miR482 Regulation of NBS-LRR Defense Genes during Fungal Pathogen Infection in Cotton

In this study, we characterized the miR482 family in cotton using existing small RNA datasets and the recently released draft genome sequence of Gossypium raimondii, a diploid cotton species whose progenitor is the putative contributor of the D_t (representing the D genome of tetraploid) genome of the cultivated tetraploid cotton species G. hirsutum and G. barbadense. Of the three ghr-miR482 members reported in G. hirsutum, ghr-miR482a has no homolog in G. raimondii, ghr-miR482b and ghr-miR482c each has a single homolog in G. raimondii. Gra-miR482d has five homologous loci (gra-miR482d, f-i) in G. raimondii and also exists in G. hirsutum (ghr-miR482d). A variant, miR482.2 that is a homolog of miR2118 in other species, is produced from several GHR-MIR482 loci in G. hirsutum. Approximately 12% of the G. raimondii NBS-LRR genes were predicted targets of various members of the gra-miR482 family. Based on the rationale that the regulatory relationship between miR482 and NBS-LRR genes will be conserved in G. raimondii and G. hirsutum, we investigated this relationship using G. hirsutum miR482 and G. raimondii NBS-LRR genes, which are not currently available in G. hirsutum. Ghr-miR482/miR482.2-mediated cleavage was confirmed for three of the four NBS-LRR genes analysed. As in tomato, miR482-mediated cleavage of NBS-LRR genes triggered production of phased secondary small RNAs in cotton. In seedlings of the susceptible cultivar Sicot71 (G. hirsutum) infected with the fungal pathogen Verticillium dahliae, the expression levels of ghr-miR482b/miR482b.2, ghr-miR482c and ghr-miR482d.2 were down-regulated, and several NBS-LRR targets of ghr-miR482c and ghr-miR482d were up-regulated. These results imply that, like tomato plants infected with viruses or bacteria, cotton plants are able to induce expression of NBS-LRR defence genes by suppression of the miRNA-mediated gene silencing pathway upon fungal pathogen attack.     

Entire nucleotide sequences of Gossypium raimondii and G. arboreum mitochondrial genomes revealed A‐genome species as cytoplasmic donor of the allotetraploid species

• Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A–G and K, and an allotetraploid genomic group, AD. Gossypium raimondii (D₅) and G. arboreum (A₂) are the putative contributors to the progenitor of G. hirsutum (AD₁), the economically important fibre‐producing cotton species.
• Mitochondrial DNA from week‐old etiolated seedlings was extracted from isolated organelles using discontinuous sucrose density gradient method. Mitochondrial genomes were sequenced, assembled, annotated and analysed in orderly.
• Gossypium raimondii (D₅) and G. arboreum (A₂) mitochondrial genomes were provided in this study. The mitochondrial genomes of two diploid species harboured circular genome of 643,914 bp (D₅) and 687,482 bp (A₂), respectively. They differ in size and number of repeat sequences, both contain illuminating triplicate sequences with 7317 and 10,246 bp, respectively, demonstrating dynamic difference and rearranged genome organisations. Comparing the D₅ and A₂ mitogenomes with mitogenomes of tetraploid Gossypium species (AD₁, G. hirsutum; AD₂, G. barbadense), a shared 11 kbp fragment loss was detected in allotetraploid species, three regions shared by G. arboreum (A₂), G. hirsutum (AD₁) and G. barbadense (AD₂), while eight regions were specific to G. raimondii (D₅). The presence/absence variations and gene‐based phylogeny supported that A‐genome is a cytoplasmic donor to the progenitor of allotetraploid species G. hirsutum and G. barbadense.
• The results present structure variations and phylogeny of Gossypium mitochondrial genome evolution.

     

Sampling nucleotide diversity in cotton

Background  

Cultivated cotton is an annual fiber crop derived mainly from two perennial species, Gossypium hirsutum L. or upland cotton, and G. barbadense L., extra long-staple fiber Pima or Egyptian cotton. These two cultivated species are among five allotetraploid species presumably derived monophyletically between G. arboreum and G. raimondii. Genomic-based approaches have been hindered by the limited variation within species. Yet, population-based methods are being used for genome-wide introgression of novel alleles from G. mustelinum and G. tomentosum into G. hirsutum using combinations of backcrossing, selfing, and inter-mating. Recombinant inbred line populations between genetics standards TM-1, (G. hirsutum) × 3-79 (G. barbadense) have been developed to allow high-density genetic mapping of traits.     

