QTL analysis of cotton fiber length in advanced backcross populations derived from a cross between <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> and <Emphasis Type="Italic">G. mustelinum</Emphasis>

Key message

QTLs for fiber length mapped in three generations of advanced backcross populations derived from crossing Gossypium hirsutum and Gossypium mustelinum showed opportunities to improve elite cottons by introgression from wild relatives.

Abstract

The molecular basis of cotton fiber length in crosses between Gossypium hirsutum and Gossypium mustelinum was dissected using 21 BC₃F₂ and 12 corresponding BC₃F_2:3 and BC₃F_2:4 families. Sixty-five quantitative trait loci (QTLs) were detected by one-way analysis of variance. The QTL numbers detected for upper-half mean length (UHM), fiber uniformity index (UI), and short fiber content (SFC) were 19, 20, and 26 respectively. Twenty-three of the 65 QTLs could be detected at least twice near adjacent markers in the same family or near the same markers across different families/generations, and 32 QTLs were detected in both one-way variance analyses and mixed model-based composite interval mapping. G. mustelinum alleles increased UHM and UI and decreased SFC for five, one, and one QTLs, respectively. In addition to the main-effect QTLs, 17 epistatic QTLs were detected which helped to elucidate the genetic basis of cotton fiber length. Significant among-family genotypic effects were detected at 18, 16, and 16 loci for UHM, UI, and SFC, respectively. Six, two, and two loci showed genotype?×?family interaction for UHM, UI and SFC, respectively, illustrating complexities that might be faced in introgression of exotic germplasm into cultivated cotton. Co-location of many QTLs for UHM, UI, and SFC accounted for correlations among these traits, and selection of these QTLs may improve the three traits simultaneously. The simple sequence repeat (SSR) markers associated with G. mustelinum QTLs will assist breeders in transferring and maintaining valuable traits from this exotic source during cultivar development.

     

Linkage disequilibrium based association mapping of fiber quality traits in <Emphasis Type="Italic">G. hirsutum</Emphasis> L. variety germplasm

Cotton is the world’s leading cash crop, but it lags behind other major crops for marker-assisted breeding due to limited polymorphisms and a genetic bottleneck through historic domestication. This underlies a need for characterization, tagging, and utilization of existing natural polymorphisms in cotton germplasm collections. Here we report genetic diversity, population characteristics, the extent of linkage disequilibrium (LD), and association mapping of fiber quality traits using 202 microsatellite marker primer pairs in 335 G. hirsutum germplasm grown in two diverse environments, Uzbekistan and Mexico. At the significance threshold (r ² ≥ 0.1), a genome-wide average of LD extended up to genetic distance of 25 cM in assayed cotton variety accessions. Genome wide LD at r ² ≥ 0.2 was reduced to ~5–6 cM, providing evidence of the potential for association mapping of agronomically important traits in cotton. Results suggest linkage, selection, inbreeding, population stratification, and genetic drift as the potential LD-generating factors in cotton. In two environments, an average of ~20 SSR markers was associated with each main fiber quality traits using a unified mixed liner model (MLM) incorporating population structure and kinship. These MLM-derived significant associations were confirmed in general linear model and structured association test, accounting for population structure and permutation-based multiple testing. Several common markers, showing the significant associations in both Uzbekistan and Mexican environments, were determined. Between 7 and 43% of the MLM-derived significant associations were supported by a minimum Bayes factor at ‘moderate to strong’ and ‘strong to very strong’ evidence levels, suggesting their usefulness for marker-assisted breeding programs and overall effectiveness of association mapping using cotton germplasm resources. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic dissection of lint yield and fiber quality traits of <Emphasis Type="Italic">G. hirsutum</Emphasis> in <Emphasis Type="Italic">G. barbadense</Emphasis> background

