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.     

Development of two loquat [Eriobotrya japonica (Thunb.) Lindl.] linkage maps based on AFLPs and SSR markers from different Rosaceae species

Loquat [Eriobotrya japonica (Thunb.) Lindl.] is a Rosaceae fruit species of growing interest as an alternative to the main fruit crops. However, only a few genetic studies have been carried out on this species. This paper reports the construction of the first genetic maps of two loquat cultivars based on AFLP and microsatellite markers from Malus, Eriobotrya, Pyrus and Prunus genera. An F₁ population consisting of 81 individuals, derived from the cross between ‘Algerie’ and ‘Zaozhong-6’ cultivars, was used to construct both maps. A total of 111 scorable simple sequence repeat (SSR) loci resulted from the testing of 440 SSR primer pairs in the analyzed progeny and the SSR transferability to Eriobotrya was found to be 74% from apple, 58% from pear and 49% from Prunus spp. In addition, 183 AFLP polymorphic bands were produced using 42 primer combinations. The ‘Algerie’ map was organized in 17 linkage groups covering a distance of 900 cM and comprising 177 loci (83 SSRs and 94 AFLPs) with an average marker distance of 5.1 cM. Self-incompatibility trait was mapped at the distal part of the LG17 linkage group, as previously reported in Malus and Pyrus. The ‘Zaozhong-6’ map covered 870 cM comprising 146 loci (64 SSRs and 82 AFLPs) with an average marker distance of 5.9 cM. The 44 SSRs and the 48 AFLPs share in common by both maps were essentially collinear and, moreover, the order of the 75% of apple and pear SSRs mapped in Eriobotrya was shown to be consistent across the Maloideae subfamily. As a whole, these maps represent a useful tool to facilitate loquat breeding and an interesting framework for map comparison in the Rosaceae.     

A genetic linkage map for an apricot (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) BC<Subscript>1</Subscript> population mapping <Emphasis Type="Italic">plum pox virus</Emphasis> resistance

Plum pox virus (sharka; PPV) can cause severe crop loss in economically important Prunus species such as peach, plum, apricot, and cherry. Of these species, certain apricot cultivars (‘Stark Early Orange’, ‘Goldrich’, ‘Harlayne’) display significant levels of resistance to the disease and are the genetic substrate for studies of several xlaboratories working cooperatively to genetically characterize and mark the resistance locus or loci for marker-assisted breeding. The goals of the work presented in this communication are the characterization of the genetics of PPV resistance in ‘Stark Early Orange’ and the development of co-dominant molecular markers for marker-assisted selection (MAS) in PPV resistance breeding. We present the first genetic linkage map for an apricot backcross population of ‘Stark Early Orange’ and the susceptible cultivar ‘Vestar’ that segregates for resistance to PPV. This map is comprised of 357 loci (330 amplified fragment length polymorphisms (AFLPs), 26 simple sequence repeats (SSRs), and 1 morphological marker for PPV resistance) assigned to eight linkage groups. Twenty-two of the mapped SSRs are shared in common with genetic reference map for Prunus (T × E; Joobeur et al. 1998) and anchor our apricot map to the general Prunus map. A PPV resistance locus was mapped in linkage group 1 and four AFLP markers segregating with the PPV resistance trait, identified through bulk segregant analysis, facilitated the development of SSRs in this region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lalli, D.A. and Salava, J. contributed equally to this work.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

Pros and cons of using genomic SSRs and EST-SSRs for resolving phylogeny of the genus Gossypium

