Cultivated Cotton Research Articles

Development of a core set of SSR markers for the characterization of Gossypium germplasm

AbstractMolecular markers such as simple sequence repeats (SSR) are a useful tool for characterizing genetic diversity of Gossypium germplasm. Genetic profiles by DNA fingerprinting of cotton accessions can only be compared among different collections if a common set of molecular markers are used by different laboratories and/or research projects. Herein, we propose and report a core set of 105 SSR markers with wide genome coverage of at least four evenly distributed markers per chromosome for the 26 tetraploid cotton chromosomes. The core marker set represents the efforts of ten research groups involved in marker development, and have been systematically evaluated for DNA polymorphism on the 12 genotypes belonging to six Gossypium species [known collectively as the cotton marker database (CMD) panel]. A total of 35 marker bins in triplex sets were arranged from the 105 markers that were each labeled with one of the three fluorescent dyes (FAM, HEX, and NED). Results from this study indicated that the core marker set was robust in revealing DNA polymorphism either between and within species. Average value of polymorphism information content (PIC) among the CMD panel was 0.65, and that within the cultivated cotton species Gossypium hirsutum was 0.29. Based on the similarity matrix and phylogenetic analysis of the CMD panel, the core marker set appeared to be sufficient in characterizing the diversity within G. hirsutum and other Gossypium species. The portability of this core marker set would facilitate the systematic characterization and the simultaneous comparison among various research efforts involved in genetic diversity analysis and germplasm resource preservation.

Selective fertilization in interspecific crosses of allotetraploid species of Gossypium

The genus Gossypium is composed of both diploid and allotetraploid species. The five allotetraploid species of Gossypium are sexually compatible, and only partial sexual barriers have been described. Natural hybrids among them do not occur or occur in situ with very low frequency in Brazil in the rare places where cultivated upland cotton ( Gossypium hirsutum L.) occurs in a sympatric range with Gossypium barbadense L. or Gossypium mustelinum Miers. We evaluated the presence of pollen competition as a prezygotic barrier in crosses between upland cotton and G. barbadense and G. mustelinum. We found that G. barbadense pollinated with a mixture of 50% upland cotton and 50% G. barbadense pollen resulted in 17.4%–31.1% interspecific hybrids, depending upon the upland cotton genotype used as pollen donor. Mixtures containing pollen from G. mustelinum and upland cotton, used to pollinate G. mustelinum in proportions of 25%:75%, 50%:50%, and 75%:25%, produced 61.3%, 22.5%, and 3.6% interspecific hybrids, respectively. These low rates of hybrid production demonstrate that pollen competition is present with G. mustelinum and G. barbadense and confirms this mechanism as a sexual barrier.

Inheritance of long staple fiber quality traits of Gossypium barbadense in G. hirsutum background using CSILs

Gossypium hirsutum is a high yield cotton species that exhibits only moderate performance in fiber qualities. A promising but challenging approach to improving its phenotypes is interspecific introgression, the transfer of valuable traits or genes from the germplasm of another species such as G. barbadense, an important cultivated extra long staple cotton species. One set of chromosome segment introgression lines (CSILs) was developed, where TM-1, the genetic standard in G. hirsutum, was used as the recipient parent and the long staple cotton G. barbadense Hai7124 was used as the donor parent by molecular marker-assisted selection (MAS) in BC(5)S(1–4) and BC(4)S(1–3) generations. After four rounds of MAS, the CSIL population was comprised of 174 lines containing 298 introgressed segments, of which 86 (49.4%) lines had single introgressed segments. The total introgressed segment length covered 2,948.7 cM with an average length of 16.7 cM and represented 83.3% of tetraploid cotton genome. The CSILs were highly varied in major fiber qualities. By integrated analysis of data collected in four environments, a total of 43 additive quantitative trait loci (QTL) and six epistatic QTL associated with fiber qualities were detected by QTL IciMapping 3.0 and multi-QTL joint analysis. Six stable QTL were detected in various environments. The CSILs developed and the analyses presented here will enhance the understanding of the genetics of fiber qualities in long staple G. barbadense and facilitate further molecular breeding to improve fiber quality in Upland cotton.