Gossypium hirsutum L. is a widely cultivated species characterized by its high yield and wide environmental adaptability, while Gossypium barbadense is well known for its superior fiber quality. In the present report, we, for the first time, developed G. hirsutum chromosome segment introgression lines (ILs) in a G. barbadense background (GhILs_Gb) and genetically dissected the inheritance of lint yield and fiber quality of G. hirsutum in G. barbadense background. The GhILs_Gb contains introgressed segments spanning 4121.20 cM, which represents 82.20% of the tetraploid cotton genome, with an average length of 18.65 cM. A total of 39 quantitative trait loci (QTLs) for six traits are identified in this IL population planted in Xinjiang. Four QTL clusters are detected. Of them, however, three clusters have deleterious effects on fiber length and strength and boll weight, and only one cluster on Chr. D9 can be used in marker-assisted selection (MAS) to increase lint percentage and decrease micronaire value in G. barbadense. QTL mapping showed that most of yield-related QTLs detected have positive effects and increase lint yield in G. barbadense, while most of fiber quality-related QTLs have deleterious effects except for micronaire. It suggested that G. hirsutum evolved to have a high lint yield. Several lines improved in lint percentage and boll size in G. barbadense by introgressed one fragment of G. hirsutum have been developed from the GhILs_Gb. The ILs developed, and the analyses presented here will enhance the understanding of the genetics of lint yield and fiber quality in G. hirsutum and facilitate further molecular breeding to improve lint yield in G. barbadense.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Biolistic transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with the <Emphasis Type="Italic">phyA</Emphasis> gene from <Emphasis Type="Italic">Aspergillus ficuum</Emphasis>

Transgenic cotton with an increased level of phytase activity was generated from cotton (Gossypium hirsutum L.) cv. ND94-7 by subjecting shoot-apex explants to particle bombardment. These tissues were transformed with plasmid pC-KSA2300 carrying a selectable marker (for kanamycin) and a target gene (phytase, or phyA, from Aspergillus ficuum). Primary plants were regenerated in a medium containing 75 mg l⁻¹ kanamycin. Of 1,534 shoot apices, 52 (3.4%) survived on this selection medium. Southern and Northern blot analyses confirmed that phyA was stably integrated and expressed in those primary transgenics. The progenies of the primary transgenic plants were found to have a 3.1- to 3.2-fold increase in root extracellular phytase activity, resulting in improved phosphorus (P) nutrition. Growth also was enhanced when they were supplied with phytate, and their P content was equivalent to that of wildtype plants supplied with inorganic phosphate. These results demonstrate that the expression of phyA in cotton plants improves their ability to utilize organic P in response to a deficiency.     

QTL mapping of anthracnose (<Emphasis Type="Italic">Colletotrichum</Emphasis> spp.) resistance in a cross between <Emphasis Type="Italic">Capsicum annuum</Emphasis> and <Emphasis Type="Italic">C. chinense</Emphasis>

Anthracnose fruit rot is an economically important disease that affects pepper production in Indonesia. Strong resistance to two causal pathogens, Colletotrichum gloeosporioides and C. capsici, was found in an accession of Capsicum chinense. The inheritance of this resistance was studied in an F₂ population derived from a cross of this accession with an Indonesian hot pepper variety (Capsicum annuum) using a quantitative trait locus (QTL) mapping approach. In laboratory tests where ripe fruits were artificially inoculated with either C. gloeosporioides or C. capsici, three resistance-related traits were scored: the infection frequency, the true lesion diameter (averaged over all lesions that actually developed), and the overall lesion diameter (averaged over all inoculation points, including those that did not develop lesions). One main QTL was identified with highly significant and large effects on all three traits after inoculation with C. gloeosporioides and on true lesion diameter after inoculation with C. capsici. Three other QTL with smaller effects were found for overall lesion diameter and true lesion diameter after inoculation with C. gloeosporioides, two of which also had an effect on infection frequency. Interestingly, the resistant parent carried a susceptible allele for a QTL for all three traits that was closely linked to the main QTL. The results with C. capsici were based on less observations and therefore less informative. Although the main QTL was shown to have an effect on true lesion diameter after inoculation with C. capsici, no significant QTL were identified for overall lesion diameter or infection frequency.     

