Dexin Liu,Xueying Liu,Yao Su,Xiao Zhang,Kai Guo,Zhonghua Teng,Jian Zhang,Dajun Liu,Zhengsheng Zhang*

College of Agronomy and Biotechnology,Southwest University,Chongqing 400716,China

ABSTRACT Naturally colored cotton fiber is environment-friendly but has monotonous color and poor fiber quality.Identification of green fiber or fuzz genes would aid in investigating the biosynthesis of green pigments in cotton fibers.In this study,we established a mapping population and found that the Lgf trait (white lint and green fuzz)from Gossypium hirsutum race latifolium is controlled by an incompletely dominant gene.The Lgf locus was mapped to a 71-kb interval on chromosome 21 containing seven genes,including a transcription factor with similarity to Arabidopsis MYB9.Harboring 13 SNPs and a 4-bp insertion/deletion in its promoter,GhMYB9 was highly up-regulated in the critical period for green pigment development in fuzz.Virus-induced gene silencing of GhMYB9 in a green-fuzz accession of G.hirsutum race latifolium TX-41 conferred white or light green fuzz.These results suggest that GhMYB9 is an important contributor to green pigments in cotton fiber and shed light on the regulatory mechanism controlling green pigmentation.

Keywords:Upland cotton Green fuzz Lgf MYB9 Green pigment

1.Introduction

Naturally colored cotton is considered environment-and health-friendly,because it synthesizes and accumulates pigments during fiber development and needs little or no processing and dyeing during fabric manufacturing [1,2].However,unstable and monotonous color,low yield,and poor fiber quality have limited commercial application of naturally colored cotton [3–5].It is difficult to breed stably colored fiber varieties with high yield and good fiber quality.Identifying the genetic and molecular mechanisms of fiber color could lead to improving both traits by genetic engineering.

Brown-and green-fiber varieties are the most widespread naturally colored cottons and are cultivated commercially[6].Genetic analysis[7]has suggested that brown fiber is controlled by six loci(Lc1–Lc6) with incomplete dominance.More recently [8–10],theLc1locus of cotton was mapped on chromosome A07 and linked to an inversion upstream of aTT2homolog.Up-regulatingGhTT2-3Aspecifically in fibers at the secondary wall-thickening stage in a white-fiber cultivar resulted in brown mature fibers with yield and quality comparable with those of the white cultivars [10].Proanthocyanidin biosynthesis and accumulation was the source of brown coloration in cotton fibers [10–12].Green fiber is apt to fade with exposure to sunshine [13],and little is known about its genetic and biochemical bases.

The seed hairs of cultivated Upland cotton are differentiated into‘‘fuzz”and‘‘lint”.TheLglocus,conferring green lint and green fuzz,was first reported inG.hirsutum[14].The color of lint and fuzz inLgis prominent at the boll opening stage but soon fades to a khaki color.A variant namedLgfwas found [7] in which the fuzz is green while the lint remains white.Seeds with a green fuzz layer were frequently observed in the indigenousG.hirsutumracelatifolium,which is considered the source of modernG.hirsutumcultivars [15,16].Conventional genetic analyses revealed thatLgandLgfwere allelic to each other and mapped to linkage group II of chromosome 15 [7].However,in our previous study [17],Lgwas mapped to the terminal region of chromosome 21,raising questions about the chromosome and position of theLgflocus.

Another limitation to genetic improvement of naturally green cotton is the lack of understanding of its composition and the biochemical mechanisms that confer its color.Previous research[18,19] has shown that cinnamic acid and its derivatives accumulated in the development of green fiber.Based on UV spectrometry and thin-layer chromatography,caffeoyl derivatives played an important role in the pigmentation of green cotton fibers [20].In that study,the phenylpropanoid metabolic pathway was involved in the pigmentation of green cotton fibers.However,the composition of naturally green fiber is complex,and the complete set of underlying biochemical mechanisms is still unknown.Other studies [10,11] suggested that cloning the underlying gene(s) would contribute to revealing the biosynthetic pathways conferring color(s),and facilitate coordinated modification (with improvement) of color and fiber quality.

In this study,we aimed to identify theLgfgene using a mapbased cloning strategy.We report (i) mapping theLgflocus using linkage and SSR markers;(ii)localizing theLgflocus to the genome ofG.hirsutumTM-1 based on the genetic map,and identification of the candidate gene containing the causative mutation by qRT-PCR and sequence analysis;and (iii) gene validation by conversion of the fuzz color from dark green to white or light green in anLgfgenotype by virus-induced gene silencing (VIGS).