Proteomic identification of differentially expressed proteins in Gossypium thurberi inoculated with cotton Verticillium dahliae

Thurber's cotton (Gossypium thurberi) is the wild relative of cultivated cotton. It is highly resistant to cotton Verticillium wilt, a disease that significantly affects cotton yield and quality. To reveal the mechanism of disease resistance in G. thurberi and to clone resistance-related genes, we used two-dimensional electrophoresis (2-DE) and tandem time-of-flight mass spectrometry (MALDI-TOF-MS) to identify differentially expressed proteins in Thurber's cotton after inoculation with Verticillium dahliae. A total of 57 different protein spots were upregulated, including 52 known proteins representing 11% of the total protein spots. These proteins are involved in resistance to stress and disease, transcriptional regulation, signal transduction, protein processing and degradation, photosynthesis, production capacity, basic metabolism, and other processes. In addition, five disease resistance proteins showed intense upregulation, indicating that resistance genes (R genes) may play a critical role in resistance to Verticillium wilt in Thurber's cotton. Our results suggest that disease and stress resistance are the combined effects of multiple co-expressed genes. This provides a basis for further, detailed investigation into the mechanisms underlying Verticillium wilt resistance of G. thurberi and for cloning essential genes into cotton cultivars to produce Verticillium wilt resistant plants.

Cotton genetic resources. A review

Since 6000 BC, cotton has been cultivated for lint fiber, which now dominates the natural textile industry worldwide. Common resources such as an integrated web database, a microsatellite database, and comparative quantitative trait loci (QTL) resources for Gossypium have accelerated the progress towards quantifying the impact of repeated human dispersals and selection regimes on various gene pools of the genus Gossypium. Out of 50 Gossypium species, four have been domesticated—two diploids and two tetraploids—for elimination of hard seed coat, improvement in lint percentage of about 40% and fiber length of 22%, larger boll size, and day-neutral reproductive habit. The major drawback of domestication is the lack of genetic diversity. This lack of genetic diversity is observed more in Gossypium hirsutum L. cultivars characterizing upland cotton than in Gossypium barbadense, typical of Pima and Egyptian cotton. Much of the genetic diversity among G. barbadense cultivars is attributed to the introgression of G. hirsutum alleles. This process highlights the importance of introgression of new alleles from accessions of all the Gossypium species into cultivated cotton species. Among the genomic resources, about 16,162 publicly available SSRs and 312 mapped cotton RFLP sequences containing simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD) markers have been surveyed on numerous mapping populations, and developed about 26 linkage maps (SSR, RFLP, AFLP, and RAPD). Reports show the identification of DNA markers associated with over 29 important traits or QTLs such as fiber quality and yield, leaf and flower morphology, trichome density and their distribution, and disease resistance. In comparative mapping studies, 432 QTLs mapped on 11 different mapping populations were aligned on a high-density reference map containing 3,475 loci. In a meta-analysis study of over 1,000 QTLs obtained from backcross population and recombinant inbred line populations derived from the same parents, most consistent meta-clusters were reported for fiber color, fineness, and length. For exploring the function of genes, the targeting induced local lesions in genomes (TILLING) approach—avoiding gene transfer process was used for identifying a brassino steroid receptor gene that is involved in fiber development. Lastly, cotton genome has been enriched with genes isolated from distantly related organisms using various transformation methods. For example, Cry1Ac, Cry1Ab, and herbicide-resistant genes were transformed in cotton that covered a vast majority of cotton acreage worldwide. Here the authors discuss investigations for improving the efficacy of transformation and regeneration systems, and for searching new genes or silencing the unwanted cotton genes using RNAi technology. We suggest initiating projects on sequencing the diploid and tetraploid genomes for exploring the extent of genetic variations, developing TILLING populations, initiating nested association mapping studies, and developing third generation genetically modified cotton, collectively setting the stage for sustaining cotton production under continually changing production conditions, climates, and human needs.