Isolation and characterization of a <Emphasis Type="Italic">GAI/RGA</Emphasis>-like gene from <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The aim of the investigation reported here was to assess the role of gibberellin in cotton fiber development. The results of experiments in which the gibberellin (GA) biosynthesis inhibitor paclobutrazol (PAC) was tested on in vitro cultured cotton ovules revealed that GA is critical in promoting cotton fiber development. Plant responses to GA are mediated by DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified from the GenBank database and analyzed with the BLAST program. The full-length cDNA was cloned from upland cotton (Gossypium hirsutum, Gh) and sequenced. A comparison of the putative protein sequence of this cDNA with all Arabidopsis DELLA proteins indicated that GhRGL is a putative ortholog of AtRGL. Over-expression of this cDNA in Arabidopsis plants resulted in the dwarfed phenotype, and the degrees of dwarfism were related to the expression levels of GhRGL. The deletion of 17 amino acids, including the DELLA domain, resulted in the dominant dwarf phenotype, demonstrating that GhRGL is a functional protein that affects plant growth. Real-time quantitative PCR results showed that GhRGL mRNA is highly expressed in the cotton ovule at the elongation stage, suggesting that GhRGL may play a regulatory role in cotton fiber elongation.     

Fine-mapping qFS07.1 controlling fiber strength in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Key message

qFS07.1 controlling fiber strength was fine-mapped to a 62.6-kb region containing four annotated genes. RT-qPCR and sequence of candidate genes identified an LRR RLK gene as the most likely candidate.

Abstract

Fiber strength is an important component of cotton fiber quality and is associated with other properties, such as fiber maturity, fineness, and length. Stable QTL qFS07.1, controlling fiber strength, had been identified on chromosome 7 in an upland cotton recombinant inbred line (RIL) population from a cross (CCRI35?×?Yumian1) described in our previous studies. To fine-map qFS07.1, an F₂ population with 2484 individual plants from a cross between recombinant line RIL014 and CCRI35 was established. A total of 1518 SSR primer pairs, including 1062, designed from chromosome 1 of the Gossypium raimondii genome and 456 from chromosome 1 of the G. arboreum genome (corresponding to the QTL region) were used to fine-map qFS07.1, and qFS07.1 was mapped into a 62.6-kb genome region which contained four annotated genes on chromosome A07 of G. hirsutum. RT-qPCR and comparative analysis of candidate genes revealed a leucine-rich repeat protein kinase (LRR RLK) family protein to be a promising candidate gene for qFS07.1. Fine mapping and identification of the candidate gene for qFS07.1 will play a vital role in marker-assisted selection (MAS) and the study of mechanism of cotton fiber development.

     

Genetic changes in plant growth and their associations with chromosomes from <Emphasis Type="Italic">Gossypium barbadense</Emphasis> L. in <Emphasis Type="Italic">G. hirsutum</Emphasis> L

Cotton (Gossypium spp.) plant growth is an important time-specific agronomic character that supports the development of squares, flowers, boll retention, and yield. With the use of a mixed linear model approach, we investigated 14 cotton chromosome substitution (CS-B) lines and their chromosome-specific F₂ hybrids for genetic changes in plant growth that was measured during the primary flowering time under two environments. The changes in additive and dominance variances for plant height and number of mainstem nodes are reported, showing that additive effects for these two traits were a key genetic component after initial flowering occurred in the field. Time-specific genetic variance components were also detected where phenotypic values observed at time t were conditioned on the events occurring at time t − 1, demonstrating new genetic variations arising at several time intervals during plant growth. Results also revealed that plant height and number of nodes shared some common influence due to additive effects during plant development. With the comparative analyzes, chromosomes associated with the genetic changes in plant growth were detected. Therefore, these results should add new understanding of the genetics underlying these time-specific traits. Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Molecular cloning of <Emphasis Type="Italic">Ve</Emphasis> promoters from <Emphasis Type="Italic">Gossypium barbadense</Emphasis> and <Emphasis Type="Italic">G. hirsutum</Emphasis> and functional analysis in Verticillium wilt resistance