The genus Gossypium is comprised of 50 diverse cotton species representing eight different genomes (A through G and K), however, phylogenetic relationship using various DNA marker types such as RAPD and SSRs was determined on limited number of cotton species. In this report, we have demonstrated the application of genomic SSRs (gSSRs) and EST-SSRs, and after combining both the data sets, for resolving the phylogenies of 36 cotton species including seven races. Out of the 100 primer pairs surveyed (50 for gSSRs and 50 for EST-SSRs), 75 produced scorable amplification products in all species. Out of these, 73 were found to be polymorphic and amplified 135 alleles ranging from 1 to 5 alleles per SSR marker (average 2.87 alleles per marker). The gSSRs amplified higher number of alleles (72) compared to the EST-SSRs (63). In total 22 highly informative SSRs with PIC values ≥0.5 were identified. Genomic SSRs containing di-while EST-SSRs containing tri-nucleotide repeats exhibited high polymorphism compared to the other nucleotide repeats containing gSSRs/EST-SSRs. Number of tandem repeats and polymorphism were positively correlated. Neither the type of chromosome nor the location of the SSRs showed association with the polymorphism. Gossypium herbaceum var. africanum (Watt) Hutch. ex and Ghose and Gossypium robinsonii F. Muell. were found the most genetically diverse, while among races of Gossypium hirsutum L. “yucatanense” and G. hirsutum “punctatum” were found genetically diverse. Of the three data sets, clustering analysis based on EST-SSRs and combined data sets, revealed parallel results reported in earlier studies. This study further confirmed that Gossypium darwinii Watt has close relationship with Gossypium barbadense L. Moreover, Gossypium raimondii Ulbr. and G. herbaceum/Gossypium arboreum L. are close living relatives of the ancestor allotetraploid species. Our studies suggest that for resolving phylogenetic relationship among the various plant species EST-SSRs could be a better choice. This information can be instrumental in transferring novel alleles or loci from the wild species into the cultivated cotton species which would set a stage for cultivating genetically diverse cultivars—a way to achieve sustainable cotton production in changing climate.     

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.     

Genetic mapping of new cotton fiber loci using EST-derived microsatellites in an interspecific recombinant inbred line cotton population

There is an immediate need for a high-density genetic map of cotton anchored with fiber genes to facilitate marker-assisted selection (MAS) for improved fiber traits. With this goal in mind, genetic mapping with a new set of microsatellite markers [comprising both simple (SSR) and complex (CSR) sequence repeat markers] was performed on 183 recombinant inbred lines (RILs) developed from the progeny of the interspecific cross Gossypium hirsutum L. cv. TM1 × Gossypium barbadense L. Pima 3-79. Microsatellite markers were developed using 1557 ESTs-containing SSRs (≥10 bp) and 5794 EST-containing CSRs (≥12 bp) obtained from ~14,000 consensus sequences derived from fiber ESTs generated from the cultivated diploid species Gossypium arboreum L. cv AKA8401. From a total of 1232 EST-derived SSR (MUSS) and CSR (MUCS) primer-pairs, 1019 (83%) successfully amplified PCR products from a survey panel of six Gossypium species; 202 (19.8%) were polymorphic between the G. hirsutum L. and G. barbadense L. parents of the interspecific mapping population. Among these polymorphic markers, only 86 (42.6%) showed significant sequence homology to annotated genes with known function. The chromosomal locations of 36 microsatellites were associated with 14 chromosomes and/or 13 chromosome arms of the cotton genome by hypoaneuploid deficiency analysis, enabling us to assign genetic linkage groups (LG) to specific chromosomes. The resulting genetic map consists of 193 loci, including 121 new fiber loci not previously mapped. These fiber loci were mapped to 19 chromosomes and 11 LG spanning 1277 cM, providing approximately 27% genome coverage. Preliminary quantitative trait loci analysis suggested that chromosomes 2, 3, 15, and 18 may harbor genes for traits related to fiber quality. These new PCR-based microsatellite markers derived from cotton fiber ESTs will facilitate the development of a high-resolution integrated genetic map of cotton for structural and functional study of fiber genes and MAS of genes that enhance fiber quality. Electronic Supplementary Material Supplementary material is available for this article at Names are necessary to report factually on available data, however, the USDA neither guarantees nor warrants the standard of products or service, and the use of the name by the USDA implies no approval of the products or service to the exclusion of others that may also be suitable.     

