Outlier Loci and Selection Signatures of Simple Sequence Repeats (SSRs) in Flax (Linum usitatissimum L.)

Genomic microsatellites (gSSRs) and expressed sequence tag-derived SSRs (EST-SSRs) have gained wide application for elucidating genetic diversity and population structure in plants. Both marker systems are assumed to be selectively neutral when making demographic inferences, but this assumption is rarely tested. In this study, three neutrality tests were assessed for identifying outlier loci among 150 SSRs (85 gSSRs and 65 EST-SSRs) that likely influence estimates of population structure in three differentiated flax sub-populations (F _ST?=?0.19). Moreover, the utility of gSSRs, EST-SSRs, and the combined sets of SSRs was also evaluated in assessing genetic diversity and population structure in flax. Six outlier loci were identified by at least two neutrality tests showing footprints of balancing selection. After removing the outlier loci, the STRUCTURE analysis and the dendrogram topology of EST-SSRs improved. Conversely, gSSRs and combined SSRs results did not change significantly, possibly as a consequence of the higher number of neutral loci assessed. Taken together, the genetic structure analyses established the superiority of gSSRs to determine the genetic relationships among flax accessions, although the combined SSRs produced the best results. Genetic diversity parameters did not differ statistically (P?>?0.05) between gSSRs and EST-SSRs, an observation partially explained by the similar number of repeat motifs. Our study provides new insights into the ability of gSSRs and EST-SSRs to measure genetic diversity and structure in flax and confirms the importance of testing for the occurrence of outlier loci to properly assess natural and breeding populations, particularly in studies considering only few loci.     

Cross-species transferability of G. arboreum-derived EST-SSRs in the diploid species of Gossypium

Diploid species with a common Gossypium origin are highly diverse in morphology and have been classified into eight genomic groups designated A–G and K. In this study, the transferability of 207 Gossypium arboreum-derived expressed sequence tag-simple sequence repeat (EST-SSR) primer pairs was examined among 25 different diploid accessions representing 7 genomes and 23 Gossypium species. We found that 124 of the 207 (60%) primer pairs produced amplification products in all 25 accessions. The remaining 83 (40%) primer pairs produced amplification in only a subset of species, ranging from 13 to 22 species, which is consistent with some genome- and species-specific amplification. The cross-species amplification of these EST-SSRs in 22 diploid species was 96.5% in 4,554 combinations (207 SSRs×22 species), indicative of a high transferability among the Gossypium species. Furthermore, a high level of polymorphism with an average number of 6.53 alleles per SSR marker was detected. No correlation was found between the repeat motif type and cross-species amplification. DNA sequencing showed that the high-level polymorphism findings was mainly due to changes in the number of repeat motifs and that the high transferability can be attributed to a higher-level conservation in the flanking regions among these diploid Gossypium species. The transferability among these different diploid species presented here can increase the efficiency of transferring genetic information across species and further enhance their introgression into cultivated cotton species by the molecular tagging of important genes existing in these diploid species using the EST-SSR markers.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Genetic diversity and relationships of diploid and tetraploid cottons revealed using AFLP

Gossypium species (± 49) represent a vast resource of genetic diversity for the improvement of cultivated cotton. To determine intra- and inter-specific genetic relationships within a diverse collection of Gossypium taxa, we employed 16 AFLP primer combinations on three diploid species, Gossypium herbaceum L. (A1), Gossypium arboreum L. (A2) and Gossypium raimondii Ulbrich (D5), and 26 AD allotetraploid accessions (Gossypium barbadense L. and Gossypium hirsutum L.). A total of 1180 major AFLP bands were observed; 368 of these (31%) were polymorphic. Genetic similarities among all taxa ranged from 0.21 (between the diploid species G. arboreum and G. raimondii) up to 0.89 (within G. barbadense). Phenetic trees based on genetic similarities (UPGMA, N-J) were consistent with known taxonomic relationships. In some cases, well-supported phylogenetic relationships, as well as evidence of genetic reticulation, could also be inferred. UPGMA trees and principal coordinate analysis based on genetic similarity matrices were used to identify genetically distinct cultivars that are potentially important sources of germplasm for cotton improvement, particularly of fiber quality traits. We show that AFLP is useful for estimating genetic relationships across a wide range of taxonomic levels, and for analyzing the evolutionary and historical development of cotton cultivars at the genomic level. Received: 17 January 2000 / Accepted: 4 May 2000     