Global warming is affecting Mediterranean ecosystems, in which Arbutus unedo represents an important species. Mesophyll protoplasts are convenient material to study plant DNA integrity, nevertheless their release from mature plants along the year has not been reported in sclerophyll species. In this respect, in the present study the chance to isolate protoplasts from leaf tissues of A. unedo in different seasons was investigated. The digestion was obtained using Macerozyme R10 (1%) and Cellulase Onozuka RS (2%), with 0.6 M of mannitol, incubated for 4 h at 25 °C in the dark. In spring, soft leaves of different ages were studied to identify the most suitable material and protoplast yields were significantly influenced, with the still expanding 4th leaf characterized by the highest amount. Protoplast release decreased during the growing season when leaves become partially hardened, while their obtainment was quite impossible in summer and winter, due to leaf hardening. In November, an increase of incubation time (16 h) and of Cellulase RS (4%) or mannitol concentration (0.8 M) was needed with chilled leaves from the field; conversely, a good source for protoplasts were the green soft leaves coming from detached and pruned branches placed in controlled environment at spring temperatures. The employment of polyvinylpyrrolidone-40 during isolation procedure is discussed. The isolated protoplasts were employed to assess the DNA integrity, by using single cell gel electrophoresis (SCGE). The X-irradiated nucleoids were significantly damaged confirming that the present approach could be used in the selection of elite material of A. unedo devoted to fruit plantation.     

Improvement of cotton fiber quality by transforming the<Emphasis Type="Italic"> acsA</Emphasis> and<Emphasis Type="Italic"> acsB</Emphasis> genes into<Emphasis Type="Italic"> Gossypium hirsutum</Emphasis> L. by means of vacuum infiltration

A novel method for the genetic transformation of cotton pollen by means of vacuum infiltration and Agrobacterium-mediated transformation is reported. The acsA and acsB genes, which are involved in cellulose synthesis in Acetobacter xylinum, were transferred into pollen grains of brown cotton with the aim of improving its fiber quality by incorporating useful prokaryotic features into the colored cotton plants. Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by vector pCAMBIA1301, which contains a reporter gene -glucuronidase (GUS), a selectable marker gene, hpt, for hygromycin resistance and the genes of interest, acsA and acsB. The integration and expression of acsA, acsB and GUS in the genome of transgenic plants were analyzed with Southern blot hybridization, PCR, histochemical GUS assay and Northern blot hybridization. We found that following pollination on the cotton stigma transformed pollen retained its capability of double-fertilization and that normal cotton seeds were produced in the cotton ovary. Of 1,039 seeds from 312 bolls pollinated with transformed pollen grains, 17 were able to germinate and grow into seedlings for more than 3 weeks in a nutrient medium containing 50 mg/l hygromycin; eight of these were transgenic plants integrated with acsA and acsB, yielding a 0.77% transformation rate. Fiber strength and length from the most positive transformants was 15% greater than those of the control (non-transformed), a significant difference, as was cellulose content between the transformed and control plants. Our study suggests that transformation through vacuum infiltration and Agrobacterium mediated transformation can be an efficient way to introduce foreign genes into the cotton pollen grain and that cotton fiber quality can be improved with the incorporation of the prokaryotic genes acsA and acsB.Communicated by D. Bartels     

<Emphasis Type="Italic">Agrobacterium</Emphasis> -mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) using a heterologous bean chitinase gene

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T₁ plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T₀ transformants and progeny plants (T₁) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Cry1C,Cry2A</Emphasis> and <Emphasis Type="Italic">Cry9C</Emphasis> genes into <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> and plant regeneration

Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation.     

Association of AFLP and SSR markers with agronomic and fibre quality traits in <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.

