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Development of New Candidate Gene and EST-Based Molecular Markers for Gossypium Species

Development of New Candidate Gene and EST-Based Molecular Markers for Gossypium Species Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2011, Article ID 894598, 9 pages doi:10.1155/2011/894598 Research Article Development of New Candidate Gene and EST-Based Molecular Markers for Gossypium Species 1 1 2 Ramesh Buyyarapu, Ramesh V. Kantety, John Z. Yu, 3 1 Sukumar Saha, andGovindC.Sharma Center for Molecular Biology, Department of Natural Resources and Environmental Sciences, Alabama A&M University, 134 ARC Building, P.O. Box 1927, Normal, AL 35762, USA Southern Plains Agricultural Research Center, USDA-ARS, 2881 F&B Road, College Station, TX 77845, USA Genetics and Precision Agriculture Research Unit, USDA-ARS, P.O. Box 5367, MS 39762, USA Correspondence should be addressed to Ramesh V. Kantety, ramesh.kantety@aamu.edu Received 31 August 2011; Revised 26 October 2011; Accepted 9 November 2011 Academic Editor: Haibao Tang Copyright © 2011 Ramesh Buyyarapu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. New source of molecular markers accelerate the efforts in improving cotton fiber traits and aid in developing high-density integrated genetic maps. We developed new markers based on candidate genes and G. arboreum EST sequences that were used for polymorphism detection followed by genetic and physical mapping. Nineteen gene-based markers were surveyed for polymorphism detection in 26 Gossypium species. Cluster analysis generated a phylogenetic tree with four major sub-clusters for 23 species while three species branched out individually. CAP method enhanced the rate of polymorphism of candidate gene-based markers between G. hirsutum and G. barbadense. Two hundred A-genome based SSR markers were designed after datamining of G. arboreum EST sequences (Mississippi Gossypium arboreum EST-SSR: MGAES). Over 70% of MGAES markers successfully produced amplicons while 65 of them demonstrated polymorphism between the parents of G. hirsutum and G. barbadense RIL population and formed 14 linkage groups. Chromosomal localization of both candidate gene-based and MGAES markers was assisted by euploid and hypoaneuploid CS-B analysis. Gene-based and MGAES markers were highly informative as they were designed from candidate genes and fiber transcriptome with a potential to be integrated into the existing cotton genetic and physical maps. 1. Introduction (A D ) cotton and with 662 loci at ∼1.96 cM intervals for t t diploid (D) genome was constructed using RFLPs, genomic Molecular markers provide valuable information in assessing SSRs, and sequence tagged sites (STS) as probes [9]. the genetic variability, generating linkage maps, enabling Advances in technology have facilitated sequencing of better understanding of the genome organization, and complete transcriptomes and genomes that are accessible deciphering quantitative trait loci (QTLs). Initial effort to through public domain databases. Increasing number of map the cotton genome using an F population utilized 705 expressed sequence tags (ESTs) for cotton facilitated the restriction fragment length polymorphism (RFLP) probes identification of simple sequence repeat (SSR) regions from that were polymorphic between G. hirsutum and G. bar- the ESTs through data mining techniques. EST-SSR markers badense and generated 41 linkage groups spanning 4675 cM reveal putative functional genes and aid in map-based [1]. Genetic variation at molecular level in cotton has been cloning of important genes [10, 11]. In cotton, several characterized using isozyme/allozyme markers [2], RFLPs [1, EST-SSRs have been recently mapped [12–15]. Cotton 3, 4], AFLPs [5, 6], and microsatellites [7, 8]in G. hirsutum fiber genes were mapped with EST-derived SSR loci using and its related species. A comprehensive comparative genetic recombinant inbred line (RIL) population derived from map with 2584 loci at ∼1.72 cM intervals for tetraploid an interspecific cross between G. hirsutum× G. barbadense 2 International Journal of Plant Genomics [16]. Other alternative mapping approaches such as whole- Table 1: Cotton genotypes used for polymorphism detection and phylogenetic tree. genome radiation hybrid (WGRH) mapping and fluorescent in situ hybridization (FISH) mapping have been utilized to No. Species/genotype Genome generate an integrated cotton genome map [17]. 1 G. hirsutum/TM-1 AD1 (CMD1) Assignment of linkage groups or markers to chromo- 2 G. barbadense/3–79 AD2 (CMD2) somes is made by use of aneuploid chromosome substitution 3 Acala maxxa AD1 (CMD3) (F Stocks) lines of G. barbadense in G. hirsutum,euploid 4 DPL 458BR AD1 (CMD4) chromosome substitution lines of G. barbadense in G. hir- sutum, and aneuploid chromosome substitution (F Stocks) 5 Paymaster 1218BR AD1 (CMD5) lines of G. tomentosum in G. hirsutum [18]. Euploid chromo- 6 Fibermax 832 AD1 (CMD6) some substitution stocks were developed by inbreeding the 7 Stoneville 4892 BR AD1 (CMD7) hemizygous monosomic and monotelodisomic substitution 8 PIMA S-6 AD2 (CMD8) stocks through backcrossing up to BC generation [19]. 9 G. arboreum A2 (CMD9) Using these available substitution lines, various molecular 10 G. raimondii D5-3 (CMD10) markers, linkage groups, and QTLs for agronomic and fiber 11 G. tomentosum AD3 (CMD11) quality traits were physically mapped to different cotton 12 G. mustelinum AD4 (CMD12) chromosomes [1, 9, 20]. 13 G. darwinii AD5 Current integrated genetic maps in cotton utilized mainly 14 G. triphyllum B2 RFLP, AFLP, genomic SSR, STS, and EST-SSRs. However, increasing the marker density with functionally expressed 15 G. sturtianum C1 genes would make the linkage maps more valuable for crop 16 G. areysianum E3 improvement programs. New sources of molecular markers 17 G. australe C3 such as cleaved amplified fragment polymorphism (CAP), 18 G. costulatum C5 EST-SSR, and single nucleotide polymorphism (SNP) mark- 19 G. pulchellum C8 ers expand the current but limited repertoire of existing 20 G. thurberi D1 molecular markers. In this study, our objective was to 21 G. armourianum D2-1 understand the genetic diversity and phylogeny of the cul- 22 G. harknessii D2-2 tivated tetraploid, and wild diploid cotton species including 23 G. klotzschianum D3K the Cotton Marker Database (CMD) panel through the 24 G. aridium D4 evaluation of candidate genes, CAP and EST-SSR marker 25 G. gossypioides D6 technologies, and to use these markers in the construction of integrated genetic and physical maps in cotton. 26 G. lobatum D7 Sequence information of candidate genes available in 27 G. trilobum D8 related species for functional genes helps in designing 28 G. turneri D10 primers to amplify and differentiate between the species. 