2.Materials and methods

2.1.Plant materials

CCRI35 and TX-41 were used as parental genotypes.TX-41 is an accession ofG.hirsutumracelatifoliumwith white fiber and green fuzz,provided by the China National Wild Cotton Plantation.CCRI35 is a commercial cultivar (G.hirsutum) with white lint and white fuzz provided by the National Medium-term Gene Bank of Cotton in China and National Cotton Germplasm Resources Platform [21,22].CCRI35 and TX-41 were crossed in winter 2014 in Sanya,Hainan,China.Individual F1plants were self-pollinated and F2seeds were harvested in summer 2015 at Southwest University(SWU),Chongqing,China.Parents and an F2segregating population were planted in winter 2015 in Sanya,Hainan,China.Plants were grown and cultivated under field conditions.The color of the fuzz was observed after the naturally opened bolls were handharvested to gin fiber.

2.2.Linkage analysis of Lgf locus

A set of 392 F2plants derived from a cross between CCRI35 and TX-41 were used to construct a genetic linkage map.Genomic DNA of parents and the segregating population were isolated from young leaves by the CTAB method described by Zhang et al.[23].The color of the fuzz was scored by visual observation after ginning of lint fiber.Simple sequence repeat(SSR)primers with prefix SWU are described in Table S1.All primers were synthesized by the Beijing Genomics Institute (BGI) in Shenzhen.Genotyping using SSR markers was performed as previously described [24].Linkage groups were constructed and ordered,treating fuzz color phenotype as a genetic marker,with JoinMap 4.0 [25].

2.3.RNA extraction and gene expression analysis

Total RNAs extracted from 22-DPA (days post-anthesis) fuzz of CCRI35 and TX-41 were used for gene expression analysis,using a rapid plant RNA extraction kit (Aidlab,Beijing,China).RNA purity and quality was monitored on 1% agarose gels.First-strand cDNA was generated from total RNA using a first-strand cDNA synthesis kit (TaKaRa,Dalian,China),according to the manufacturer’s instructions.Quantitative real-time PCRs were performed in 20 μL reactions with SYBR Green Supermix (Bio-Rad,CA,USA) as described by Liu et al.[22].Gene expression levels were determined based on three biological replicates each with three technical replicates.The positive control in the qRT-PCR reactions wasGhUBQ14[26].Primers used in qRT-PCR are described in Table S1.

2.4.Virus-induced gene silencing (VIGS) of GhMYB9 in TX-41

To knock down the expression ofGhMYB9,a 223-bp fragment ofGhMYB9was amplified from cDNA derived from TX-41 fuzz at 22 DPA.The PCR product was cloned into the PDM19-T vector(TaKaRa) with the restriction enzymesKpnI andSmalI,and then sequenced by BGI.The fragment digested byKpnI andSmalI was further recombined into the TRV2-GoCEN vector constructed by our laboratory [22],and a VIGS vector named TRV2-GoCEN&MYB9 was produced.In addition to the TRV2-GoCEN&MYB9 experimental treatment,a negative control vector was constructed.A 242-bp fragment of the YFP gene was amplified and cloned into the PMD19-T vector (TaKaRa) with the same restriction enzyme.The VIGS vector TRV2-GoCEN &Yellow Fluorescent Protein (YFP) was constructed followed the method described for the TRV2-GoCEN &MYB9 vector.The primers are described in Table S1.

The two VIGS vectors,TRV2-GoCEN&MYB9 and TRV2-GoCEN&YFP,were transformed intoAgrobacteriumGV3101 competent cells by electroporation and then plated on Luria–Bertani agar containing 50 μg mL-1of kanamycin and 100 μg mL-1of rifampicin.Cotton plants(TX-41)were used at about one week after germination,with mature cotyledons but no true leaves.Agrobacteriumcells were harvested at an OD600of 0.5 and resuspended in half the volume of the original culture with 10 mmol L-1MES,10 mmol L-1MgCl2,and 200 mmol L-1acetosyringone.Seedling cotyledons were infiltrated with a 1:1 mixture ofAgrobacteriumcarrying TRV1 and TRV2-GoCEN&MYB9 in six plants and the negative control,TRV1 and TRV2-GoCEN&YFP in 8 plants,using a 1 mL syringe.The inoculated plants were held at 22 °C in darkness for 48 h and then transferred to a 16 h light/8 h dark cycle in a growth chamber at 26 °C.