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.     

A genetic bottleneck in the ’evolution under domestication’ of upland cotton Gossypium hirsutum L. examined using DNA fingerprinting

Reliable information about the evolutionary and genetic relationships of various germplasm resources is essential to the establishment of rational strategies for crop improvement. We used AFLPs to study the genetic relationships of 43 cultivars of Gossypium hirsutum representative of the genomic composition of modern ’Upland’ cotton. The study also included representatives of the related tetraploid species Gossypium barbadense, as well as the diploid species Gossypium raimondii, Gossypium incanum, Gossypium herbaceum and Gossypium arboreum. We tested 20 primer combinations that resulted in a total of 3,178 fragments. At the species level, and above, genetic similarities based on AFLPs were in agreement with the known taxonomic relationships. Similarity indices ranged from 0.25 to 0.99. Representatives of the G. hirsutum germplasm resources utilized in North America, including secondary accessions collected by breeders in Central America (’Acala’, ’Tuxtla’, ’Kekchi’) and the southwestern US (’Hopi Moencopi’), formed a single cluster with exceedingly limited genetic diversity (with many pairwise similarity indices >0.96) We concluded that these accessions were derived from the same genetic pool. The early maturing or ’latifolium’ or ’Mexican Highlands’ cultigens from which these cultivars were derived appear to have had extremely limited genetic diversity, perhaps as a result of a severe genetic bottleneck resulting from the selection pressures of domestication. Outside of the major G. hirsutum cluster, well-supported phylogenies were inferred. Inside this cluster, phylogenies were obscured by limited diversity, reticulation and lineage sorting. The implications of these findings for cotton improvement are discussed. Received: 23 May 2000 / Accepted: 23 January 2001     

Population genomics reveals a fine‐scale recombination landscape for genetic improvement of cotton

Recombination breaks up ancestral linkage disequilibrium, creates combinations of alleles, affects the efficiency of natural selection, and plays a major role in crop domestication and improvement. However, there is little knowledge regarding the variation in the population‐scaled recombination rate in cotton. We constructed recombination maps and characterized the difference in the genomic landscape of the population‐scaled recombination rate between Gossypium hirsutum and G. arboreum and sub‐genomes based on the 381 sequenced G. hirsutum and 215 G. arboreum accessions. Comparative genomics identified large structural variations and syntenic genes in the recombination regions, suggesting that recombination was related to structural variation and occurred preferentially in the distal chromosomal regions. Correlation analysis indicated that recombination was only slightly affected by geographical distribution and breeding period. A genome‐wide association study (GWAS) was performed with 15 agronomic traits using 267 cotton accessions and identified 163 quantitative trait loci (QTL) and an important candidate gene (Ghir_COL2) for early maturity traits. Comparative analysis of recombination and a GWAS revealed that the QTL of fibre quality traits tended to be more common in high‐recombination regions than were those of yield and early maturity traits. These results provide insights into the population‐scaled recombination landscape, suggesting that recombination contributed to the domestication and improvement of cotton, which provides a useful reference for studying recombination in other species.     

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     

Inter-Specific Differences in Cotton for Nutrient Partitioning from Subtending Leaves to Reproductive Parts at Various Developmental Stages: Consequences for Fruit Growth and Yield

A field study was carried out to unravel the inter-specific differences in cotton for the partitioning of N, P, K, S, Ca, Mg, Na and Cl from the subtending leaves to the reproductive parts of Gossypium hirsutum, G. barbadense and G. arboreum at various developmental stages. Results revealed significant differences among the species for the various parameters studied. Overall there was a greater fresh and dry matter yield of various reproductive parts and subtending leaves of G. hirsutum and G. barbadense than G. arboreum, although the leaf photosynthetic rate was similar. Age-dependent increase in leaf area/leaf mass ratio indicated a greater partitioning of earlier acquired assimilates to the growth of reproductive parts. Results indicated greater partitioning of N, P, S and Ca during later reproductive growth (from boll production to its opening) in G. hirsutum and G. barbadense but during earlier reproductive growth in G. arboreum (from bud up to flower formation) as was evident by decreased subtending leaf/reproductive parts ratio. It is concluded that better N, P, S and Ca partitioning ability of G. hirsutum and G. barbadense at the onset of boll development played a major role in the better yield and good quality fiber characteristics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessing genetic diversity in <Emphasis Type="Italic">Gossypium arboreum</Emphasis> L. cultivars using genomic and EST-derived microsatellites