29 G. schwendimanii D11 CAP markers are extensively used in human and animal 30 G. stocksii E1 sciences while they were not exploited well in plant sciences 31 G. somalense E2 for mapping [21]. CAP is an effective technology that uses 32 G. bickii G1 PCR and restriction digestion to elucidate the polymorphism at nucleotide level without the knowledge of the sequence information of a marker. Using the abundant sequence and (2) other geographically diverse tetraploid species and information available for G. arboreum (diploid) fiber ESTs wild diploid accessions (Table 1). Leaf samples were also col- in GenBank, we designed over 700 nonredundant primer lected from USDA-ARS Mississippi State, MS, for aneuploid pairs based on the identification of the SSR regions, and chromosome substitution lines (BC F )of G. barbadense in 0 1 tested 200 primer pairs using the two major cultivated G. hirsutum and seventeen euploid chromosome substitution tetraploid species G. hirsutum (TM-1) and G. barbadense lines of G. barbadense in G. hirsutum. Genomic DNA of 186 (3–79), the parents for RIL population used in this study. individual RILs derived from a cross between TM-1 x 3–79 Polymorphic markers were used for genetic mapping using were provided by USDA-ARS, Southern Plains Agricultural an RIL population, and were further physically localized Research Center, Crop Germplasm Research Unit, College through the use of monosomic, monotelosomic aneuploid, Station, TX. and euploid chromosome substitution lines of G. barbadense in G. hirsutum genetic background. 2.2. DNA Extraction. Young leaf tissues were lyophilized and the DNA was extracted using the Yu lab method at 2. Materials and Methods USDA-ARS, College Station, TX [23]. DNA quality was 2.1. Genetic Materials. Leaf samples were collected from the evaluated using 0.7% agarose gel electrophoresis at 40 V for Cotton Germplasm Research Unit greenhouses at USDA- 3 hours. Genomic DNA was also quantified using TKO 100 ARS, College Station, TX, for (1) Cotton Marker Database fluorometer and further diluted to a working concentration (CMD) panel (12 genotypes of six Gossypium species) [22] of 50 ng/µL for use in polymerase chain reaction (PCR). International Journal of Plant Genomics 3 2.3. Gene-Based Markers. G. arboreum EST sequences in 2.5. Data Analysis. Polymorphic data was scored as binary GenBank were compared with non-redundant (nr) protein values (1 as presence of fragment, 0 as absence) and used database to derive the putative gene function using BLASTX for the calculation of PIC value [27]. Binary data was also program. ESTs that had significant homology with func- used to generate a phylogram using cluster analysis with tional genes in Arabidopsis, Oryza, and others were selected SAS v9.1 (SAS Inc, Cary, NC). Similarly, binary data from for polymorphism screening. Forty-seven primer pairs were RIL population for polymorphic fragments were used to synthesized (Sigma Genosys, The Woodlands, TX) based on create linkage groups using MapManager QTX software these candidate genes that have functional significance in [28]. Recombination frequencies were converted into linkage cotton (See Supplementary File-1 in supplementary material distances using Haldane function [29]. The maximum available on line at doi:10.1155/2011/894598). Primers were linkage distance below 50 cM between any two markers and evaluated for amplification using PCR at two annealing an LOD (logarithm of odds) score of 4 and above were conditions (T = 50 C and 60–55 touchdown). Ampli- considered optimal to qualify as a linkage group. fiedproductsweresurveyedfor polymorphism using 6% polyacrylamide gel electrophoresis (PAGE) and scored in 3. Results and Discussion binary fashion for each fragment size. The data was used to calculate the Polymorphism Information Content (PIC) 3.1. Candidate Gene-Based Markers. The thrust of this effort value. Cluster analysis was conducted with nearest neighbor- was to expand the very limited base of markers that are hood joining method in classifying the binary data derived utilized in characterizing genetic variability in Gossypium. to generate phylogenetic tree to assess the evolutionary Comparative genetic approaches have been proved successful relationships [24] among the five tetraploid and 21 diploid for characterizing genomes and mapping important traits species. PCR products of the monomorphic markers between based on the sequence information in related plant species G. hirsutum and G. barbadense were subjected to digestion [30]. Candidate genes in G. arboreum EST sequences were using RsaI, MspI, HhaI, and HaeIII restriction endonucleases identified by comparison with distant species using BLASTX andsurveyedfor polymorphism using PAGE fordetection to reveal the gene function information and such ESTs were of CAP markers. Fragment-based and CAP-based markers used to design primers. Nineteen candidate gene markers were subsequently tested for chromosomal localization in (40%) were successfully amplified out of 47 gene-based aneuploid and euploid chromosome substitution lines. markers screened across the 32 genotypes from 26 diverse Gossypium species tested in this study. PAGE fragment 2.4. EST-SSR-Based Markers. G. arboreum ESTs (38,893) analysis for amplified products identified 13 markers that were collected from GenBank and were searched for the were polymorphic among these cotton species (68%). Binary presence of SSR sequences, followed by masking. The masked fragment data for these markers were used to calculate PIC ESTs were clustered using StackPack v2.1 (Electric Genetics, value for each marker that ranged from 0.794 to 0.998 Reston, VA) software to reduce the redundancy. The non- (Table 2). redundant (NR) sequences that contain an SSR motif were Though these gene-based markers were highly polymor- selected for further analysis as described by Kantety et al. phic across multiple cotton species, the fragment polymor- [25]. A subset of NR-ESTs mainly expressed in fibers (725) phism rates detected using direct amplicons were very low were identified with having a repeat length 18 or more. for the two cultivated tetraploids versus G. hirsutum and Among this subset SSR containing NR-ESTs, we designed G. barbadense. Only one polymorphic marker was identified 200 primer-pairs for further analysis. They were designated between G. hirsutum TM-1 (CMD-1) and G. barbadense as Mississippi Gossypium arboreum EST-SSRs (MGAES). The PIMA 3–79 (CMD-2). This limitation led us to explore design of the primers was based on the sequence information additional avenues by restriction digestion of the large PCR flanking the SSR region with an estimated product size of fragments to survey for polymorphism at a higher resolution. ∼200–300 base pairs using Primer3 software [26]and were CAP markers have been used in marker-assisted selection synthesized at Sigma-Genosys (Sigma-Aldrich, Saint Louis, process and mapping genetic loci of interest [21, 31]. Large MO). The primer sequences, EST