3.Results

3.1.Genetic analysis of green fuzz

Accession TX-41,classified asG.hirsutumracelatifolium,has varying intensities of green fuzz and white lint,a trait calledLgf(Fig.1a).To investigate the inheritance of the green fuzz trait,a mapping population including 392 F2plants was established by crossing TX-41 with the high-yielding Upland cotton cultivar CCRI35 with white fuzz and white lint (Fig.1b).The fuzz of the F1hybrid was light green,suggesting that the green fuzz was an incompletely dominant trait.In the F2population,96 plants showed dark green fuzz,189 light green,and 107 white,fitting a 1:2:1 segregation ratio (χ2=1.12,P>0.05).This result suggested that the green fuzz trait was controlled by a single incompletely dominant gene.

3.2.Genetic mapping of Lgf

Fig.1.Lint and fuzz phenotypes in the two mapping parents.(a) Lint with white color in TX-41.(b) Fuzz with green color in TX-41.(c) Lint with white color in CCRI35.(d)Fuzz with white color in CCRI35.

Lgfhad been identified as an allele at theLg(green fuzz and green lint) locus [7],and we previously [17] mappedLgto the end of chromosome 21 (Dtsub-genome) ofG.hirsutum.IfLgfwas allelic withLg,then both should lie on chromosome 21.First,based on the genetic map constructed by Wang et al.[27],135 SSR markers (from cottongen:http://www.cottongen.org) located on chromosome 21 were screened for polymorphism between TX-41 and CCRI35,and two(NAU4004 and NAU2151)showed polymorphism,for a polymorphism rate of 1.5%.When these SSRs were used to genotype the 92 F2plants,both were linked to theLgflocus,indicating that this locus too was located on chromosome 21.To increase the number of markers on chromosome 21 of theLgfmapping population,1023 SSR markers developed from chromosome 7(corresponding to chromosomes 11 and 21 of tetraploid cotton)in our previous study [27],were used to screen for polymorphism between the parents.Thirty-five(3.4%) SSR markers showed polymorphism.They were used to genotype the 92 F2plants and 23 new SSR markers were localized on chromosome 21.These,with NAU4004 and NAU2151,were used to genotype the other 300 F2plants.A genetic map was constructed,spanning 183.7 cM with a mean interval of 7.65 cM between adjacent markers (Fig.2a).TheLgflocus co-segregated with SWU1713 in the 0.28-cM interval between markers NAU2151 and SWU1715 (Fig.2b).Based on the physical position of NAU2151 and SWU1715,theLgflocus was localized to a 67-kb interval on chromosome 7 ofG.raimondii[28],corresponding to a 71-kb interval on chromosome D11 ofG.hirsutumTM-1 [29].The intervals containingLgfwere predicted to contain 7 open reading frames(ORFs)in the respective referenceG.raimondiiandG.hirsutumgenomes (Fig.2c;Table 1).

Table 1 Functional annotation and associated information of candidate genes within the 71 kb genomic interval containing Lgf on chromosome 21 of Upland cotton.

3.3.GhMYB9 confers green fuzz in Upland cotton

Under field conditions and liquid culture,the green color of lint was clearly observed at 25 DPA[30],but it was not clear in fuzz.To investigate the timing of green and white fuzz,we further observed the phenotype of cotton fuzz in TX-41 and CCRI35 from 14 to 28 DPA,finding that the accumulation of green pigment occurred mainly after 21 DPA in TX-41 but not in CCRI35 (Fig.3a).Based on this observation,we performed expression analysis to help identify the causal gene in TX-41 among the 7 ORFs in the interval containingLgf.In developing cotton fuzz at 22 DPA,three ORFs of seven (ORF1,ORF4,and ORF7) were predominantly expressed,but only ORF7 showed significantly higher expression in greenthan in white-fuzz plants (Fig.3b).ORF7 (GH_D11G3607),namedMYB9,is annotated as an R2R3-MYB transcription factor that had been shown[8,10]to regulate pigmentation metabolism in cotton,via control byGhTT2-3Aof proanthocyanidin(PA)biosynthesis and brown fibers.This finding suggested that up-regulation ofGhMYB9in TX-41 was responsible for the green fuzz.