Cotton genome mapping with new microsatellites from Acala ‘Maxxa’ BAC-ends

Fine mapping and positional cloning will eventually improve with the anchoring of additional markers derived from genomic clones such as BACs. From 2,603 new BAC-end genomic sequences from Gossypium hirsutum Acala ‘Maxxa’, 1,316 PCR primer pairs (designated as MUSB) were designed to flank microsatellite or simple sequence repeat motif sequences. Most (1164 or 88%) MUSB primer pairs successfully amplified DNA from three species of cotton with an average of three amplicons per marker and 365 markers (21%) were polymorphic between G. hirsutum and G. barbadense. An interspecific RIL population developed from the above two entries was used to map 433 marker loci and 46 linkage groups with a genetic distance of 2,126.3 cM covering approximately 45% of the cotton genome and an average distance between two loci of 4.9 cM. Based on genome-specific chromosomes identified in G. hirsutum tetraploid (A and D), 56.9% of the coverage was located on the A subgenome while 39.7% was assigned to the D subgenome in the genetic map, suggesting that the A subgenome may be more polymorphic and recombinationally active than originally thought. The linkage groups were assigned to 23 of the 26 chromosomes. This is the first genetic map in which the linkage groups A01 and A02/D03 have been assigned to specific chromosomes. In addition the MUSB-derived markers from BAC-end sequences markers allows fine genetic and QTL mapping of important traits and for the first time provides reconciliation of the genetic and physical maps. Limited QTL analyses suggested that loci on chromosomes 2, 3, 12, 15 and 18 may affect variation in fiber quality traits. The original BAC clones containing the newly mapped MUSB that tag the QTLs provide critical DNA regions for the discovery of gene sequences involved in biological processes such as fiber development and pest resistance in cotton. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Development and characterization of simple sequence repeats for flowering dogwood (Cornus florida L.)

Abundant, codominant simple sequence repeats (SSRs) markers can be used for constructing genetic linkage maps and in marker-assisted breeding programs. Enrichment methods for SSR motifs were optimized with the ultimate aim of developing numerous loci in flowering dogwood (C. florida L.) genome. Small insert libraries using four motifs (GT, CT, TGG, and AAC) were constructed with C. florida ‘Cherokee Brave’ deoxyribonucleic acid (DNA). Colony polymerase chain reaction (PCR) of 2,208 selected clones with three primers we reported previously indicated that 47% or 1,034 of the clones harbored one of the four targeted SSR motifs. Sequencing the putative positive clones confirmed that nearly 99% (1,021 of 1,034) of them contained the desired motifs. Of the 871 unique SSR loci, 617 were dinucleotide repeats (70.8%), and 254 were trinucleotide or longer repeats (29.2%). In total, 379 SSR loci had perfect structure, 237 had interrupted, and 255 had compound structure. Primer pairs were designed from 351 unique sequences. The ability of the 351 SSR primer pairs to amplify specific loci was evaluated with genomic DNA of ‘Appalachian Spring’ and ‘Cherokee Brave’. Of these primers, 311 successfully amplified product(s) with ‘Cherokee Brave’ DNA, 21 produced weak or faint products, and 19 did not amplify any products. Additionally, 218 of the 311 primers pairs revealed polymorphisms between the two cultivars, and 20 out of 218 primers detected an average of 13.7 alleles from 38 selected Cornus species and hybrids. These SSR loci constitute a valuable resource of ideal markers for both genetic linkage mapping and gene tagging of flowering dogwood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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     

Development of 28 EST‐SSR markers based on transcriptome sequences of Gobiobotia filifer and cross‐species amplification

Gobiobotia filifer is a small benthic fish distributed in Yangtze River Basin. The abundance of G. filifer increased after impoundment of Xiluodu Dam and Xiangjiaba Dam. The state of population structure and changes of genetic diversity before and after impoundment of Xiluodu Dam and Xiangjiaba Dam were interesting issues. However, efficient molecular markers were rare, which will limit us to solve above problems. Twenty‐eight expressed sequence tag SSRs (EST‐SSRs) were successfully identified and verified as stable amplification and polymorphic loci by polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The number of alleles at these EST‐SSR loci ranged from 3 to 14, the polymorphism information content values were 0.125–0.897, and the observed and expected heterozygosities were 0.0–0.857 and 0.132–0.928, respectively. Cross‐species amplification of the 28 loci developed in this study was examined in seven individuals of each of the 7 taxa. The amplification efficiency of 28 EST‐SSRs primer pairs is related to the distance of genetic relationship between cross‐species with G. filifer, and same subfamily species (Xenophysogobio boulengeri and Xenophysogobio nudicorpa) showed the highest (50%) amplification efficiency. These EST‐SSR markers could be used to analyse genetic diversity and population structure of G. filifer and related species.     