sources, and their putative amplicon sizes for many gene-based markers provided an function were summarized in Supplementary File-2. The opportunity to employ CAP technique to detect nucleotide MGAES primers were verified against all the SSR marker level polymorphism for TM-1 and PIMA 3–79. To enable primer sequences available at Cotton Microsatellite Database higher restriction site choices in these amplicons, we used (CMD, http://www.cottonmarker.org/) for redundancy and RsaI, MspI, HhaI, and HaeIII enzymes that detect four base sequence homology using BLAST search. These MGAES and restriction site recognition motifs. CAP technique identified gene-based primer sequences will be submitted to CMD for eleven polymorphic markers (58%) of the 19 tested on TM-1 cotton research community use. MGAES primers were first and PIMA 3–79 suggesting the potential for CAP technology amplified on the RIL parents: G. hirsutum and G. barbadense, as a useful resource for identifying genetic variation. One ◦ ◦ at annealing conditions of 50 Cand 55 C; and surveyed fragment-based and five CAP-based markers were localized for fragment length polymorphism using 6% PAGE. Poly- to cotton chromosome or chromosome-arm using the morphic markers were then identified to genotype the RIL euploid CS-B lines. Our results suggest that CAP-based population for construction of genetic linkage groups. The marker technology is a robust approach for detection of amplified markers were also used for physical mapping onto variation in closely related species and provides an alternative chromosomes and chromosome arms. to cost-intensive SNP-based approaches. 4 International Journal of Plant Genomics Table 2: Polymorphism information content and chromosomal localization of gene-based markers by CAP technique. Marker Putative gene PIC value Restriction enzyme Chromosome BG2926 Actin gene 0.949 — — BG7042 S-adenosyl-L-methionine decarboxylase proenzyme 0.997 — — BG7067 Low MW heat shock protein gene (Gmhsp17.6-L) 0.988 — — BG7092 Glyceraldehyde-3-phosphate dehydrogenase 0.981 — — BG7164 Mitogen-activated protein kinase (MAPK) 0.917 — — BG7197 Auxin induced basic helix-loop-helix transcription factor 0.934 — — BG7211 DNA-binding protein (WRKY 1) 0.967 RsaI 11 short arm BG7213 Zinc finger protein (TIM9) 0.908 — — BG7215 Acyl CoA independent ceramide 0.912 RsaI6 BG7226 Potassium transporter HAK3p 0.967 — — BG7238 Photolyase/blue light photoreceptor phr2 0.849 — — BG7314 Copalyl diphosphate synthase 1 0.954 HaeIII 14 short arm, 25 BG7356 Omega-3 fatty acid desaturase (FAD3) 0.902 — — BG7405 Transcription factor (Hap5a) 0.967 HhaI 10, 16, 22 Short arm BG7411 Ubiquitin extension protein 0.849 — — BG7428 Cinnamic acid 4-hydroxylase 0.794 — — BG7443 Small heat stress protein 0.952 MspI16 BG7446 G-protein beta subunit 0.84 — — BG7485 Flavonoid 3 -hydroxylase 0.998 HhaI16 Euploid CS-B lines were annotated on the basis of the origin of New World tetraploids from the Old World diploids chromosome pair substituted for the complete chromo- [38]. somesorchromosomearmsof G. hirsutum monosomic In this study, a total of 76 fragments from nineteen can- or monotelodisomics [19, 32]. If a polymorphic marker didate gene-based markers were observed across the diverse between G. hirsutum and G. barbadense showed similar panel of 32 cotton genotypes. Binary data derived from fragment patterns to that of G. barbadense in a euploid the fragment analysis was used to generate a phylogenetic CS-B line, then that marker was concluded to be localized tree providing the evolutionary relationships among the 26 to particular substituted chromosome or arm. In this cotton species by cluster analysis using maximum likelihood manner, both dominant and recessive alleles were physically method (Figure 1). Cluster analysis resulted in a dendrogram mapped using euploid CS-B lines. One amplicon length comprising four major clusters grouping 23 species except polymorphism and five CAP-based markers were localized that G. bickii (G1), G. pulchellum (C3), and G. australe to seven chromosomes or arms using the euploid CS-B (C8) branched out individually. As the dendrogram was lines (Table 2). As these markers were based on homologous derived based on the maximum likelihood method, it gene sequences, there is a possibility of having multiple provides the evolutionary relationships based on combined copies in the tetraploid cotton genomes. Therefore this study genetic lineages of the candidate genes used in this study. adds a new set of gene-based markers with their specific Many genotypes from tetraploid species formed into two chromosomal location and help in assessing the evolutionary clades while the diploid species form the remaining. Species relationships among the 26 Gossypium species. belonging to the same genome are grouped together to form subclusters in the dendrogram and was evident in grouping of tetraploids as wells as other diploid species. Though the 3.2. Phylogenetic Analysis. Gossypium genus includes five number of genes used in this study is not an exhaustive data tetraploid species from AD –AD genomes and approxi- 1 5 set, the evolutionary relationships among the 26 species were mately forty-five diploid species from genome groups A–G mostly congruent with earlier studies [37]. The dendrogram and K [33]. Of these all five tetraploid species and twenty-one also supported the theory that G. darwinii (AD ), another diploid species belonging to A(1), B(1), C(4), D(11), E(3), tetraploid species endemic to Galapagos Islands is closely and G(1) genomes were included in this study. Relationships related to G. barbadense (AD )[39, 40]. among these cotton genome groups were studied earlier using polymorphisms exhibited in chloroplast genome [34], ribosomal genes [35], and Adh genes [36]. These studies 3.3. G. arboretum EST-SSR Markers. Availability of abun- showed close relationships among the species belonging to dant EST information in public databases and detection the same geographical origin [37] besides explaining the procedures to identify SSR regions in ESTs have established International Journal of Plant Genomics 5 G. hirsutum TM-1 AD1 CMD1 G. hirsutum Acala maxxa AD1 CMD3 G. hirsutum DPL 458BR AD1 CMD4 G. hirsutum Fibermax AD1 CMD6 AD1 CMD7 G. hirsutum Stoneville 4892BR AD2 CMD2 G. barbadense PIMA3-79 AD5 G. darwinii AD4 CMD12 G. mustelinum AD1 CMD5 G. hirsutum Paymaster G. barbadense PIMA S-6 AD2 CMD8 G. klotschianum D3K E3 G. areysianum A2 CMD9 G. arboreum G. tomentosum AD3 CMD11 G. lobatum D7 G. aridium D4 G. stocksii D6 E2 G. gossypioides G. somalense D8 G. trilobum E1 G. turneri C5 G. costulatum D11 G. schwendimanii D10 G. raimondii D5-3 CMD10 G. thurberi D1 G. armourianum D2-1 G. harknessii D2-2 G. bickii G1 G. australe C3 G. pulchellum C8 G. triphyllum B2 G. sturtianum C1 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Average distance between clusters Figure 1: Phylogenetic relationships among the 32 Gossypium genotypes (Table 1) based on the fragment size polymorphism. EST-SSRs as an informative resource for genetic mapping detected using G. arboreum-based EST-SSRs suggested the [7, 12, 14, 25, 41]. Despite the earlier efforts by Han et al. potential of cross-species transferability of these markers [15]and Part et al.