A genetic map of an Australian wildGossypiumC genome and assignment of homoeologies with tetraploid cultivated cotton

Genetic diversity for traits such as fibre quality or disease resistance to microorganisms is limited in the elite cotton germplasm; consequently, cotton breeders are looking for novel alleles in the secondary or even in the tertiary gene pools. The wild Australian Gossypium species (tertiary gene pool) represent an alternative source of novel alleles. However, to use these species efficiently, enabling tools are required. Chromosome-specific molecular markers are particularly useful tools to track the transmission of this exotic genetic material into the cultivated cotton during introgression. In this study, we report the construction of a genetic linkage map of the Australian wild C-genome species Gossypium sturtianum. The map, based on an F(2) population of 114 individuals, contains 291 AFLP loci. The map spans 1697 cM with an average distance of 5.8 cM between markers. To associate C-genome chromosomes with the A and D subgenomes of cultivated cotton, 29 SSR and RFLP-STS markers were assigned to chromosomes using cultivated cotton mapped marker information. Polymorphisms were revealed by 51 AFLP primer combinations and 38 RFLP-STS and 115 SSR cotton mapped markers. The utility of transferring RFLP-STS and SSR cotton mapped markers to other Gossypium species shows the usefulness of a comparative approach as a source of markers and for aligning the genetic map of G. sturtianum with the cultivated species in the future. This also indicates that the overall structure of the G. sturtianum linkage groups is similar to that of the A and D subgenomes of cotton at the gross structural level. Applications of the map for the Australia wild C-genome species and cotton breeding are discussed.

Characterization of PROFILIN genes from allotetraploid (Gossypium hirsutum) cotton and its diploid progenitors and expression analysis in cotton genotypes differing in fiber characteristics

The actin-binding protein profilin (PRF) plays an important role in cell growth and expansion by regulating the organization of the actin filaments. Recent studies have reported association between fiber elongation in cultivated cotton (Gossypium hirsutum) and PRF expression. In the present study, we cloned four genomic clones from allotetraploid cotton (G. hirsutum) and its putative diploid progenitors (G. arboreum and G. raimondii) designated GhPRF1_A, GhPRF1_D, GaPRF1, and GrPRF1 encoding cotton PRF and characterized their genomic structure, phylogenetic relationships and promoter structure. Sequence analysis of the coding regions of all clones resulted in a single protein product which revealed more than 80% similarity to most plant PRFs and a typical organization with an actin-binding and a polybasic phospholipid binding motif at the carboxy terminus. DNA blot hybridization suggested that PRF gene is present with more than one copy in the allotetraploid species G. hirsutum. Expression analysis performed in various organs of cultivated cotton revealed that the PRF gene was preferentially expressed in cotton fibers. Very low levels of expression were observed in whole flowers, while PRF transcripts were not detected in other organs examined. Furthermore, higher levels of expression were observed at the early stages of cotton fiber development (at 10days post anthesis), indicative that this gene may play a major role in the early stages of cotton fiber development. Quantitation of the expression by real-time PCR revealed higher expression levels in a G. hirsutum variety with higher fiber percentage compared to a variety with lower percentage. In addition, higher levels of expression were found in cultivated allotetraploid G. barbadense cotton species with higher fiber length in comparison to cultivated allotetraploid G. hirsutum.

Alien chromosome transmission and somatic elimination in monosomic addition lines of Gossypium australe F. Muell in G. hirsutum L.

The efficiency of using monosomic alien addition lines (MAALs) to introgress agronomical traits of interest carried by wild diploid Gossypium species into the main cultivated cotton species G. hirsutum depends on the opportunities of confronting the alien chromosome with the recipient background genome at each generation and on the occurrence of translocations and homoeologous recombinations. The selfed-progeny of five MAALs of G. australe in G. hirsutum was screened with SSR markers to determine the transmission frequency of the alien chromosome and monitor its integrity. Three MAALs revealed a transmission frequency significantly lower than the expected ratio and one MAAL presented an exclusive transmission of the additional chromosome. In these four MAAL the alien chromosome was transmitted almost unaltered. With the fifth MAAL the alien chromosome was normally transmitted but was altered in half of the plants carrying it. In one MAAL, normally carrying brown fiber, the emergence of some plants carrying white and brown fiber revealed the somatic elimination of the additional chromosome. The loss of this chromosome seems to be triggered by its deletion.

