthologs. Of the 1536 SNP markers, 1054 were placed on a genetic map. In addition, 598 EST-SSR markers identified in A. hypogaea assemblies were included in the map along with 37 disease resistance gene candidate (RGC) and 35 other previously published markers. In total, 1724 markers spanning 1081.3 cM over 10 linkage groups were mapped. Gene sequences that provided mapped markers were annotated using similarity searches in three different databases, and gene ontology descriptions were determined using the Medicago Gene Atlas and TAIR databases. Synteny analysis between A. duranensis, Medicago and Glycine revealed significant stretches of conserved gene clusters spread across the peanut genome. A higher level of colinearity was detected between A. duranensis and Glycine than with Medicago.ConclusionsThe first high-density, gene-based linkage map for A. duranensis was generated that can serve as a reference map for both wild and cultivated Arachis species. The markers developed here are valuable resources for the peanut, and more broadly, to the legume research community. The A-genome map will have utility for fine mapping in other peanut species and has already had application for mapping a nematode resistance gene that was introgressed into A. hypogaea from A. cardenasii.

Analysis of complete nucleotide sequences of 12 Gossypium chloroplast genomes: origin and evolution of allotetraploids.

BackgroundCotton (Gossypium spp.) is a model system for the analysis of polyploidization. Although ascertaining the donor species of allotetraploid cotton has been intensively studied, sequence comparison of Gossypium chloroplast genomes is still of interest to understand the mechanisms underlining the evolution of Gossypium allotetraploids, while it is generally accepted that the parents were A- and D-genome containing species. Here we performed a comparative analysis of 13 Gossypium chloroplast genomes, twelve of which are presented here for the first time.Methodology/Principal FindingsThe size of 12 chloroplast genomes under study varied from 159,959 bp to 160,433 bp. The chromosomes were highly similar having >98% sequence identity. They encoded the same set of 112 unique genes which occurred in a uniform order with only slightly different boundary junctions. Divergence due to indels as well as substitutions was examined separately for genome, coding and noncoding sequences. The genome divergence was estimated as 0.374% to 0.583% between allotetraploid species and A-genome, and 0.159% to 0.454% within allotetraploids. Forty protein-coding genes were completely identical at the protein level, and 20 intergenic sequences were completely conserved. The 9 allotetraploids shared 5 insertions and 9 deletions in whole genome, and 7-bp substitutions in protein-coding genes. The phylogenetic tree confirmed a close relationship between allotetraploids and the ancestor of A-genome, and the allotetraploids were divided into four separate groups. Progenitor allotetraploid cotton originated 0.43–0.68 million years ago (MYA).ConclusionDespite high degree of conservation between the Gossypium chloroplast genomes, sequence variations among species could still be detected. Gossypium chloroplast genomes preferred for 5-bp indels and 1–3-bp indels are mainly attributed to the SSR polymorphisms. This study supports that the common ancestor of diploid A-genome species in Gossypium is the maternal source of extant allotetraploid species and allotetraploids have a monophyletic origin. G. hirsutum AD1 lineages have experienced more sequence variations than other allotetraploids in intergenic regions. The available complete nucleotide sequences of 12 Gossypium chloroplast genomes should facilitate studies to uncover the molecular mechanisms of compartmental co-evolution and speciation of Gossypium allotetraploids.

Assessing the monophyly of polyploid Gossypium species

The origin and monophyly of the polyploid cotton (Gossypium) species has been largely accepted, despite the lack of explicit phylogenetic evidence. Recent studies in other polyploid systems have demonstrated that multiple origins for polyploid species are much more common than once thought, raising the possibility that Gossypium polyploids also had multiple origins, as postulated by some authors. To test the monophyly of polyploid cotton, we sequenced a 2.8-kb intergenic region from all diploid species belonging to the genome groups from which the polyploid originates. The resulting phylogenetic analyses strongly support a single origin of polyploid cotton involving a D-genome ancestor related to Gossypium raimondii and an A-genome ancestor that was sister to both extant A-genome species.