Molecular markers linked to QTL contributing to agronomic and fibre quality traits would be useful for cotton improvement. We have attempted to tag yield and fibre quality traits with AFLP and SSR markers using F₂ and F₃ populations of a cross between two Gossypium hirsutum varieties, PS56-4 and RS2013. Out of 50 AFLP primer combinations and 177 SSR primer pairs tested, 32 AFLP and four SSR primers were chosen for genotyping F₂ individuals. Marker-trait associations were studied for eight agronomic and five fibre quality traits through simple and multiple regression analysis (MRA) using a set of 92 AFLP polymorphic loci and four SSR markers. Simple linear regression analysis (SLRA) identified 23 markers for eight different traits whereas multiple regression analysis identified 30 markers for at least one of the 13 traits. SSR marker BNL 3502 was consistently identified to be associated with fibre strength. While all the markers identified in SLRA were also detected in MRA, as many as 16 of the 30 markers were identified to be associated with respective traits in both F₂ and F₃ generations. The markers explained up to 41 per cent of phenotypic variation for individual traits. A number of markers were found to be associated with multiple traits suggesting clustering of QTLs for fibre quality traits in cotton.     

Phenolic compounds and somatic embryogenesis in cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Studies of phenolic compounds were performed during cell suspension cultures in relation with the induction of embryogenic structures in two cultivars of cotton. Coker 312 produced embryogenic structures, unlike R405-2000 which was found to be a non-embryogenic cultivar. Embryogenesis induction in Coker 312 was strongly linked to a higher content of caffeic, ferulic and salicylic acids and to the appearance of p-coumaric acid, benzoic acid, trans-resveratrol, catechin and naringenin.     

Functional characterization of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> profilin 1 gene (<Emphasis Type="Italic">GhPFN1</Emphasis>) in tobacco suspension cells

Cotton fiber is an extremely long plant cell. Fiber elongation is a complex process and the genes that are crucial for elongation are largely unknown. We previously cloned a cDNA encoding an isoform of cotton profilin and found that the gene (designated GhPFN1) was preferentially expressed in cotton fibers. In the present study, we have further analyzed the expression pattern of GhPFN1 during fiber development and studied its cellular function using tobacco suspension cells as an experimental system. We report that expression of GhPFN1 is tightly associated with fast elongation of cotton fibers whose growth requires an intact actin cytoskeleton. Overexpression of GhPFN1 in the transgenic tobacco cells was correlated with the formation of elongated cells that contained thicker and longer microfilament cables. Quantitative analyses revealed a 2.5–3.6 fold increase in total profilin levels and a 1.6–2.6 fold increase in the F-actin levels in six independent transgenic lines. In addition to the effect on cell elongation, we also observed delayed cell cycle progression and a slightly lower mitotic index in the transgenic cells. Based on these data, we propose that GhPFN1 may play a critical role in the rapid elongation of cotton fibers by promoting actin polymerization. Hai-Yun Wang and Yi Yu contributed equally to this work.     

QTL mapping of flag leaf-related traits in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

QTL controlling flag leaf length, flag leaf width, flag leaf area and flag leaf angle were mapped in wheat.

Abstract

This study aimed to advance our understanding of the genetic mechanisms underlying morphological traits of the flag leaves of wheat (Triticum aestivum L.). A recombinant inbred line (RIL) population derived from ND3331 and the Tibetan semi-wild wheat Zang1817 was used to identify quantitative trait loci (QTLs) controlling flag leaf length (FLL), flag leaf width (FLW), flag leaf area (FLA), and flag leaf angle (FLANG). Using an available simple sequence repeat genetic linkage map, 23 putative QTLs for FLL, FLW, FLA, and FLANG were detected on chromosomes 1B, 2B, 3A, 3D, 4B, 5A, 6B, 7B, and 7D. Individual QTL explained 4.3–68.52% of the phenotypic variance in different environments. Four QTLs for FLL, two for FLW, four for FLA, and five for FLANG were detected in at least two environments. Positive alleles of 17 QTLs for flag leaf-related traits originated from ND3331 and 6 originated from Zang1817. QTLs with pleiotropic effects or multiple linked QTL were also identified on chromosomes 1B, 4B, and 5A; these are potential target regions for fine-mapping and marker-assisted selection in wheat breeding programs.