Mapping resistance gene analogs (RGAs) in cultivated tetraploid cotton using RGA-AFLP analysis

Diseases cause significant losses in cotton production throughout the US Cotton Belt. Growing resistant cultivars can significantly improve cotton yields and effectively reduce production inputs. Disease resistance (R) genes have been isolated in numerous plant species and the R genes with domains of nucleotide binding sites (NB) and leucine rich repeats (LRR) represent the largest R gene family. Degenerate primers designed based on conserved motifs of plant disease resistance genes were used alone or in combination with AFLP primers to analyze disease resistance gene analogs (RGAs) in a recombinant inbred line (RIL) population of Pima (Gossypium barbadense) 3–79 and Upland cotton (G. hirsutum) line NM 24016. Eighty-eight polymorphic RGA markers were amplified by 8 pairs of RGA degenerate primers, while 131 polymorphic RGA-AFLP markers were produced from six pairs of RGA-AFLP primer combinations. Of the 219 polymorphic RGA and RGA-AFLP markers that were identified, 212 were assigned to 18 chromosomes and linkage groups based on existing SSR markers that are on known chromosomes. However, the RGA and RGA-AFLP markers are not evenly distributed among chromosomes in that 189 RGA and RGA-AFLP markers (88%) are assigned onto three “giant” chromosomes, i.e., C6, C12, and C15, suggesting RGA clusters in the cotton genome. Several RGA and RGA-AFLP markers were mapped to the same linkage group carrying a root-knot nematode resistance gene. The identification and mapping of RGA and RGA-AFLP markers provide a framework to facilitate marker-assisted selection of disease resistance in cotton breeding and to understand the physical relationship of cotton resistance genes.

Delineation of interspecific epistasis on fiber quality traits in Gossypium hirsutum by ADAA analysis of intermated G. barbadense chromosome substitution lines

Genetic diversity is the foundation of any crop improvement program, but the most cultivated Upland cotton [Gossypium hirsutum L., 2n = 52, genomic formula 2(AD)(1)] has a very narrow gene pool resulting from its evolutionary origin and domestication history. Cultivars of this cotton species (G. hirsutum L.) are prized for their combination of exceptional yield, other agronomic traits, and good fiber properties, whereas the other cultivated 52-chromosome species, G. barbadense L. [2n = 52, genomic formula 2(AD)(2)], is widely regarded as having the opposite attributes. It has exceptionally good fiber qualities, but generally lower yield and less desirable agronomic traits. Breeders have long aspired to combine the best attributes of G. hirsutum and G. barbadense, but have had limited success. F(1) hybrids are readily created and largely fertile, so the limited success may be due to cryptic biological and technical challenges associated with the conventional methods of interspecific introgression. We have developed a complementary alternative approach for introgression based on chromosome substitution line, followed by increasingly sophisticated genetic analyses of chromosome-derived families to describe the inheritance and breeding values of the chromosome substitution lines. Here, we analyze fiber quality traits of progeny families from a partial diallel crossing scheme among selected chromosome substitution lines (CS-B lines). The results provide a more detailed and precise QTL dissection of fiber traits, and an opportunity to examine allelic interaction effects between two substituted chromosomes versus one substituted chromosome. This approach creates new germplasm based on pair wise combinations of quasi-isogenic chromosome substitutions. The relative genetic simplicity of two-chromosome interactions departs significantly from complex or RIL-based populations, in which huge numbers of loci are segregating in all 26 chromosome pairs. Data were analyzed according to the ADAA genetic model, which revealed significant additive, dominance, and additive-by-additive epistasis effects on all of the fiber quality traits associated with the substituted chromosome or chromosome arm of CS-B lines. Fiber of line 3-79, the donor parent for the substituted chromosomes, had the highest Upper Half Mean length (UHM), uniformity ratio, strength, elongation, and lowest micronaire among all parents and hybrids. CS-B16 and CS-B25 had significant additive effects for all fiber traits. Assuming a uniform genetic background of the CS-B lines, the comparative analysis of the double-heterozygous hybrid combinations (CS-B × CS-B) versus their respective single heterozygous combinations (CS-B × TM-1) demonstrated that interspecific epistatic effects between the genes in the chromosomes played a major role in most of the fiber quality traits. Results showed that fiber of several hybrids including CS-B16 × CS-B22Lo, CS-B16 × CS-B25 and CS-B16 × TM-1 had significantly greater dominance effects for elongation and hybrid CS-B16 × CS-B17 had higher fiber strength than their parental lines. Multiple antagonistic genetic effects were also present for fiber quality traits associated with most of the substituted chromosomes and chromosome arms. Results from this study highlight the vital importance of epistasis in fiber quality traits and detected novel effects of some cryptic beneficial alleles affecting fiber quality on the 3-79 chromosomes, whose effects were not detected in the 3-79 parental lines.