Development and mapping of SNP assays in allotetraploid cotton

A narrow germplasm base and a complex allotetraploid genome have made the discovery of single nucleotide polymorphism (SNP) markers difficult in cotton (Gossypium hirsutum). To generate sequence for SNP discovery, we conducted a genome reduction experiment (EcoRI, BafI double digest, followed by adapter ligation, biotin–streptavidin purification, and agarose gel separation) on two accessions of G. hirsutum and two accessions of G. barbadense. From the genome reduction experiment, a total of 2.04 million genomic sequence reads were assembled into contigs with an N₅₀ of 508 bp and analyzed for SNPs. A previously generated assembly of expressed sequence tags (ESTs) provided an additional source for SNP discovery. Using highly conservative parameters (minimum coverage of 8× at each SNP and 20% minor allele frequency), a total of 11,834 and 1,679 non-genic SNPs were identified between accessions of G. hirsutum and G. barbadense in genome reduction assemblies, respectively. An additional 4,327 genic SNPs were also identified between accessions of G. hirsutum in the EST assembly. KBioscience KASPar assays were designed for a portion of the intra-specific G. hirsutum SNPs. From 704 non-genic and 348 genic markers developed, a total of 367 (267 non-genic, 100 genic) mapped in a segregating F₂ population (Acala Maxxa × TX2094) using the Fluidigm EP1 system. A G. hirsutum genetic linkage map of 1,688 cM was constructed based entirely on these new SNP markers. Of the genic-based SNPs, we were able to identify within which genome (‘A’ or ‘D’) each SNP resided using diploid species sequence data. Genetic maps generated by these newly identified markers are being used to locate quantitative, economically important regions within the cotton genome.     

Molecular dissection of interspecific variation between Gossypium hirsutum and Gossypium barbadense (cotton) by a backcross-self approach: I. Fiber elongation

The current study is the first installment of an effort to explore the secondary gene pool for the enhancement of Upland cotton (Gossypium hirsutum L.) germplasm. We developed advanced-generation backcross populations by first crossing G. hirsutum cv. Tamcot 2111 and G. barbadense cv. Pima S6, then independently backcrossing F₁ plants to the G. hirsutum parent for three cycles. Genome-wide mapping revealed introgressed alleles at an average of 7.3% of loci in each BC₃F₁ plant, collectively representing G. barbadense introgression over about 70% of the genome. Twenty-four BC₃F₁ plants were selfed to generate 24 BC₃F₂ families of 22–172 plants per family (totaling 2,976 plants), which were field-tested for fiber elongation and genetically mapped. One-way analysis of variance detected 22 non-overlapping quantitative trail loci (QTLs) distributed over 15 different chromosomes. The percentage of variance explained by individual loci ranged from 8% to 28%. Although the G. barbadense parent has lower fiber elongation than the G. hirsutum parent, the G. barbadense allele contributed to increased fiber elongation at 64% of the QTLs. Two-way analysis of variance detected significant (P<0.001) among-family genotype effects and genotype×family interactions in two and eight regions, respectively, suggesting that the phenotypic effects of some introgressed chromosomal segments are dependent upon the presence/absence of other chromosomal segments.Electronic Supplementary Material Supplementary material is available for this article at     

Wide coverage of the tetraploid cotton genome using newly developed microsatellite markers

Microsatellite [simple-sequence repeat (SSR)] markers were developed and positioned on the genetic map of tetraploid cotton. Three hundred and ninety-two unique microsatellite sequences, all but two containing a (CA/GT) repeat, were isolated, and the deduced primers were used to screen for polymorphism between the Gossypium hirsutum and G. barbadense parents of the mapping population analyzed in our laboratory. The observed rate of polymorphism was 56%. The 204 polymorphic SSRs revealed 261 segregating bands, which ultimately gave rise to 233 mapped loci. The updated status of our genetic map is now of 1,160 loci and 5,519 cM, with an average distance between two loci of 4.8 cM. The presence of a total of 466 microsatellite loci, with an average distance of 12 cM between two SSR loci, now provides wide coverage of the genome of tetraploid cotton and thus represents a powerful means for the production of a consensus map and for the effective tracking of QTLs.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00122-004-1612-1Communicated by C. Möllers     

Identification and mapping of microsatellite markers linked to a root-knot nematode resistance gene (rkn1) in Acala NemX cotton (Gossypium hirsutum L.)