The cultivated diploid, Gossypium arboreum L., (A genome) is an invaluable genetic resource for improving modern tetraploid cotton (G. hirsutum L. and G. barbadense L.) cultivars. The objective of this research is to select a set of informative and robust microsatellites for studying genetic relationships among accessions of geographically diverse G. arboreum cultivars. From more than 1,500 previously developed simple sequence repeat (SSR) markers, 115 genomic (BNL) and EST-derived (MUCS and MUSS) markers were used to evaluate the allelic diversity of a core panel of G. arboreum accessions. These SSR data enabled advanced genome analyses. A set of 25 SSRs were selected based both upon their high level of informativeness (PIC ≥ 0.50) and the production of clear PCR bands on agarose gels. Subsequently, 96 accessions representing a wide spectrum of diversity of G. arboreum cultivars were analyzed with these markers. The 25 SSR loci revealed 75 allelic variants (polymorphisms) ranging from 2 to 4 alleles per locus. The Neighborjoining (NJ) method, based on genetic dissimilarities, revealed that cultivars from geographically adjacent countries tend to cluster together. Outcomes of this research should be useful in decreasing redundancy of effort and in constructing a core collection of G. arboreum, important for efficient use of this genetic resource in cotton breeding.     

Sl‐lncRNA15492 interacts with Sl‐miR482a and affects Solanum lycopersicum immunity against Phytophthora infestans

Long non‐coding RNAs (lncRNAs) are involved in the resistance of plants to infection by pathogens via interactions with microRNAs (miRNAs). Long non‐coding RNAs are cleaved by miRNAs to produce phased small interfering RNAs (phasiRNAs), which, as competing endogenous RNAs (ceRNAs), function as decoys for mature miRNAs, thus inhibiting their expression, and contain pre‐miRNA sequences to produce mature miRNAs. However, whether lncRNAs and miRNAs mediate other molecular mechanisms during plant resistance to pathogens is unknown. In this study, as a positive regulator, Sl‐lncRNA15492 from tomato (Solanum lycopersicum Zaofen No. 2) plants affected tomato resistance to Phytophthora infestans. Gain‐ and loss‐of‐function experiments and RNA ligase‐mediated 5′‐amplification of cDNA ends (RLM‐5′ RACE) also revealed that Sl‐miR482a was negatively involved in tomato resistance by targeting Sl‐NBS‐LRR genes and that silencing of Sl‐NBS‐LRR1 decreased tomato resistance. Sl‐lncRNA15492 inhibited the expression of mature Sl‐miR482a, whose precursor was located within the antisense sequence of Sl‐lncRNA15492. Further degradome analysis and additional RLM‐5′ RACE experiments verified that mature Sl‐miR482a could also cleave Sl‐lncRNA15492. These results provide a mechanism by which lncRNAs might inhibit precursor miRNA expression through antisense strands of lncRNAs, and demonstrate that Sl‐lncRNA15492 and Sl‐miR482a mutually inhibit the maintenance of Sl‐NBS‐LRR1 homeostasis during tomato resistance to P. infestans.     