Establishment of a microsatellite panel covering the sugi (Cryptomeria japonica) genome,and its application for localization of a male-sterile gene (ms-2)

A microsatellite (simple sequence repeat; SSR) panel for Cryptomeria japonica was established, using both newly developed and previously reported markers, to construct a frame of linkage map and facilitate localization of important genes in this species. In this study, 32 new expressed sequence tag SSRs (EST-SSRs) and 12 new genomic SSRs (gSSRs) were developed. Their average polymorphism information content (PIC) values were 0.549 and 0.776, respectively. The markers were mapped onto a high-density linkage map. The SSR panel that was established to cover the genome consisted of 46 gSSRs and 47 EST-SSRs. The number of SSR markers in each linkage group, the average map distance between loci within a linkage group, and the average PIC values in each linkage group ranged from 6 to 13, 6.77 to 19.88 and 0.475 to 0.712, respectively. The utility of the SSR panel was tested by using it to localize a male-sterile gene, ms-2. The ms-2 locus was successfully localized on the linkage group 5 using 33 SSR markers (three SSRs per linkage group) which were selected from the SSR panel based on the existence of polymorphisms and the absence of null alleles in the mapping population for ms-2. A partial linkage map surrounding the ms-2 locus was then constructed using a further 57 single nucleotide polymorphisms and three SSRs, to facilitate future development of markers tightly linked to the ms-2 locus for use in marker-assisted selection. The SSR panel covering the C. japonica genome will allow researchers to localize important genes efficiently.     

Development of chromosome-specific markers with high polymorphism for allotetraploid cotton based on genome-wide characterization of simple sequence repeats in diploid cottons (Gossypium arboreum L. and Gossypium raimondii Ulbrich)

Background

Tetraploid cotton contains two sets of homologous chromosomes, the At- and Dt-subgenomes. Consequently, many markers in cotton were mapped to multiple positions during linkage genetic map construction, posing a challenge to anchoring linkage groups and mapping economically-important genes to particular chromosomes. Chromosome-specific markers could solve this problem. Recently, the genomes of two diploid species were sequenced whose progenitors were putative contributors of the At- and Dt-subgenomes to tetraploid cotton. These sequences provide a powerful tool for developing chromosome-specific markers given the high level of synteny among tetraploid and diploid cotton genomes. In this study, simple sequence repeats (SSRs) on each chromosome in the two diploid genomes were characterized. Chromosome-specific SSRs were developed by comparative analysis and proved to distinguish chromosomes.

Results

A total of 200,744 and 142,409 SSRs were detected on the 13 chromosomes of Gossypium arboreum L. and Gossypium raimondii Ulbrich, respectively. Chromosome-specific SSRs were obtained by comparing SSR flanking sequences from each chromosome with those from the other 25 chromosomes. The average was 7,996 per chromosome. To confirm their chromosome specificity, these SSRs were used to distinguish two homologous chromosomes in tetraploid cotton through linkage group construction. The chromosome-specific SSRs and previously-reported chromosome markers were grouped together, and no marker mapped to another homologous chromosome, proving that the chromosome-specific SSRs were unique and could distinguish homologous chromosomes in tetraploid cotton. Because longer dinucleotide AT-rich repeats were the most polymorphic in previous reports, the SSRs on each chromosome were sorted by motif type and repeat length for convenient selection. The primer sequences of all chromosome-specific SSRs were also made publicly available.

Conclusion

Chromosome-specific SSRs are efficient tools for chromosome identification by anchoring linkage groups to particular chromosomes during genetic mapping and are especially useful in mapping of qualitative-trait genes or quantitative trait loci with just a few markers. The SSRs reported here will facilitate a number of genetic and genomic studies in cotton, including construction of high-density genetic maps, positional gene cloning, fingerprinting, and genetic diversity and comparative evolutionary analyses among Gossypium species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1265-2) contains supplementary material, which is available to authorized users.     

Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from <Emphasis Type="Italic">Gossypium bickii</Emphasis>

The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated ∼2000 genomic and ∼800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.     