Chloroplast genome of tetraploid cultivated cotton Gossypium barbadense L. and G. hirsutum L.

A detailed in-lab or wet comparison of the chloroplast genomes of two tetraploid cultivated cotton species Gossypium barbadense L. and G. hirsutum L. revealed the presence of some polymorphisms. Comparison of the chloroplast DNA (cpDNA) amplified by PCR after agarose gel electrophoresis showed only one obvious amplified fragment length difference (AFLD). DNA sequences of this AFLD region unraveled the presence of 17 and 51 base pairs (bp) long insertion/deletion as direct repeats in G. hirsutum as well as an insertion/deletion of 129 bp in G. barbadense. All three polymorphisms were accompanied by many direct, indirect, inverted and simple sequence repeats in A+T rich regions that could be used as species-specific markers.

Elucidation of thermotolerance diversity in cotton (Gossypium hirsutum L.) using physio-molecular approaches

Cotton (Gossypium hirsutum) is an important cash crop, but high temperature during its growing season is one of the major factors that limit its productivity. This problem compels plant breeders to breed for heat tolerance, which can help to overcome this challenge. It is very important to make a comprehensive screening of heat-tolerant genotypes so that only the best are chosen. Here we report the combined use of several techniques that can help breeders to screen their germplasm. Twelve cultivated cotton genotypes were evaluated for thermotolerance, using assays that included electrolyte leakage, chlorophyll accumulation and protein profiling, as well as RAPDs to assess genetic diversity. Two genotypes (B-557 and NIAB-78) showed tolerant behavior in three thermotolerance assays. RAPD analysis results showed maximum similarity in a range of 86.7-66.7% between the genotypes MNH-554 and CIM-443. We conclude that combined use should be made of relative electrolyte leakage, chlorophyll stability and differential display with SDS-PAGE to aid in screening for stress tolerance. RAPD-based diversity analysis will further help to improve the efficiency of breeding programs.

Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons

MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expression comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression levels in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two cultivated cotton cultivars, Pima Phy 76 ( Gossypium barbadense) and Acala 1517-99 ( Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA-AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirmation using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies.

Isolation of five new monosomic alien addition lines of Gossypium australe F. Muell in G. hirsutum L. by SSR and GISH analyses

With 2 figures and 3 tables Abstract Gossypium australe F. Muell (2n = 2x = 26) is a wild perennial species possessing agronomic useful traits that would be interesting to introgress into G. hirsutum L. (2n = 4x = 52), the main cultivated cotton species. To isolate monosomic alien addition lines (MAALs) of G. australe in G. hirsutum, the [2(G. hirsutum × G. australe) × G. hirsutum] pentaploid (2n = 5x = 65) was backcrossed as male parent to G. hirsutum. Analysis of 42 BC1 plants and seven alien addition lines, already available, with 150 SSR markers developed from G. hirsutum revealed a cross‐species amplification rate of 100% and a polymorphism rate of 56%. Eighty polymorphic SSR markers generated 87 G. australe‐specific loci that have been assigned by a hierarchical cluster analysis to 13 linkage groups corresponding to the 13 chromosomes of G. australe. Analysis by SSR markers and genomic in situ hybridization of the self‐progeny of disomic alien addition lines, backcross progeny of the pentaploid, allowed the isolation of five new MAALs.

A draft physical map of a D-genome cotton species (Gossypium raimondii).