Diversity of the Ty-1 copia retrotransposon Tos17 in rice (Oryza sativa L.) and the AA genome of the Oryza genus

Retrotransposons are mobile genetic elements, ubiquitous in Eukaryotic genomes, which have proven to be major genetic tools in determining phylogeny and structuring genetic diversity, notably in plants. We investigate here the diversity of the Ty1-copia retrotransposon Tos17 in the cultivated rice of Asian origin (Oryza sativa L.) and related AA genome species of the Oryza genus, to contribute understanding of the complex evolutionary history in this group of species through that of the element in the lineages. In that aim, we used a combination of Southern hybridization with a reverse transcriptase (RT) probe and an adapter-PCR mediated amplification, which allowed the sequencing of the genomic regions flanking Tos17 insertions. This analysis was carried out in a collection of 47 A-genome Oryza species accessions and 202 accessions of a core collection of Oryza sativa L. representative of the diversity of the species. Our Southern hybridization results show that Tos17 is present in all the accessions of the A-genome Oryza species, except for the South American species O. glumaepatula and the African species O. glaberrima and O. breviligulata. In O. sativa, the number of putative copies of Tos17 per accession ranged from 1 to 11 and multivariate analysis based on presence/absence of putative copies yielded a varietal clustering which is consistent with the isozyme classification of rice. Adapter PCR amplification and sequencing of flanking regions of Tos17 insertions in A-genome species other than O. sativa, followed by anchoring on the Nipponbare genome sequence, revealed 13 insertion sites of Tos17 in the surveyed O. rufipogon and O. longistaminata accessions, including one shared by both species. In O. sativa, the same approach revealed 25 insertions in the 6 varietal groups. Four insertion sites located on chromosomes 1, 2, 10, and 11 were found orthologous in O. rufipogon and O. sativa. The chromosome 1 insertion was also shared between O. rufipogon and O. longistaminata. The presence of Tos17 at three insertion sites was confirmed by retrotransposon-based insertion polymorphism (RBIP) in a sample of O. sativa accessions. The first insertion, located on chromosome 3 was only found in two japonica accessions from the Bhutan region while the second insertion, located on chromosome 10 was specific to the varietal groups 1, 2, and 5. The third insertion located on chromosome 7 corresponds to the only insertion shown active in rice so far, notably in cv. Nipponbare, where it has been extensively used for insertion mutagenesis. This copy was only found in a few varieties of the japonica group 6 and in one group 5 accession. Taken together, these results confirm that Tos17 was probably present in the ancestor of A-genome species and that some copies of the element remained active in some Oryza lineages--notably in O. rufipogon and O. longistaminata--as well as in the indica and japonica O. sativa L. lineages.

Molecular biogeographic study of recently described B- and A-genomeArachisspecies, also providing new insights into the origins of cultivated peanut

The cultivated peanut Arachis hypogaea is a tetraploid, likely derived from A- and B-genome species. Reproductive isolation of the cultigen has resulted in limited genetic variability for important traits. Artificial hybridizations using selected diploid parents have introduced alleles from wild species, but improved understanding of recently classified B-genome accessions would aid future introgression work. To this end, 154 cDNA probes were used to produce 1887 RFLP bands scored on 18 recently classified or potential B-genome accessions and 16 previously identified species. One group of B-genome species consisted of Arachis batizocoi, Arachis cruziana, Arachis krapovickasii, and one potential additional species; a second consisted of Arachis ipaënsis, Arachis magna, and Arachis gregoryi. Twelve uncharacterized accessions grouped with A-genome species. Many RFLP markers diagnostic of A. batizocoi group specificity mapped to linkage group pair 2/12, suggesting selection or genetic control of chromosome pairing. The combination of Arachis duranensis and A. ipaënsis most closely reconstituted the marker haplotype of A. hypogaea, but differences allow for other progenitors or genetic rearrangements after polyploidization. From 2 to 30 alleles per locus were present, demonstrating section Arachis wild species variation of potential use for expanding the cultigen's genetic basis.