Generation of ESTs for flowering gene discovery and SSR marker development in upland cotton

Background

Upland cotton, Gossypium hirsutum L., is one of the world''s most important economic crops. In the absence of the entire genomic sequence, a large number of expressed sequence tag (EST) resources of upland cotton have been generated and used in several studies. However, information about the flower development of this species is rare.

Methodology/Principal Findings

To clarify the molecular mechanism of flower development in upland cotton, 22,915 high-quality ESTs were generated and assembled into 14,373 unique sequences consisting of 4,563 contigs and 9,810 singletons from a normalized and full-length cDNA library constructed from pooled RNA isolated from shoot apexes, squares, and flowers. Comparative analysis indicated that 5,352 unique sequences had no high-degree matches to the cotton public database. Functional annotation showed that several upland cotton homologs with flowering-related genes were identified in our library. The majority of these genes were specifically expressed in flowering-related tissues. Three GhSEP (G. hirsutum L. SEPALLATA) genes determining floral organ development were cloned, and quantitative real-time PCR (qRT-PCR) revealed that these genes were expressed preferentially in squares or flowers. Furthermore, 670 new putative microsatellites with flanking sequences sufficient for primer design were identified from the 645 unigenes. Twenty-five EST–simple sequence repeats were randomly selected for validation and transferability testing in 17 Gossypium species. Of these, 23 were identified as true-to-type simple sequence repeat loci and were highly transferable among Gossypium species.

Conclusions/Significance

A high-quality, normalized, full-length cDNA library with a total of 14,373 unique ESTs was generated to provide sequence information for gene discovery and marker development related to upland cotton flower development. These EST resources form a valuable foundation for gene expression profiling analysis, functional analysis of newly discovered genes, genetic linkage, and quantitative trait loci analysis.     

Isolation, characterization and mapping of genes differentially expressed during fibre development between Gossypium hirsutum and G. barbadense by cDNA-SRAP

Gossypium hirsutum and G. barbadense are two cultivated tetraploid cotton species with differences in fibre quality. The fibre of G. barbadense is longer, stronger and finer than that of G. hirsutum. To isolate genes expressed differently between the two species during fibre development, cDNA-SRAP (sequence-related amplified polymorphism) was applied. This technique was used to analyse genes at different stages of fibre development in G. hirsutum cv. Emian22 and G. barbadense acc. 3-79, the parents of our interspecific mapping population. A total of 4096 SRAP primer combinations were used to screen polymorphism between the DNA of the parents, and 275 highly polymorphic primers were picked out to analyse DNA and RNA from leaves and fibres at different developmental stages of the parents. A total of 168 DNA fragments were isolated from gels and sequenced: 54, 30, 38 and 41 from fibres of 5, 10, 15 and 20 days post-anthesis, respectively, and five from multi stages. To genetically map these sequences, 104 sequence-specific primers were developed and were used to screened polymorphism between the mapping parents. Finally, six markers were mapped on six chromosomes of our backbone interspecific genetic map. This work can give us a primary knowledge of differences in mechanism of fibre development between G. hirsutum and G. barbadense.     

Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium

Although lily is the second largest flower crop in cutting flower commodity, only six simple sequence repeats SSRs have been reported. Thus, we developed expressed sequence tag derived-SSRs (EST-SSRs) for the Lilium genus. Among 2,235 unique ESTs, 754 ESTs contained SSR motifs, among which 165 ESTs were amenable to primer design. Among these 165 EST-SSRs, 131 EST-SSRs showed amplification in at least one Lilium species, and 76 EST-SSRs showed amplification in at least nine species. Of the 76 EST-SSRs, 47 showed amplification in all Lilium species analyzed. Using 10 breeding lines, we selected 21 EST-SSRs that had the highest number of alleles and polymorphism information content. The polymorphism information content values of these selected EST-SSRs ranged from 0.49 to 0.94 with an average of 0.76, which are higher than other plant species. The phylogenetic dendrogram derived from the amplification profiles of the 21 high polymorphic EST-SSRs was congruent with the genetic background of the 84 selected lily accessions and hybrids, which are available in commerce. Thus, the developed EST-SSRs will be very useful in germplasm management, genetic diversity analysis, cultivar finger printing, and molecular breeding in the lily.     