[16] to characterize cotton genome using among diploid and tetraploid species [42]. Many of these EST-SSRs, the molecular variation in the coding regions MGAES markers were derived from the fiber expressed ESTs of many fiber expressed genes was not yet fully utilized to thus making them more valuable in breeding programs assist the marker-assisted selection of important fiber traits. and marker-assisted selection for fiber-associated traits. Two hundred primer pairs were designed specifically from Polymorphism detected for each fragment in 186 RILs was fiber related ESTs were used for polymorphism detection scored initially in ternary fashion and then converted into and mapping in this study. Except 27 individual primer binary fashion by treating the heterozygous alleles as missing sequences, the rest of the MGAES markers were new and values. MapManager program was used for constructing nonrepetitive based on BLAST homology search from earlier linkage groups. Fourteen linkage groups were generated studies of Han et al. [15]and Park et al.[41]. Though spanning ∼399 cM with minimum two markers for linkage these EST-SSRs were derived from a diploid progenitor group and with an LOD score threshold of 4. These linkage (genome A )oftetraploidspecies (A D ), 147 markers groups and polymorphic markers can be incorporated into 1−2 t t (74%) out of 200 primer pairs were successfully amplified existing genetic maps to generate an integrated genetic map in tetraploid cultivars G. hirsutum TM-1 and G. barbadense for cotton. PIMA 3–79 suggesting considerable homology exists of the tetraploid cotton with the diploid ancestral species. Sixty- 3.4. Chromosomal Localization of MGAES Markers. Physical five MGAES markers (44%) were polymorphic between TM- mapping of the polymorphic markers was facilitated using 1 and PIMA 3–79 indicating the merit of these markers aneuploid (Figure 2) and euploid chromosome substitution due to high rate of polymorphism compared to earlier lines (Figure 3). Sixteen markers were localized to different studies [15]. Though these G. arboreum EST-SSR markers chromosomes using euploid CS-B lines while 14 markers were highly polymorphic between the G. hirsutum and were localized using aneuploid CS-B lines. Missing a poly- G. barbadense species, we observed very low polymorphism morphic locus in a specific aneuploid (BC F ) accession 0 1 rate within each species. High polymorphic rate between determines the chromosomal localization of a dominant the species could also be attributed to amplification of marker to that corresponding specific chromosome or genotypes under stringent PCR conditions to avoid non- chromosome arm. Results derived from both aneuploid and specific amplification. High levels of polymorphism were euploid CS-B lines served as cross-reference to each other. 6 International Journal of Plant Genomics Table 3: Genetic and physical mapping of G. arboreum-based EST-SSR markers. (—: physical mapping was inconclusive; X: unlinked marker). Annealing Aneuploid CS-B chromosome Marker Euploid CS-B chromosome localization Linkage group temperature ( C) localization MGAES-2 55 — — LG 14 MGAES-3 55 CS-B 01 Te7Sh LG 14 MGAES-5 55 — — X MGAES-10 50 CS-B 15Sh — X MGAES-11 50 — Te9Lo, H9 LG 9 MGAES-21 55 XX Te20Lo, H20 X MGAES-22 55 CS-B 01 — X MGAES-25 55 — — LG 5 MGAES-27 50 — — X MGAES-28 55 CS-B 10 H3Sub, Te11Lo, H11, NTN12-11 LG 7 MGAES-40 50 CS-B26Lo X MGAES-41 55 — — X MGAES-43 50 — — X MGAES-44 55 — — LG 5 MGAES-49 55 — — LG 6 MGAES-51 55 — — LG 6 MGAES-57 55 NTN17-11 H3Sub X MGAES-58 55 — H3Sub LG 10 MGAES-64 55 CS-B 11Sh Te11Lo, H11, NTN12-11 LG 7 MGAES-72 55 — — X MGAES-73 55 — — X MGAES-78 55 CS-B 11Sh Te11Lo, H11 LG 7 MGAES-80 55 — — X MGAES-81 50 — — X MGAES-82 55 — — X MGAES-83 55 CS-B04, CS-B 14Sh, NTN4-15, NTN10-19 — X MGAES-87 55 CS-B 11Sh Te11Lo, H10 LG 4 MGAES-91 55 — — X MGAES-95 55 CS-B 02 Te2Lo, H3SuB X MGAES-104 50 — — X MGAES-105 55 — Te15, Te20Lo, H3Sub LG 5 MGAES-106 55 CS-B26Lo H12, NTN12-11 LG 3 MGAES-107 55 — Te7Lo X MGAES-111 50 — — X MGAES-122 50 CS-B 11Sh, NTN17-11 — LG 12 MGAES-126 50 — — LG 8 MGAES-130 55 NTN4-15 H3Sub LG 13 MGAES-135 55 CS-B 18 Te18Lo, H18 LG 1 MGAES-141 55 — — LG 2 MGAES-142 55 — H10 X MGAES-143 55 — — LG 11 MGAES-153 50 CS-B 01 — X MGAES-157 55 — — X MGAES-160 50 — — X MGAES-161 50 — — X MGAES-165 50 — — X MGAES-194 55 — — LG 2 MGAES-200 50 — — X International Journal of Plant Genomics 7 L 1234 5 6 7 8 910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 L Figure 2: Chromosomal localization of EST-SSR marker MGAES-64 to Te11Lo (lane 10), H11 (lane 28), NTN12-11 (lane 38), and G. barbadense (lane 42) lines using aneuploid CS-B lines. L 1 24 3 2 5 4 7 6 9 8 11101312151417161918212023 22 Figure 3: Chromosomal localization of EST-SSR marker MGAES-64 to CS-B 11Sh using euploid CS-B lines: sample 8: CS-B 11Sh, sample 24: G. barbadense (3–79). AFLP: Amplified fragment length polymorphism For example, MGAES-64 marker was localized to H11 and Te11Lo aneuploid CS-B accessions where the accessions were SNP: Single nucleotide polymorphism CMD: Cotton Marker Database. deficient for chromosome 11 and its long arm, respectively; in both accessions the missing G. hirsutum fragment or locus has been observed confirming the localization of the marker Acknowledgments to chromosome 11 and its long arm (Figure 2). Using euploid chromosome substitution lines, the same This paper was supported by USDA-CSREES Grant no. MGAES-64 marker has been localized to CS-B11Sh acces- ALAX-011-706 to R. V. Kantety, ALAX-011-206 to G. C. sion, where a pair of chromosomes from G. barbadense Sharma, and NSF Plant Genome Research award no. 0703470 was substituted for the long-arm deficient ditelosomic lines to R. V. Kantety. The authors would like to acknowl- of G. hirsutum; polymorphic fragment corresponding to edge the informatics and review support provided by Mr. G. barbadense was only observed in CS-B11Sh explaining Venkateswara Rao Sripathi and other anonymous reviewers its localization to chromosome 11 long arm (Figure 3). who assisted in improving the paper. This is journal article Polymorphic markers, linkage group information, euploid, no. 629 of Alabama A&M University Agricultural Experi- and aneuploid CS-B chromosome localization were shown ment Station. in Table 3. However, we observed incongruency in localizing some markers to just a single chromosome using euploid References and aneuploid CS-B analysis. This needs to be further investigated as it might be a result of duplicated loci or [1] A. J. 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Development of New Candidate Gene and EST-Based Molecular Markers for Gossypium Species