Distribution of 5S and 18S–28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors

The most widely cultivated species of cotton,Gossypium hirsutum, is a disomic tetraploid (2n=4x=52). It has been proposed previously that extant A- and D-genome species are most closely related to the diploid progenitors of the tetraploid. We used fluorescent in situ hybridization (FISH) to determine the distribution of 5S and 18S-28S rDNA loci in the A-genome speciesG. herbaceum andG. arboreum, the D-genome speciesG. raimondii andG. thurberi, and the AD tetraploidG. hirsutum. High signal-to-noise, single-label FISH was used to enumerate rDNA loci, and simultaneous, dual-label FISH was used to determine the syntenic relationships of 5S rDNA loci relative to 18S–28S rDNA loci. These techniques provided greater sensitivity than our previous methods and permitted detection of six newG. hirsutum 18S–28S rDNA loci, bringing the total number of observed loci to 11. Differences in the intensity of the hybrizization signal at these loci allowed us to designate them as major, intermediate, or minor 18–28S loci. Using genomic painting with labeled A-genome DNA, five 18S–28S loci were localized to theG. hirsutum A-subgenome and six to the D-subgenome. Four of the 11 18S–28S rDNA loci inG. hirsutum could not be accounted for in its presumed diploid progenitors, as both A-genome species has three loci and both D-genome species had four.G. hirsutum has two 5S rDNA loci, both of which are syntenic to major 18S–28S rDNA loci. All four of the diploid genomes wer examined contained a single 5S locus. InG. herbaceum (A₁) andG. thurberi (D₁), the 5S locus is syntenic to a major 18S–28S locus, but inG. arboreum (A₂) andG. raimondii (D₅), the proposed D-genome progenitor ofG. hirsutum, the 5S loci are syntenic tominor and intermediate 18S–28S loci, respecitively. The multiplicity, variation in size and site number, and lack of additivity between the tetraploid species and its putative diploid ancestors indicate that the behavior of rDNA loci in cotton is nondogmatic, and considerably more complex and dynamic than previously envisioned. The relative variability of 18S–28S rDNA loci versus 5S rDNA loci suggests that the behavior of tandem repearts can differ widely. Edited by: R. Appels     

Genetic diversity and geographic pattern in early South American cotton domestication

Amplified fragment length polymorphism fingerprinting was applied to survey the genetic diversity of primitive South American Gossypium barbadense cotton for establishing a possible link to its pre-Columbian expansion. New germplasm was collected along coastal Peru and over an Andean transect in areas where most of the archaeological evidence relating to cotton domestication has been recorded. Gene bank material of three diploid (G. raimondii, G. arboreum, and G. herbaceum) and four allotetraploid cotton species (G. hirsutum, G. mustelinum, G. tomentosum and additional G. barbadense) was added for inter- and intra-specific comparison. Eight primer combinations yielded 340 polymorphic bands among the 131 accessions. The obtained neighbor joining and unweighted pair-group method with arithmetic means are in full agreement with the known cytogenetics of the tetraploid cottons and their diploid genome donors. The four tetraploid species are clearly distinct based on taxonomic classification. The genetic diversity within G. barbadense reveals geographic patterns. The locally maintained cottons from coastal Peru display a distinct genetic diversity that mirrors their primitive agro-morphological traits. Accessions from the northernmost coast of Peru and from southwestern (SW) Ecuador cluster basal to the east-of-Andes accessions. The remaining accessions from Bolivia, Brazil, Columbia, Venezuela, and the Caribbean and Pacific islands cluster with the east-of-Andes accessions. Northwestern Peru/SW Ecuador (the area flanking the Guayaquil gulf) appears to be the center of the primitive domesticated G. barbadense cotton from where it spread over the Andes and expanded into its pre-Columbian range.This publication is dedicated to Prof. Dr. Drs.h.c. Gerhard Röbbelen on the occasion of his 75th birthday     

GbEXPATR,a species‐specific expansin,enhances cotton fibre elongation through cell wall restructuring