To identify mutation(s) inGhMYB9that caused the green fuzz,we cloned the gene in genomic DNA and its ORF in cDNA from TX-41 and CCRI35 based on a draft genome ofG.hirsutum[29].The results showed thatGhMYB9had two introns and three exons,but there were no differences inGhMYB9sequence between the two mapping parents,TX-41 and CCRI35.Sequencing theGhMYB9promoter showed a 4-bp insertion located -412 bp upstream of the translation start site on TX-41,and 13 SNPs among the two mapping parents (Fig.3c).These polymorphisms may explain the higher expression ofGhMYB9in TX-41 than in CCRI35.

3.4.Silent GhMYB9 transforming green fuzz into white

To verify the function ofGhMYB9in controlling green fuzz pigmentation in cotton,we silencedGhMYB9expression using VIGS,an easy and effective way to test gene function in cotton[22,31,32].To overcome the photoperiod sensitivity of TX-41 fromG.hirsutumracelatifolium,we isolated the 3′-end 387-bp fragment ofGhMYB9from TX-41 and inserted it into our previous VIGS vector pTRV2-CNE,silencing with which led to early flowering and growth termination in modern cultivars and Upland cotton races[22].As a negative control,we cloned the 242-bp fragment from the YFP gene and inserted it into pTRV2-CNE.Early flowering in the fifth node and termination of growth at 30–40 cm height were observed in the VIGS of TX-41 plants,but wild-type plants produced exclusively vegetative growth,suggesting normal development in the VIGS system (Fig.4a).The level ofGhMYB9transcript was dramatically reduced in the pTRV2-CNE-MYB9-containing lines,compared with the negative control pTRV2-CNEYFPin 22-DPA fuzz(Fig.4d).At boll opening,we harvested the fiber and seeds of individual plants and found that the intense green color in fuzz was greatly reduced to light green or white inGhMYB9down-regulated plants,and that all lint was white (Fig.4b and c).Thus,down-regulation ofGhMYB9transformed the intense green fuzz in TX-41 to light green or white,suggesting that the expression ofGhMYB9was at least partly responsible for the green fuzz in TX-41.

Fig.2.Linkage and physical maps of the Lgf locus in Gossypium hirsutum.(a) Genetic map of the Lgf locus on chromosome 21 in cotton.(b)The Lgf locus was mapped in the middle of the 0.28 cM interval between NAU2151 and SWU1715.(c)Delimitation of Lgf to a 71 kb interval in Gossypium hirsutum containing seven predicted genes.Arrows indicate putative genes predicted in the TM-1 genome [29].

4.Discussion

Fig.3.Candidate gene GhMYB9 confirmed for Lgf.(a)Phenotypic comparison of green pigment development in fuzz between CCRI35 and TX-41 from 14 to 28 DPA.The series of red squares are magnified frames in the in-focus region of the fuzz.Scale bars,2 cm and 5 mm(the red magnified frames).DPA,days post-anthesis.(b)Relative expression of eight candidate genes between white and green fuzz on 22 DPA.Transcript abundance was measured by RT-qPCR.**,P<0.05,values are mean±SD of three biological replicates.(c)Nucleotide polymorphisms in promoter and structural regions of GhMYB9 in CCRI35 and TX-41.Scale bar,500 bp.

A series of fiber pigment mutants have been found in cotton that include brown and green fiber.Lg,with green lint and fuzz at boll opening,was incompletely dominant inG.hirsutum[14].A mutant calledLgfwas identified with intensities of green fuzz and white lint [33].In that study,one mapping parent withLgfwas selected fromG.hirsutumracelatifolium,andLgfwas incompletely dominant to white fuzz.Lgfhad previously [33] been identified as allelic toLg,which was mapped on linkage group II (chromosome 15).However,in the present study,we mappedLgfofG.hirsutumracelatifoliuminto a 0.28-cM interval on chromosome 21 using many SSR markers,a more reliable result than the previous placement on chromosome 15 based on a few morphological markers.Our recent study [17] mapped theLglocus to the same region of chromosome 21 (Dtsub-genome) ofG.hirsutum,and these results supported that theLgandLgfwere multiple alleles on chromosome 21.

Fig.4.Functional characterization of GhMYB9 by virus induced gene silencing (VIGS).(a) Phenotypes of TX-41 after GhMYB9&YFP, GhYFP&CEN silencing by VIGS,showing silenced CEN plants with early flowering and determinate growth.Scale bars,20 cm.(b)Representative seed with fuzz and fiber from VIGS experiment,showing the reversion from dark green to white or light green fuzz after silencing of GhMYB9.i,ii,and iii are magnified frames in the in-focus region in the red squares of the fuzz from iv,v,and vi,respectively.Scale bars,1.5 cm(i,ii,and iii)or 1 mm(iv,v,and vi).(c)The proportions of reversion of fuzz color from dark green in silencing GhMYB9 treatment.(d)Relative transcript levels of candidate genes in the GhCEN, GhMYB9&YFP,and GhYFP&CEN silenced lines confirmed the effective knockdown of GhMYB9 on 22 DPA.Transcript abundance was measured by qRT-PCR.**, P <0.05.Values are mean ± SD of three biological replicates.