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Copyright © 2011 Ramesh Buyyarapu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2011, Article ID 894598, 9 pages doi:10.1155/2011/894598 Research Article Development of New Candidate Gene and EST-Based Molecular Markers for Gossypium Species 1 1 2 Ramesh Buyyarapu, Ramesh V. Kantety, John Z. Yu, 3 1 Sukumar Saha, andGovindC.Sharma Center for Molecular Biology, Department of Natural Resources and Environmental Sciences, Alabama A&M University, 134 ARC Building, P.O. Box 1927, Normal, AL 35762, USA Southern Plains Agricultural Research Center, USDA-ARS, 2881 F&B Road, College Station, TX 77845, USA Genetics and Precision Agriculture Research Unit, USDA-ARS, P.O. Box 5367, MS 39762, USA Correspondence should be addressed to Ramesh V. Kantety, ramesh.kantety@aamu.edu Received 31 August 2011; Revised 26 October 2011; Accepted 9 November 2011 Academic Editor: Haibao Tang Copyright © 2011 Ramesh Buyyarapu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. New source of molecular markers accelerate the efforts in improving cotton fiber traits and aid in developing high-density integrated genetic maps. We developed new markers based on candidate genes and G. arboreum EST sequences that were used for polymorphism detection followed by genetic and physical mapping. Nineteen gene-based markers were surveyed for polymorphism detection in 26 Gossypium species. Cluster analysis generated a phylogenetic tree with four major sub-clusters for 23 species while three species branched out individually. CAP method enhanced the rate of polymorphism of candidate gene-based markers between G. hirsutum and G. barbadense. Two hundred A-genome based SSR markers were designed after datamining of G. arboreum EST sequences (Mississippi Gossypium arboreum EST-SSR: MGAES). Over 70% of MGAES markers successfully produced amplicons while 65 of them demonstrated polymorphism between the parents of G. hirsutum and G. barbadense RIL population and formed 14 linkage groups. Chromosomal localization of both candidate gene-based and MGAES markers was assisted by euploid and hypoaneuploid CS-B analysis. Gene-based and MGAES markers were highly informative as they were designed from candidate genes and fiber transcriptome with a potential to be integrated into the existing cotton genetic and physical maps. 1. Introduction (A D ) cotton and with 662 loci at ∼1.96 cM intervals for t t diploid (D) genome was constructed using RFLPs, genomic Molecular markers provide valuable information in assessing SSRs, and sequence tagged sites (STS) as probes [9]. the genetic variability, generating linkage maps, enabling Advances in technology have facilitated sequencing of better understanding of the genome organization, and complete transcriptomes and genomes that are accessible deciphering quantitative trait loci (QTLs). Initial effort to through public domain databases. Increasing number of map the cotton genome using an F population utilized 705 expressed sequence tags (ESTs) for cotton facilitated the restriction fragment length polymorphism (RFLP) probes identification of simple sequence repeat (SSR) regions from that were polymorphic between G. hirsutum and G. bar- the ESTs through data mining techniques. EST-SSR markers badense and generated 41 linkage groups spanning 4675 cM reveal putative functional genes and aid in map-based [1]. Genetic variation at molecular level in cotton has been cloning of important genes [10, 11]. In cotton, several characterized using isozyme/allozyme markers [2], RFLPs [1, EST-SSRs have been recently mapped [12–15]. Cotton 3, 4], AFLPs [5, 6], and microsatellites [7, 8]in G. hirsutum fiber genes were mapped with EST-derived SSR loci using and its related species. A comprehensive comparative genetic recombinant inbred line (RIL) population derived from map with 2584 loci at ∼1.72 cM intervals for tetraploid an interspecific cross between G. hirsutum× G. barbadense 2 International Journal of Plant Genomics [16]. Other alternative mapping approaches such as whole- Table 1: Cotton genotypes used for polymorphism detection and phylogenetic tree. genome radiation hybrid (WGRH) mapping and fluorescent in situ hybridization (FISH) mapping have been utilized to No. Species/genotype Genome generate an integrated cotton genome map [17]. 1 G. hirsutum/TM-1 AD1 (CMD1) Assignment of linkage groups or markers to chromo- 2 G. barbadense/3–79 AD2 (CMD2) somes is made by use of aneuploid chromosome substitution 3 Acala maxxa AD1 (CMD3) (F Stocks) lines of G. barbadense in G. hirsutum,euploid 4 DPL 458BR AD1 (CMD4) chromosome substitution lines of G. barbadense in G. hir- sutum, and aneuploid chromosome substitution (F Stocks) 5 Paymaster 1218BR AD1 (CMD5) lines of G. tomentosum in G. hirsutum [18]. Euploid chromo- 6 Fibermax 832 AD1 (CMD6) some substitution stocks were developed by inbreeding the 7 Stoneville 4892 BR AD1 (CMD7) hemizygous monosomic and monotelodisomic substitution 8 PIMA S-6 AD2 (CMD8) stocks through backcrossing up to BC generation [19]. 9 G. arboreum A2 (CMD9) Using these available substitution lines, various molecular 10 G. raimondii D5-3 (CMD10) markers, linkage groups, and QTLs for agronomic and fiber 11 G. tomentosum AD3 (CMD11) quality traits were physically mapped to different cotton 12 G. mustelinum AD4 (CMD12) chromosomes [1, 9, 20]. 13 G. darwinii AD5 Current integrated genetic maps in cotton utilized mainly 14 G. triphyllum B2 RFLP, AFLP, genomic SSR, STS, and EST-SSRs. However, increasing the marker density with functionally expressed 15 G. sturtianum C1 genes would make the linkage maps more valuable for crop 16 G. areysianum E3 improvement programs. New sources of molecular markers 17 G. australe C3 such as cleaved amplified fragment polymorphism (CAP), 18 G. costulatum C5 EST-SSR, and single nucleotide polymorphism (SNP) mark- 19 G. pulchellum C8 ers expand the current but limited repertoire of existing 20 G. thurberi D1 molecular markers. In this study, our objective was to 21 G. armourianum D2-1 understand the genetic diversity and phylogeny of the cul- 22 G. harknessii D2-2 tivated tetraploid, and wild diploid cotton species including 23 G. klotzschianum D3K the Cotton Marker Database (CMD) panel through the 24 G. aridium D4 evaluation of candidate genes, CAP and EST-SSR marker 25 G. gossypioides D6 technologies, and to use these markers in the construction of integrated genetic and physical maps in cotton. 26 G. lobatum D7 Sequence information of candidate genes available in 27 G. trilobum D8 related species for functional genes helps in designing 28 G. turneri D10 primers to amplify and differentiate between the species. 