BackgroundGenetically anchored physical maps of large eukaryotic genomes have proven useful both for their intrinsic merit and as an adjunct to genome sequencing. Cultivated tetraploid cottons, Gossypium hirsutum and G. barbadense, share a common ancestor formed by a merger of the A and D genomes about 1-2 million years ago. Toward the long-term goal of characterizing the spectrum of diversity among cotton genomes, the worldwide cotton community has prioritized the D genome progenitor Gossypium raimondii for complete sequencing.ResultsA whole genome physical map of G. raimondii, the putative D genome ancestral species of tetraploid cottons was assembled, integrating genetically-anchored overgo hybridization probes, agarose based fingerprints and 'high information content fingerprinting' (HICF). A total of 13,662 BAC-end sequences and 2,828 DNA probes were used in genetically anchoring 1585 contigs to a cotton consensus genetic map, and 370 and 438 contigs, respectively to Arabidopsis thaliana (AT) and Vitis vinifera (VV) whole genome sequences.ConclusionSeveral lines of evidence suggest that the G. raimondii genome is comprised of two qualitatively different components. Much of the gene rich component is aligned to the Arabidopsis and Vitis vinifera genomes and shows promise for utilizing translational genomic approaches in understanding this important genome and its resident genes. The integrated genetic-physical map is of value both in assembling and validating a planned reference sequence.

Mining, characterization, and exploitation of EST-derived microsatellites in Gossypium barbadense

Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. However, the number of expressed sequence tags (ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species. In this study, EST-SSR distribution from G. barbadense was characterized and new G. barbadense-derived EST-SSR markers were determined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G. barbadense. By mining 9697 non-redundant ESTs, a total of 638 SSR loci derived from 595 ESTs were observed. In G. barbadense, the frequency of ESTs containing SSRs was 6.13%, with an average of 1 SSR in every 10.4 kb of EST sequence. Furthermore, trinucleotide was found to be the most abundant repeat type among 2–6-nucleotide repeat types. It accounted for 26.6% of the total, followed by the hexanucleotide (26.0%) and pentanucleotide repeats (25.9%). Among all the repeat motifs, (AAG)n accounted for the highest proportion. EST-SSR primer pairs were developed using the Primer3 program, and the redundant primers were removed using the virtual PCR approach. As a result, 380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G. hirsutum ‘TM-1’ and G. barbadense ‘Hai7124’ for constructing linkage groups in cultivated allotetraploid cotton. Out of these, 98 (25.8%) primer pairs detected polymorphisms. Finally, 95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map; of these, 42 were mapped into the A subgenome and 53 into the D subgenome. The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.

Physiological Response to Salt (NaCl) Stress in Selected Cultivated Tetraploid Cottons

In the southwestern and western Cotton Belt of the U.S. soil salinity can reduce cotton productivity and quality. This study was conducted to determine the physiological responses of six genotypes including five Upland cotton (Gossypium hirsutumL.) cultivars and one Pima cotton line (G. barbadenseL.) to NaCl under greenhouse conditions. Seeds were germinated and grown for 14 days prior to salt treatment (daily 100 ml of 200 mM NaCl) for 21 days. Compared with the control (daily 100 ml tap water), the NaCl treatment significantly reduced plant height, leaf area, fresh weight, and dry weight. The NaCl stress also significantly increased leaf chlorophyll content, but did not affect leaf fluorescence. Of the six genotypes, Pima 57-4 and SG 747 had the most growth reduction, and were most sensitive to NaCl; DP 33B, JinR 422 and Acala Phy 72 had the least growth reduction and were most NaCl tolerant. Although all the six genotypes under the salt treatment had significantly higher Na and Cl accumulation in leaves, SG 747 and Pima 57-4 accumulated more Na and Cl than DP 33B. Increases in leaf N, Zn, and Mn concentrations were also observed in the NaCl-treated plants. While leaf P, Ca, and S concentrations remained unchanged overall in the genotypes tested, leaf K, Mg, Fe, and Cu concentrations significantly decreased during salt stress. Reduction in plant height is a simple, easy, sensitive, non-destructive measurement to evaluate salt tolerance in cotton.