Phylogenetic diversity and relationship among Gossypium germplasm using SSRs markers

Gossypium species represent a vast resource of genetic multiplicity for the improvement of cultivated cotton. To determine genetic diversity and relationships within a diverse collection of Gossypium, we employed 120 SSR primers on 20 diploid species representing seven basic genome groups of the genus Gossypium, five AD allotetraploid cotton accessions while T. populnea served as an outgroup species. Out of 120 SSR primers, 49 pairs are polymorphic, which produced a total of 99 distinct alleles with an average of 2.0 alleles per primer pair. A total of 1139 major SSR bands were observed. Genetic similarities among all the diploid species ranged from 0.582 (between G. herbaceum and G. trilobum) up to 0.969 (between G. arboreum and G. herbaceum). Phylogenetic trees based on genetic similarities were consistent with known taxonomic relationships. The results also indicated that G. raimondii is the closest living relative of the ancestral D-genome donor of tetraploid species and the A-genome donor is much similar to the present-day G. herbaceum and G. arboreum. Ancient tetraploid cotton species were formed by hybridizing and chromosome doubling between them, then different tetraploid cotton species appeared by further geographical and genetic isolation and separating differentiation. The results showed that SSRs could be an ideal means for the identification of the genetic diversity and relationship of cotton resources at the genomic level.     

Bin mapping of genomic and EST-derived SSRs in melon (Cucumis melo L.)

We report the development of 158 primer pairs flanking SSR motifs in genomic (gSSR) and EST (EST-SSR) melon sequences, all yielding polymorphic bands in melon germplasm, except one that was polymorphic only in Cucurbita species. A similar polymorphism level was found among EST-SSRs and gSSRs, between dimeric and trimeric EST-SSRs, and between EST-SSRs placed in the open reading frame or any of the 5′- or 3′-untranslated regions. Correlation between SSR length and polymorphism was only found for dinucleotide EST-SSRs located within the untranslated regions, but not for trinucleotide EST-SSRs. Transferability of EST-SSRs to Cucurbita species was assayed and 12.7% of the primer pairs amplified at least in one species, although only 5.4% were polymorphic. A set of 14 double haploid lines from the cross between the cultivar “Piel de Sapo” and the accession PI161375 were selected for the bin mapping approach in melon. One hundred and twenty-one SSR markers were newly mapped. The position of 46 SSR loci was also verified by genotyping the complete population. A final bin-map was constructed including 80 RFLPs, 212 SSRs, 3 SNPs and the Nsv locus, distributed in 122 bins with an average bin length of 10.2 cM and a maximum bin length of 33 cM. Map density was 4.2 cM/marker or 5.9 cM/SSR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic diversity and population structure in the US Upland cotton (Gossypium hirsutum L.)

Key message

Genetic diversity and population structure in the US Upland cotton was established and core sets of allelic richness were identified for developing association mapping populations in cotton.

Abstract

Elite plant breeding programs could likely benefit from the unexploited standing genetic variation of obsolete cultivars without the yield drag typically associated with wild accessions. A set of 381 accessions comprising 378 Upland (Gossypium hirsutum L.) and 3 G. barbadense L. accessions of the United States cotton belt were genotyped using 120 genome-wide SSR markers to establish the genetic diversity and population structure in tetraploid cotton. These accessions represent more than 100 years of Upland cotton breeding in the United States. Genetic diversity analysis identified a total of 546 alleles across 141 marker loci. Twenty-two percent of the alleles in Upland accessions were unique, specific to a single accession. Population structure analysis revealed extensive admixture and identified five subgroups corresponding to Southeastern, Midsouth, Southwest, and Western zones of cotton growing areas in the United States, with the three accessions of G. barbadense forming a separate cluster. Phylogenetic analysis supported the subgroups identified by STRUCTURE. Average genetic distance between G. hirsutum accessions was 0.195 indicating low levels of genetic diversity in Upland cotton germplasm pool. The results from both population structure and phylogenetic analysis were in agreement with pedigree information, although there were a few exceptions. Further, core sets of different sizes representing different levels of allelic richness in Upland cotton were identified. Establishment of genetic diversity, population structure, and identification of core sets from this study could be useful for genetic and genomic analysis and systematic utilization of the standing genetic variation in Upland cotton.     