Host-plant resistance is the most economic and effective strategy for root-knot nematode (RKN) Meloidogyne incognita control in cotton (Gossypium hirsutum L.). Molecular markers linked to resistance are important for incorporating resistance genes into elite cultivars. To screen for microsatellite markers (SSR) closely linked to RKN resistance in G. hirsutum cv. Acala NemX, F₁, F₂, BC₁F₁, and F_2:7 recombinant inbred lines (RILs) from intraspecific crosses and an F₂ from an interspecific cross with G. barbadense cv. Pima S-7 were used. Screening of 284 SSR markers, which cover all the known identified chromosomes and most linkage groups of cotton, was performed by bulked segregant analysis, revealing informative SSRs. The informative SSRs were then mapped on the above populations. One co-dominant SSR marker CIR316 was identified tightly linked to a major resistance gene (designated as rkn1), producing amplified DNA fragments of approximately 221 bp (CIR316a) and 210 bp (CIR316c) in Acala NemX and susceptible Acala SJ-2, respectively. The linkage between CIR316a marker and resistance gene rkn1 in Acala NemX had an estimated distance of 2.1–3.3 cM depending on the population used. Additional markers, including BNL1231 with loose linkage to rkn1 (map distance 25.1–27.4 cM), BNL1066, and CIR003 allowed the rkn1 gene to be mapped to cotton linkage group A03. This is the first report in cotton with a closely linked major gene locus determining nematode resistance, and informative SSRs may be used for marker-assisted selection.     

Assaying polymorphism at DNA level for genetic diversity diagnostics of the safflower (Carthamus tinctorius L.) world germplasm resources

Carthamus tinctorius (2n = 2x = 24), commonly known as safflower, is widely cultivated in agricultural production systems of Asia, Europe, Australia, and the Americas as a source of high quality vegetable and industrial oil. Twenty-two RAPD primers, 18 SSR primers, and 10 AFLP primer combinations were used to assess: (1) the genetic diversity of 85 accessions (originating from 24 countries) representing global germplasm variability of safflower and (2) the interrelationships among safflower ‘centers of similarity’ or ‘regional gene pools’ proposed earlier. The RAPD and SSR primers and AFLP primer combinations revealed 57.6, 68.0, and 71.2% polymorphism, respectively, among 111, 72, and 330 genetic loci amplified from the accessions. The sum of effective number of alleles (66.44), resolving power (59.16), and marker index (51.3) explicitly revealed the relative superiority of AFLP as a marker system in uncovering variation in safflower. Overall, AFLP markers could recognize ‘centers of similarity’ or ‘regional gene pools’. Analysis of molecular variance and Shannon’s information index provided corroborating evidences for the present and previous studies that concluded fragmentation of safflower gene pool into many gene pools. Divergent directional selection is likely to have played an important role in shaping the diversity. From the practical applications standpoint, the diversity of Iran–Afghanistan gene pool is very high, equivalent to the total diversity of the species. The Far East gene pool is the least diverse. The present comprehensive input, first of its own kind in safflower, will assist marker based improvement programmes in the crop.     

Wide-cross whole-genome radiation hybrid mapping of the cotton (Gossypium barbadense L.) genome

Whole-genome radiation hybrid mapping has been applied extensively to human and certain animal species, but little to plants. We recently demonstrated an alternative mapping approach in cotton (Gossypium hirsutum L.), based on segmentation by 5-krad γ-irradiation and derivation of wide-cross whole-genome radiation hybrids (WWRHs). However, limitations observed at the 5-krad level suggested that higher doses might be advantageous. Here, we describe the development of an improved second-generation WWRH panel after higher dose irradiation and compare the resulting map to the 5-krad map. The genome of G. hirsutum (n=26) was used to rescue the radiation-segmented genome of G. barbadense (n=26) introduced via 8- and 12-krad γ-irradiated pollen. Viable seedlings were not recovered after 12-krad irradiation, but 8-krad irradiation permitted plant recovery and construction of a 92-member WWRH mapping panel. Assessment of 31 SSR marker loci from four chromosomes revealed that the 8-krad panel has a marker retention frequency of ca. 76%, which is approximately equivalent to the rate of loss in a low-dose animal radiation hybrid panel. Retention frequencies of loci did not depart significantly from independence when compared between the A and D subgenomes, or according to positions along individual chromosomes. WWRH maps of chromosomes 10 and 17 were generated by the maximum likelihood RHMAP program and the general retention model. The resulting maps bolster evidence that WWRH mapping complements traditional linkage mapping and works in cotton, and that the 8-krad panel complements the 5-krad panel by offering higher rates of chromosome breakages, lower marker retention frequency, and more retention patterns. Electronic Supplementary Material Supplementary material is available for this article at     