Cotton provides us the most important natural fibre. High fibre quality is the major goal of cotton breeding, and introducing genes conferring longer, finer and stronger fibre from Gossypium barbadense to Gossypium hirsutum is an important breeding strategy. We previously analysed the G. barbadense fibre development mechanism by gene expression profiling and found two homoeologous fibre‐specific α‐expansins from G. barbadense, GbEXPA2 and GbEXPATR. GbEXPA2 (from the D_T genome) is a classical α‐expansin, while its homoeolog, GbEXPATR (A_T genome), encodes a truncated protein lacking the normal C‐terminal polysaccharide‐binding domain of other α‐expansins and is specifically expressed in G. barbadense. Silencing EXPA in G. hirsutum induced shorter fibres with thicker cell walls. GbEXPA2 overexpression in G. hirsutum had no effect on mature fibre length, but produced fibres with a slightly thicker wall and increased crystalline cellulose content. Interestingly, GbEXPATR overexpression resulted in longer, finer and stronger fibres coupled with significantly thinner cell walls. The longer and thinner fibre was associated with lower expression of a number of secondary wall‐associated genes, especially chitinase‐like genes, and walls with lower cellulose levels but higher noncellulosic polysaccharides which advocated that a delay in the transition to secondary wall synthesis might be responsible for better fibre. In conclusion, we propose that α‐expansins play a critical role in fibre development by loosening the cell wall; furthermore, a truncated form, GbEXPATR, has a more dramatic effect through reorganizing secondary wall synthesis and metabolism and should be a candidate gene for developing G. hirsutum cultivars with superior fibre quality.     

Introgression of the low-gossypol seed & high-gossypol plant trait in upland cotton: Analysis of [(Gossypium hirsutum?×?G. raimondii)2?×?G. sturtianum] trispecific hybrid and selected derivatives using mapped SSRs

In order to select genotypes of Gossypium hirsutum genetically balanced and expressing the low-gossypol seed & high-gossypol plant trait introgressed from the Australian wild diploid species G. sturtianum, the [(G. hirsutum × G. raimondii)2 × G. sturtianum] triple hybrid was backcrossed to G. hirsutum and autopollinated to produce backcross and selfed progenies. Two hundred and six mapped SSR markers of G. hirsutum were used to monitor the introgression of SSR alleles specific to G. sturtianum and G. raimondii in the selected progenies. A high level of heterozygosity, varying from 25 to 100%, was observed for all G. sturtianum-specific SSR markers conserved in the most advanced progenies. These results indicate the existence of segregation distortion factors that are associated with the genes controlling the researched trait. This study represents a starting point to map the genes involved in the expression of the trait and better understand its genetic determinism.     

Duplication,divergence and persistence in the Phytochrome photoreceptor gene family of cottons (<Emphasis Type="Italic">Gossypium</Emphasis> spp.)

Background  

Phytochromes are a family of red/far-red photoreceptors that regulate a number of important developmental traits in cotton (Gossypium spp.), including plant architecture, fiber development, and photoperiodic flowering. Little is known about the composition and evolution of the phytochrome gene family in diploid (G. herbaceum, G. raimondii) or allotetraploid (G. hirsutum, G. barbadense) cotton species. The objective of this study was to obtain a preliminary inventory and molecular-evolutionary characterization of the phytochrome gene family in cotton.     

Integrative placement and orientation of non-redundant SSR loci in cotton linkage groups by deficiency analysis

A combination of previously mapped and unmapped non-redundant SSR loci, using 381 primer pairs were chromosomally and sub-chromosomally localized by deficiency analysis of two sets of quasi-isogenic interspecific Gossypium hirsutum L. hypoaneuploid F₁ hybrids involving Gossypium barbadense L. and Gossypium tomentosum (Nuttall ex Seemann). Polymorphisms were detected for 369 SSR primer pairs. A total of 318 SSR loci were rendered deficient by the available hypoaneuploid stocks, which included primary monosomics (2n = 51), monotelodisomics and duplication-deficient (segmental trisomic–monosomic) (2n = 52) types. Chromosomal associations were newly determined for 123 SSR loci, of which 90, 106 and 73 were polymorphic in G. tomentosum, G. barbadense, and both sets, respectively. The deficiency tests independently confirmed the recent identifications of linkage groups (LG) A01, A02, A03 and D08 to be chromosome (Chr)-13, Chr-8, Chr-11 and Chr-19, respectively, and collectively delimited LG D02 and D03 to Chr-21 and 24, and their homeologs to Chr-8 and 11. Segmental homeology was detected between Chr-2 and Chr-17 loci, adding to evidence of segmental homeology between Chr-2 and 3 versus Chr-14 and 17. The 318 non-redundant SSR loci localized in this study will enhance the construction of linkage maps and QTL identification in molecular marker assisted selection since the confirmed and newly discovered SSR loci can serve as anchor loci for their respective chromosomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.