DNA Polymorphisms of Genes Involved in Fiber Development in a Selected Set of Cultivated Tetraploid Cotton

ABSTRACTThe lack of genetic diversity within cultivated upland cotton (Gossypium hirsutum L.) has hindered the construction of genomewide linkage maps and their applications in genetics and breeding. The objective of this investigation was to develop candidate gene markers for fiber quality and yield on the basis of approximately 90 genes implicated in fiber development. Polymorphisms using sequence‐tagged site (STS) and single nucleotide polymorphism (SNP) markers based on single strand conformation polymorphism (SSCP) and cleaved amplified polymorphism (CAP) were evaluated among three upland and five Pima cotton (G. barbadense L.) genotypes. Of the 90 primer pairs, 75 resulted in polymerase chain reaction amplifications, including 11 that yielded polymorphic STS markers. Of the 48 primer pairs that produced polymorphic SSCP markers, 27 yielded interspecific polymorphism, while 15 yielded both inter‐ and intraspecific polymorphisms. Six pairs yielded only intraspecific polymorphisms. A total of 18 SNPs, including four indels, were identified in seven of the 15 fiber gene fragments on the basis of direct DNA sequencing, and the average length was 350 bp, with a mean of 1.3 SNPs per fragment. The average rate of SNPs per nucleotide was 0.34%, and 0.31% and 0.41% in coding and noncoding regions, respectively. Eight of the 15 SNPs were interspecific and 78% were nucleotide substitutions, with the four indels contributing to interspecific polymorphism. Six selected SNPs were confirmed by restriction enzyme digestion. The high level of SSCP polymorphism observed within a selected set of agronomically improved lines of upland cotton suggests that the use of SSCP will greatly facilitate genomewide mapping in upland cotton.

A Target Region Amplified Polymorphism Marker for Fertility Restorer Gene Rf1 and Chromosomal Localization of Rf1 and Rf2 in Cotton

ABSTRACTCytoplasmic male sterility (CMS), a maternally inherited trait characterized as an inability to produce functional pollen, is an important biological system for economically producing hybrid seed to enhance crop yield and studying cytoplasmic and nuclear gene interactions. In cultivated tetraploid cotton (Gossypium hirsutum L.), male fertility in two systems CMS‐D2 and CMS‐D8 is restored by two restorer genes Rf1 and Rf2, respectively. The objectives of the present study were to identify additional molecular markers for the two restorer genes and to determine their chromosomal location in the cotton genome. Two backcross (BC1F1) populations were developed with D2 and D8 restorers containing their respective cytoplasms as female in crosses with the same Upland cotton maintainer as male and recurrent parent. One pentatricopeptide repeat (PPR)–based target region amplified polymorphism (TRAP) marker was developed to be tightly linked to the Rf1 gene with a genetic distance of 0.8 cM, while three more simple sequence repeat (SSR) markers (NAU2232‐550/650, NAU2232‐750/850, and NAU 2801‐250) were identified to be closely linked to Rf2 Using two common SSR markers, a consensus linkage group was constructed to include both Rf1 and Rf2 loci that were anchored in a 14.0‐cM region by the two PPR gene‐based markers. Based on four chromosome‐anchored SSR markers, Rf1 and Rf2 were localized on chromosome D5 within a genetic distance of 1.4 cM, providing an incentive for further investigations of this Rf1/Rf2–containing region.

In situ conservation and genetic diversity of three populations of Gossypium mustelinum Miers ex Watt

Gossypium mustelinum Miers ex Watt is a native from Northeastern Brazil and belongs to the primary gene pool of the cultivated cotton. The unique places where the species was known to occur were visited to plan ex situ and in situ preservation. Two populations at Caico became extinct, and only eleven individuals were found. At Jaguarari, although one population became extinct, a new one was localized with approximately 500 adult plants, where a fence avoided cattle feeding. The population from Macurure consisted of 28 plants protected from animals by thorns of Bromelia laciniosa Mart. ex Schult. f. Thirteen SSR primer pairs amplified 40 alleles, among which 30% were exclusive of one of the populations. The genetic differences between populations represented 58.3% of the total genetic variation observed. The high genetic distances are likely to be caused by geographical isolation as well as by the small number of individuals which contribute to new generations, and consequential genetic drift.