Expansion of MIR482/2118 by a class‐II transposable element in cotton

Some plant microRNA (miRNA) families contain multiple members generating identical or highly similar mature miRNA variants. Mechanisms underlying the expansion of miRNA families remain elusive, although tandem and/or segmental duplications have been proposed. In this study of two tetraploid cottons, Gossypium hirsutum and Gossypium barbadense, and their extant diploid progenitors, Gossypium arboreum and Gossypium raimondii, we investigated the gain and loss of members of the miR482/2118 superfamily, which modulates the expression of nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) disease resistance genes. We found significant expansion of MIR482/2118d in G. barbadense, G. hirsutum and G. raimondii, but not in G. arboreum. Several newly expanded MIR482/2118d loci have mutated to produce different miR482/2118 variants with altered target‐gene specificity. Based on detailed analysis of sequences flanking these MIR482/2118 loci, we found that this expansion of MIR482/2118d and its derivatives resulted from an initial capture of an MIR482/2118d by a class‐II DNA transposable element (TE) in G. raimondii prior to the tetraploidization event, followed by transposition to new genomic locations in G. barbadense, G. hirsutum and G. raimondii. The ‘GosTE’ involved in the capture and proliferation of MIR482/2118d and its derivatives belongs to the PIF/Harbinger superfamily, generating a 3‐bp target site duplication upon insertion at new locations. All orthologous MIR482/2118 loci in the two diploids were retained in the two tetraploids, but mutation(s) in miR482/2118 were observed across all four species as well as in different cultivars of both G. barbadense and G. hirsutum, suggesting a dynamic co‐evolution of miR482/2118 and its NBS‐LRR targets. Our results provide fresh insights into the mechanisms contributing to MIRNA proliferation and enrich our knowledge on TEs.     

Identification,characterisation and mapping of simple sequence repeat (SSR) markers from raspberry root and bud ESTs

Raspberry breeding is a long, slow process in this highly heterozygous out-breeder. Selections for complex traits like fruit quality are broad-based and few simple methodologies and resources are available for glasshouse and field screening for key pest and disease resistances. Additionally, the timescale for selection of favourable agronomic traits requires data from different seasons and environmental locations before any breeder selection can proceed to finished cultivar. Genetic linkage mapping offers the possibility of a more knowledge-based approach to breeding through linking favourable traits to markers and candidate genes on genetic linkage maps. To further increase the usefulness of existing maps, a set of 25 polymorphic SSRs derived from expressed sequences (EST-SSRs) have been developed in red raspberry (Rubus idaeus). Two different types of expressed sequences were targeted. One type was derived from a root cDNA library as a first step in assessing sequences which may be involved in root vigour and root rot disease resistance and the second type were ESTs from a gene discovery project examining bud dormancy release and seasonality. The SSRs detect between 2 and 4 alleles per locus and were assigned to linkage groups on the existing ‘Glen Moy’ × ‘Latham’ map following genotyping of 188 progeny and examined for association with previously mapped QTL. The loci were also tested on a diverse range of Rubus species to determine transferability and usefulness for germplasm diversity studies and the introgression of favourable alleles.     

Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (<Emphasis Type="Italic">Vaccinium macrocarpon</Emphasis> Ait.)

The American cranberry (Vaccinium macrocarpon Ait.) is a major commercial fruit crop in North America, but limited genetic resources have been developed for the species. Furthermore, the paucity of codominant DNA markers has hampered the advance of genetic research in cranberry and the Ericaceae family in general. Therefore, we used Roche 454 sequencing technology to perform low-coverage whole genome shotgun sequencing of the cranberry cultivar ‘HyRed’. After de novo assembly, the obtained sequence covered 266.3 Mb of the estimated 540–590 Mb in cranberry genome. A total of 107,244 SSR loci were detected with an overall density across the genome of 403 SSR/Mb. The AG repeat was the most frequent motif in cranberry accounting for 35% of all SSRs and together with AAG and AAAT accounted for 46% of all loci discovered. To validate the SSR loci, we designed 96 primer-pairs using contig sequence data containing perfect SSR repeats, and studied the genetic diversity of 25 cranberry genotypes. We identified 48 polymorphic SSR loci with 2–15 alleles per locus for a total of 323 alleles in the 25 cranberry genotypes. Genetic clustering by principal coordinates and genetic structure analyzes confirmed the heterogeneous nature of cranberries. The parentage composition of several hybrid cultivars was evident from the structure analyzes. Whole genome shotgun 454 sequencing was a cost-effective and efficient way to identify numerous SSR repeats in the cranberry sequence for marker development.