<Emphasis Type="Italic">SnRK2</Emphasis> Homologs in <Emphasis Type="Italic">Gossypium</Emphasis> and <Emphasis Type="Italic">GhSnRK2.6</Emphasis> Improved Salt Tolerance in Transgenic Upland Cotton and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SnRK2s are a large family of plant-specific protein kinases, which play important roles in multiple abiotic stress responses in various plant species. But the family in Gossypium has not been well studied. Here, we identified 13, 10, and 13 members of the SnRK2 family from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively, and analyzed the locations of SnRK2 homologs in chromosomes based on genome data of cotton species. Phylogenetic tree analysis of SnRK2 proteins showed that these families were classified into three groups. All SnRK2 genes were comprised of nine exons and eight introns, and the exon distributions and the intron phase of homolog genes among different cotton species were analogous. Moreover, GhSnRK2.6 was overexpressed in Arabidopsis and upland cotton, respectively. Under salt treatment, overexpressed Arabidopsis could maintain higher biomass accumulation than wild-type plants, and GhSnRK2.6 overexpression in cotton exhibited higher germination rate than the control. So, the gene GhSnRK2.6 could be utilized in cotton breeding for salt tolerance.     

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.     

GENETIC DIVERSITY AND ORIGIN OF THE HAWAIIAN ISLANDS COTTON,GOSSYPIUM TOMENTOSUM

Gossypium tomentosum is the only member of the cotton genus endemic to the Hawaiian archipelago. It is morphologically distinct from other allopolyploid Gossypium species, and its phylogenetic relationships with them are uncertain. Chloroplast and ribosomal DNA restriction site variation were used to estimate the phylogeny of the allopolyploids. Gossypium mustelinum is resolved as sister to the remaining allopolyploid species, which include two species-pairs, G. barbadense-G. darwinii and G. hirsutum (including G. lanceolatum)-G. tomentosum. This indication that G. tomentosum is sister to G. hirsutum is supported by allozyme data. Gossypium tomentosum is proposed, based on biogeographic evidence and molecular data, to have originated by transoceanic dispersal from a Mesoamerican progenitor. Few restriction site variants were observed among the allopolyploids, suggesting that present lineages diverged relatively rapidly following polyploidization. Allozyme analysis of 30 G. tomentosum accessions collected from seven islands revealed relatively low levels of genetic diversity: 11 of 50 loci were polymorphic, mean number of alleles per locus was 1.24, and mean panmictic heterozygosity was 0.033. Little geographic patterning of allelic distributions was observed. Despite historical cultivation of G. barbadense and G. hirsutum in Hawaii and the existence of their naturalized derivatives, no allozyme evidence of interspecific introgression into G. tomentosum was detected.     

Development and Characterization of Polymorphic EST-SSR and Genomic SSR Markers for Tibetan Annual Wild Barley

Tibetan annual wild barley is rich in genetic variation. This study was aimed at the exploitation of new SSRs for the genetic diversity and phylogenetic analysis of wild barley by data mining. We developed 49 novel EST-SSRs and confirmed 20 genomic SSRs for 80 Tibetan annual wild barley and 16 cultivated barley accessions. A total of 213 alleles were generated from 69 loci with an average of 3.14 alleles per locus. The trimeric repeats were the most abundant motifs (40.82%) among the EST-SSRs, while the majority of the genomic SSRs were di-nuleotide repeats. The polymorphic information content (PIC) ranged from 0.08 to 0.75 with a mean of 0.46. Besides this, the expected heterozygosity (He) ranged from 0.0854 to 0.7842 with an average of 0.5279. Overall, the polymorphism of genomic SSRs was higher than that of EST-SSRs. Furthermore, the number of alleles and the PIC of wild barley were both higher than that of cultivated barley, being 3.12 vs 2.59 and 0.44 vs 0.37. Indicating more polymorphism existed in the Tibetan wild barley than in cultivated barley. The 96 accessions were divided into eight subpopulations based on 69 SSR markers, and the cultivated genotypes can be clearly separated from wild barleys. A total of 47 SSR-containing EST unigenes showed significant similarities to the known genes. These EST-SSR markers have potential for application in germplasm appraisal, genetic diversity and population structure analysis, facilitating marker-assisted breeding and crop improvement in barley.