29 G. schwendimanii D11 CAP markers are extensively used in human and animal 30 G. stocksii E1 sciences while they were not exploited well in plant sciences 31 G. somalense E2 for mapping [21]. CAP is an effective technology that uses 32 G. bickii G1 PCR and restriction digestion to elucidate the polymorphism at nucleotide level without the knowledge of the sequence information of a marker. Using the abundant sequence and (2) other geographically diverse tetraploid species and information available for G. arboreum (diploid) fiber ESTs wild diploid accessions (Table 1). Leaf samples were also col- in GenBank, we designed over 700 nonredundant primer lected from USDA-ARS Mississippi State, MS, for aneuploid pairs based on the identification of the SSR regions, and chromosome substitution lines (BC F )of G. barbadense in 0 1 tested 200 primer pairs using the two major cultivated G. hirsutum and seventeen euploid chromosome substitution tetraploid species G. hirsutum (TM-1) and G. barbadense lines of G. barbadense in G. hirsutum. Genomic DNA of 186 (3–79), the parents for RIL population used in this study. individual RILs derived from a cross between TM-1 x 3–79 Polymorphic markers were used for genetic mapping using were provided by USDA-ARS, Southern Plains Agricultural an RIL population, and were further physically localized Research Center, Crop Germplasm Research Unit, College through the use of monosomic, monotelosomic aneuploid, Station, TX. and euploid chromosome substitution lines of G. barbadense in G. hirsutum genetic background. 2.2. DNA Extraction. Young leaf tissues were lyophilized and the DNA was extracted using the Yu lab method at 2. Materials and Methods USDA-ARS, College Station, TX [23]. DNA quality was 2.1. Genetic Materials. Leaf samples were collected from the evaluated using 0.7% agarose gel electrophoresis at 40 V for Cotton Germplasm Research Unit greenhouses at USDA- 3 hours. Genomic DNA was also quantified using TKO 100 ARS, College Station, TX, for (1) Cotton Marker Database fluorometer and further diluted to a working concentration (CMD) panel (12 genotypes of six Gossypium species) [22] of 50 ng/µL for use in polymerase chain reaction (PCR). International Journal of Plant Genomics 3 2.3. Gene-Based Markers. G. arboreum EST sequences in 2.5. Data Analysis. Polymorphic data was scored as binary GenBank were compared with non-redundant (nr) protein values (1 as presence of fragment, 0 as absence) and used database to derive the putative gene function using BLASTX for the calculation of PIC value [27]. Binary data was also program. ESTs that had significant homology with func- used to generate a phylogram using cluster analysis with tional genes in Arabidopsis, Oryza, and others were selected SAS v9.1 (SAS Inc, Cary, NC). Similarly, binary data from for polymorphism screening. Forty-seven primer pairs were RIL population for polymorphic fragments were used to synthesized (Sigma Genosys, The Woodlands, TX) based on create linkage groups using MapManager QTX software these candidate genes that have functional significance in [28]. Recombination frequencies were converted into linkage cotton (See Supplementary File-1 in supplementary material distances using Haldane function [29]. The maximum available on line at doi:10.1155/2011/894598). Primers were linkage distance below 50 cM between any two markers and evaluated for amplification using PCR at two annealing an LOD (logarithm of odds) score of 4 and above were conditions (T = 50 C and 60–55 touchdown). Ampli- considered optimal to qualify as a linkage group. fiedproductsweresurveyedfor polymorphism using 6% polyacrylamide gel electrophoresis (PAGE) and scored in 3. Results and Discussion binary fashion for each fragment size. The data was used to calculate the Polymorphism Information Content (PIC) 3.1. Candidate Gene-Based Markers. The thrust of this effort value. Cluster analysis was conducted with nearest neighbor- was to expand the very limited base of markers that are hood joining method in classifying the binary data derived utilized in characterizing genetic variability in Gossypium. to generate phylogenetic tree to assess the evolutionary Comparative genetic approaches have been proved successful relationships [24] among the five tetraploid and 21 diploid for characterizing genomes and mapping important traits species. PCR products of the monomorphic markers between based on the sequence information in related plant species G. hirsutum and G. barbadense were subjected to digestion [30]. Candidate genes in G. arboreum EST sequences were using RsaI, MspI, HhaI, and HaeIII restriction endonucleases identified by comparison with distant species using BLASTX andsurveyedfor polymorphism using PAGE fordetection to reveal the gene function information and such ESTs were of CAP markers. Fragment-based and CAP-based markers used to design primers. Nineteen candidate gene markers were subsequently tested for chromosomal localization in (40%) were successfully amplified out of 47 gene-based aneuploid and euploid chromosome substitution lines. markers screened across the 32 genotypes from 26 diverse Gossypium species tested in this study. PAGE fragment 2.4. EST-SSR-Based Markers. G. arboreum ESTs (38,893) analysis for amplified products identified 13 markers that were collected from GenBank and were searched for the were polymorphic among these cotton species (68%). Binary presence of SSR sequences, followed by masking. The masked fragment data for these markers were used to calculate PIC ESTs were clustered using StackPack v2.1 (Electric Genetics, value for each marker that ranged from 0.794 to 0.998 Reston, VA) software to reduce the redundancy. The non- (Table 2). redundant (NR) sequences that contain an SSR motif were Though these gene-based markers were highly polymor- selected for further analysis as described by Kantety et al. phic across multiple cotton species, the fragment polymor- [25]. A subset of NR-ESTs mainly expressed in fibers (725) phism rates detected using direct amplicons were very low were identified with having a repeat length 18 or more. for the two cultivated tetraploids versus G. hirsutum and Among this subset SSR containing NR-ESTs, we designed G. barbadense. Only one polymorphic marker was identified 200 primer-pairs for further analysis. They were designated between G. hirsutum TM-1 (CMD-1) and G. barbadense as Mississippi Gossypium arboreum EST-SSRs (MGAES). The PIMA 3–79 (CMD-2). This limitation led us to explore design of the primers was based on the sequence information additional avenues by restriction digestion of the large PCR flanking the SSR region with an estimated product size of fragments to survey for polymorphism at a higher resolution. ∼200–300 base pairs using Primer3 software [26]and were CAP markers have been used in marker-assisted selection synthesized at Sigma-Genosys (Sigma-Aldrich, Saint Louis, process and mapping genetic loci of interest [21, 31]. Large MO). The primer sequences, EST sources, and their