In the present study,the interval containingLgf,defined by flanking SSR markers NAU2151 and SWU1715,was predicted to contain seven genes on a reference genome ofG.hirsutum[29].One of the putative genes,GH_D11G3607,shows high similarity with an R2R3-typeMYB9[34].R2R3-type MYB genes had been shown [35] to affect phenylpropanoid metabolism inA.thaliana.Overexpression ofAtMYB75/PAP1andAtMYB90/PAP2resulted in accumulation of anthocyanins.GhPAP1Din Upland cotton encoded the red-plant geneR1,overexpression of which resulted in increased anthocyanin accumulation in transgenic tobacco and cotton [36].GhMYB9was predicted to contain three exons and two introns,with a 711-bp coding region and is structurally similar to theArabidopsis MYB9gene.MYB9 proteins fromArabidopsis,tomato,apple,grape,potato,and rice all contained conservative domains of a clade of GDSL-motif esterases and showed conserved expression patterns[37].A 4-bp insertion upstream of the translation start site and 13 SNPs were significantly associated with gene expression (Fig.3).Down-regulation ofGhMYB9by VIGS transformed the normally intensely green fuzz in TX-41 to light green or white(Fig.4).We conclude thatGH_D11G3607plays a vital role in the development of green fuzz pigment in cotton.

Green fiber color is generally unstable and associated with low fiber quality,a property that severely restricts its large-scale production and utilization [5,7].Understanding the molecular mechanisms controlling pigment formation in green fiber is very important for developing cotton cultivars with stable green-colored fibers and commercially acceptable fiber quality.In the present study,the color of green and white fuzz was visually distinguishable at approximately 21 DPA.In agreement with the expression and VIGS results obtained,promoter polymorphism in TX-41 led to higher expression ofGhMYB9and increased green pigment accumulation in fuzz.This result contributes insights into the regulatory mechanism of green pigment.InArabidopsis thaliana,myb9mutations led to a significant reduction in suberin monomers and altered levels of other seed coat-associated metabolites[37].The fibers of the green-lint mutant of cotton were suberized[38]and contained large amounts of wax[39].Chemical and gas chromatography–mass spectrometry analysis showed the presence of suberin polymers with 65%of their total monomers in the isolated cell walls from green fibers[40,41],with the major aliphatic monomers being 22-hydroxydocosanoic acid(70%)and docosanedoic acid(25%)with lengths of C22and C24[42].Transcriptome sequencing and metabolome analysis revealed that the cutin,suberin,and wax biosynthesis pathways were increased in green fiber compared to white fiber at 24 DPA,and cytochromeP45086A1(CYP86A1)andP45086B1(CYP86B1)genes required for fatty acid w-hydroxylase were up-regulated in green fibers[43].Overall,our results suggest thatGhMYB9acts as a key regulator of green pigment formation by activating the suberin biosynthesis pathway.

In conclusion,we report the candidate gene of the green fuzz with white fiber trait in Upland cotton.TheGhMYB9gene plays a pivotal role in green pigment formation.The cloning ofGhMYB9may assist in elucidating the relationship between green pigment formation and fiber quality traits and ultimately in increasing the stability of green color and fiber quality and/or productivity.

CRediT authorship contribution statement

Zhengsheng Zhang and Dexin Liu conceived and designed the research.Zhengsheng Zhang made crosses for the mapping populations.Xueying Liu,Yao Su,and Xiao Zhang isolated DNA,conducted DNA marker genotyping,and developed the genetic map.Xueying Liu and Dexin Liu performed gene cloning and data analysis.Dajun Liu,Jian Zhang,and Zhonghua Teng contributed to growing the experimental materials and the data of field experiment.Dexin Liu and Kai Guo isolated RNA and conducted qRT-PCR analysis,and designed and executed the VIGS experiment.Dexin Liu and Zhengsheng Zhang wrote the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Genetically Modified Organisms Breeding Major Project of China(2016ZX08005005-001),the National Natural Science Foundation of China(31701471),and the Fundamental Research Funds for the Central Universities(SWU118093).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.09.009.