putative amplicon sizes for many gene-based markers provided an function were summarized in Supplementary File-2. The opportunity to employ CAP technique to detect nucleotide MGAES primers were verified against all the SSR marker level polymorphism for TM-1 and PIMA 3–79. To enable primer sequences available at Cotton Microsatellite Database higher restriction site choices in these amplicons, we used (CMD, http://www.cottonmarker.org/) for redundancy and RsaI, MspI, HhaI, and HaeIII enzymes that detect four base sequence homology using BLAST search. These MGAES and restriction site recognition motifs. CAP technique identified gene-based primer sequences will be submitted to CMD for eleven polymorphic markers (58%) of the 19 tested on TM-1 cotton research community use. MGAES primers were first and PIMA 3–79 suggesting the potential for CAP technology amplified on the RIL parents: G. hirsutum and G. barbadense, as a useful resource for identifying genetic variation. One ◦ ◦ at annealing conditions of 50 Cand 55 C; and surveyed fragment-based and five CAP-based markers were localized for fragment length polymorphism using 6% PAGE. Poly- to cotton chromosome or chromosome-arm using the morphic markers were then identified to genotype the RIL euploid CS-B lines. Our results suggest that CAP-based population for construction of genetic linkage groups. The marker technology is a robust approach for detection of amplified markers were also used for physical mapping onto variation in closely related species and provides an alternative chromosomes and chromosome arms. to cost-intensive SNP-based approaches. 4 International Journal of Plant Genomics Table 2: Polymorphism information content and chromosomal localization of gene-based markers by CAP technique. Marker Putative gene PIC value Restriction enzyme Chromosome BG2926 Actin gene 0.949 — — BG7042 S-adenosyl-L-methionine decarboxylase proenzyme 0.997 — — BG7067 Low MW heat shock protein gene (Gmhsp17.6-L) 0.988 — — BG7092 Glyceraldehyde-3-phosphate dehydrogenase 0.981 — — BG7164 Mitogen-activated protein kinase (MAPK) 0.917 — — BG7197 Auxin induced basic helix-loop-helix transcription factor 0.934 — — BG7211 DNA-binding protein (WRKY 1) 0.967 RsaI 11 short arm BG7213 Zinc finger protein (TIM9) 0.908 — — BG7215 Acyl CoA independent ceramide 0.912 RsaI6 BG7226 Potassium transporter HAK3p 0.967 — — BG7238 Photolyase/blue light photoreceptor phr2 0.849 — — BG7314 Copalyl diphosphate synthase 1 0.954 HaeIII 14 short arm, 25 BG7356 Omega-3 fatty acid desaturase (FAD3) 0.902 — — BG7405 Transcription factor (Hap5a) 0.967 HhaI 10, 16, 22 Short arm BG7411 Ubiquitin extension protein 0.849 — — BG7428 Cinnamic acid 4-hydroxylase 0.794 — — BG7443 Small heat stress protein 0.952 MspI16 BG7446 G-protein beta subunit 0.84 — — BG7485 Flavonoid 3 -hydroxylase 0.998 HhaI16 Euploid CS-B lines were annotated on the basis of the origin of New World tetraploids from the Old World diploids chromosome pair substituted for the complete chromo- [38]. somesorchromosomearmsof G. hirsutum monosomic In this study, a total of 76 fragments from nineteen can- or monotelodisomics [19, 32]. If a polymorphic marker didate gene-based markers were observed across the diverse between G. hirsutum and G. barbadense showed similar panel of 32 cotton genotypes. Binary data derived from fragment patterns to that of G. barbadense in a euploid the fragment analysis was used to generate a phylogenetic CS-B line, then that marker was concluded to be localized tree providing the evolutionary relationships among the 26 to particular substituted chromosome or arm. In this cotton species by cluster analysis using maximum likelihood manner, both dominant and recessive alleles were physically method (Figure 1). Cluster analysis resulted in a dendrogram mapped using euploid CS-B lines. One amplicon length comprising four major clusters grouping 23 species except polymorphism and five CAP-based markers were localized that G. bickii (G1), G. pulchellum (C3), and G. australe to seven chromosomes or arms using the euploid CS-B (C8) branched out individually. As the dendrogram was lines (Table 2). As these markers were based on homologous derived based on the maximum likelihood method, it gene sequences, there is a possibility of having multiple provides the evolutionary relationships based on combined copies in the tetraploid cotton genomes. Therefore this study genetic lineages of the candidate genes used in this study. adds a new set of gene-based markers with their specific Many genotypes from tetraploid species formed into two chromosomal location and help in assessing the evolutionary clades while the diploid species form the remaining. Species relationships among the 26 Gossypium species. belonging to the same genome are grouped together to form subclusters in the dendrogram and was evident in grouping of tetraploids as wells as other diploid species. Though the 3.2. Phylogenetic Analysis. Gossypium genus includes five number of genes used in this study is not an exhaustive data tetraploid species from AD –AD genomes and approxi- 1 5 set, the evolutionary relationships among the 26 species were mately forty-five diploid species from genome groups A–G mostly congruent with earlier studies [37]. The dendrogram and K [33]. Of these all five tetraploid species and twenty-one also supported the theory that G. darwinii (AD ), another diploid species belonging to A(1), B(1), C(4), D(11), E(3), tetraploid species endemic to Galapagos Islands is closely and G(1) genomes were included in this study. Relationships related to G. barbadense (AD )[39, 40]. among these cotton genome groups were studied earlier using polymorphisms exhibited in chloroplast genome [34], ribosomal genes [35], and Adh genes [36]. These studies 3.3. G. arboretum EST-SSR Markers. Availability of abun- showed close relationships among the species belonging to dant EST information in public databases and detection the same geographical origin [37] besides explaining the procedures to identify SSR regions in ESTs have established International Journal of Plant Genomics 5 G. hirsutum TM-1 AD1 CMD1 G. hirsutum Acala maxxa AD1 CMD3 G. hirsutum DPL 458BR AD1 CMD4 G. hirsutum Fibermax AD1 CMD6 AD1 CMD7 G. hirsutum Stoneville 4892BR AD2 CMD2 G. barbadense PIMA3-79 AD5 G. darwinii AD4 CMD12 G. mustelinum AD1 CMD5 G. hirsutum Paymaster G. barbadense PIMA S-6 AD2 CMD8 G. klotschianum D3K E3 G. areysianum A2 CMD9 G. arboreum G. tomentosum AD3 CMD11 G. lobatum D7 G. aridium D4 G. stocksii D6 E2 G. gossypioides G. somalense D8 G. trilobum E1 G. turneri C5 G. costulatum D11 G. schwendimanii D10 G. raimondii D5-3 CMD10 G. thurberi D1 G. armourianum D2-1 G. harknessii D2-2 G. bickii G1 G. australe C3 G. pulchellum C8 G. triphyllum B2 G. sturtianum C1 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Average distance between clusters Figure 1: Phylogenetic relationships among the 32 Gossypium genotypes (Table 1) based on the fragment size polymorphism. EST-SSRs as an informative resource for genetic mapping detected using G. arboreum-based EST-SSRs suggested the [7, 12, 14, 25, 41]. Despite the earlier efforts by Han et al. potential of cross-species transferability of these markers [15]and Part et al.[16] to characterize cotton genome using among diploid and tetraploid species [42]. Many of these EST-SSRs, the molecular variation in the coding regions MGAES markers were derived from the fiber expressed ESTs of many fiber expressed genes was not yet fully utilized to thus making them more valuable in breeding programs assist the marker-assisted selection of important fiber traits. and marker-assisted selection for fiber-associated traits. Two hundred primer pairs were designed specifically from Polymorphism detected for each fragment in 186 RILs was fiber related ESTs were used for polymorphism detection scored initially in ternary fashion and then converted into and mapping in this study. Except 27 individual primer binary fashion by treating the heterozygous alleles as missing sequences, the rest of the MGAES markers were new and values. MapManager program was used for constructing nonrepetitive based on BLAST homology search from earlier linkage groups. Fourteen linkage groups were generated studies of Han et al. [15]and Park et al.[41]. Though spanning ∼399 cM with minimum two markers for linkage these EST-SSRs were derived from a diploid progenitor group and with an LOD score threshold of 4. These linkage (genome A )oftetraploidspecies (A D ), 147 markers groups and polymorphic markers can be incorporated into 1−2 t t (74%) out of 200 primer pairs were successfully amplified existing genetic maps to generate an integrated genetic map in tetraploid cultivars G. hirsutum TM-1 and G. barbadense for cotton. PIMA 3–79 suggesting considerable homology exists of the tetraploid cotton with the diploid ancestral species. Sixty- 3.4. Chromosomal Localization of MGAES Markers. Physical five MGAES markers (44%) were polymorphic between TM- mapping of the polymorphic markers was facilitated using 1 and PIMA 3–79 indicating the merit of these markers aneuploid (Figure 2) and euploid chromosome substitution due to high rate of polymorphism compared to earlier lines (Figure 3). Sixteen markers were localized to different studies [15]. Though these G. arboreum EST-SSR markers chromosomes using euploid CS-B lines while 14 markers were highly polymorphic between the G. hirsutum and were localized using aneuploid CS-B lines. Missing a poly- G. barbadense species, we observed very low polymorphism morphic locus in a specific aneuploid (BC F ) accession 0 1 rate within each species. High polymorphic rate between determines the chromosomal localization of a dominant the species could also be attributed to amplification of marker to that corresponding specific chromosome or genotypes under stringent PCR conditions to avoid non- chromosome arm. Results derived from both aneuploid and specific amplification. High levels of polymorphism were euploid CS-B lines served as cross-reference to each other. 6 International Journal of Plant Genomics Table 3: Genetic and physical mapping of G. arboreum-based EST-SSR markers. (—: physical mapping was inconclusive; X: unlinked marker). Annealing Aneuploid CS-B chromosome Marker Euploid CS-B chromosome localization Linkage group temperature ( C) localization MGAES-2 55 — — LG 14 MGAES-3 55 CS-B 01 Te7Sh LG 14 MGAES-5 55 — — X MGAES-10 50 CS-B 15Sh — X MGAES-11 50 — Te9Lo, H9 LG 9 MGAES-21 55 XX Te20Lo, H20 X MGAES-22 55 CS-B 01 — X MGAES-25 55 — — LG 5 MGAES-27 50 — — X MGAES-28 55 CS-B 10 H3Sub, Te11Lo, H11, NTN12-11 LG 7 MGAES-40 50 CS-B26Lo X MGAES-41 55 — — X MGAES-43 50 — — X MGAES-44 55 — — LG 5 MGAES-49 55 — — LG 6 MGAES-51 55 — — LG 6 MGAES-57 55 NTN17-11 H3Sub X MGAES-58 55 — H3Sub LG 10 MGAES-64 55 CS-B 11Sh Te11Lo, H11, NTN12-11 LG 7 MGAES-72 55 — — X MGAES-73 55 — — X MGAES-78 55 CS-B 11Sh Te11Lo, H11 LG 7 MGAES-80 55 — — X MGAES-81 50 — — X MGAES-82 55 — — X MGAES-83 55 CS-B04, CS-B 14Sh, NTN4-15, NTN10-19 — X MGAES-87 55 CS-B 11Sh Te11Lo, H10 LG 4 MGAES-91 55 — — X MGAES-95 55 CS-B 02 Te2Lo, H3SuB X MGAES-104 50 — — X MGAES-105 55 — Te15, Te20Lo, H3Sub LG 5 MGAES-106 55 CS-B26Lo H12, NTN12-11 LG 3 MGAES-107 55 — Te7Lo X MGAES-111 50 — — X MGAES-122 50 CS-B 11Sh, NTN17-11 — LG 12 MGAES-126 50 — — LG 8 MGAES-130 55 NTN4-15 H3Sub LG 13 MGAES-135 55 CS-B 18 Te18Lo, H18 LG 1 MGAES-141 55 — — LG 2 MGAES-142 55 — H10 X MGAES-143 55 — — LG 11 MGAES-153 50 CS-B 01 — X MGAES-157 55 — — X MGAES-160 50 — — X MGAES-161 50 — — X MGAES-165 50 — — X MGAES-194 55 — — LG 2 MGAES-200 50 — — X International Journal of Plant Genomics 7 L 1234 5 6 7 8 910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 L Figure 2: Chromosomal localization of EST-SSR marker MGAES-64 to Te11Lo (lane 10), H11 (lane 28), NTN12-11 (lane 38), and G. barbadense (lane 42) lines using aneuploid CS-B lines. L 1 24 3 2 5 4 7 6 9 8 11101312151417161918212023 22 Figure 3: Chromosomal localization of EST-SSR marker MGAES-64 to CS-B 11Sh using euploid CS-B lines: sample 8: CS-B 11Sh, sample 24: G. barbadense (3–79). AFLP: Amplified fragment length polymorphism For example, MGAES-64 marker was localized to H11 and Te11Lo aneuploid CS-B accessions where the accessions were SNP: Single nucleotide polymorphism CMD: Cotton Marker Database. deficient for chromosome 11 and its long arm, respectively; in both accessions the missing G. hirsutum fragment or locus has been observed confirming the localization of the marker Acknowledgments to chromosome 11 and its long arm (Figure 2). Using euploid chromosome substitution lines, the same This paper was supported by USDA-CSREES Grant no. MGAES-64 marker has been localized to CS-B11Sh acces- ALAX-011-706 to R. V. Kantety, ALAX-011-206 to G. C. sion, where a pair of chromosomes from G. barbadense Sharma, and NSF Plant Genome Research award no. 0703470 was substituted for the long-arm deficient ditelosomic lines to R. V. Kantety. The authors would like to acknowl- of G. hirsutum; polymorphic fragment corresponding to edge the informatics and review support provided by Mr. G. barbadense was only observed in CS-B11Sh explaining Venkateswara Rao Sripathi and other anonymous reviewers its localization to chromosome 11 long arm (Figure 3). who assisted in improving the paper. This is journal article Polymorphic markers, linkage group information, euploid, no. 629 of Alabama A&M University Agricultural Experi- and aneuploid CS-B chromosome localization were shown ment Station. in Table 3. However, we observed incongruency in localizing some markers to just a single chromosome using euploid References and aneuploid CS-B analysis. This needs to be further investigated as it might be a result of duplicated loci or [1] A. J. 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