Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L

Abstract

Simultaneous improvement of fibre quality, early-flowering, early-maturity and productivity in Upland cotton (G. hirsutum) is a challenging task for conventional breeding. The influence of red/far-red light ratio on the fibre length prompted us to examine the phenotypic effects of RNA interference (RNAi) of the cotton PHYA1 gene. Here we show a suppression of up to ~70% for the PHYA1 transcript, and compensatory overexpression of up to ~20-fold in the remaining phytochromes in somatically regenerated PHYA1 RNAi cotton plants. Two independent transformants of three generations exhibited vigorous root and vegetative growth, early-flowering, significantly improved upper half mean fibre length and an improvement in other major fibre characteristics. Small decreases in lint traits were observed but seed cotton yield was increased an average 10-17% compared with controls. RNAi-associated phenotypes were heritable and transferable via sexual hybridization. These results should aid in the development of early-maturing and productive Upland cultivars with superior fibre quality.

ARTICLE

Received 15 Jul 2013 | Accepted 4 Dec 2013 | Published 16 Jan 2014

DOI: 10.1038/ncomms4062

Phytochrome RNAi enhances major bre quality and agronomic traits of the cotton Gossypium hirsutum L

Ibrokhim Y. Abdurakhmonov1,w, Zabardast T. Buriev1,w, Sukumar Saha2, Johnie N. Jenkins2, Abdusattor Abdukarimov1,w & Alan E. Pepper3

Simultaneous improvement of bre quality, early-owering, early-maturity and productivity in Upland cotton (G. hirsutum) is a challenging task for conventional breeding. The inuence of red/far-red light ratio on the bre length prompted us to examine the phenotypic effects of RNA interference (RNAi) of the cotton PHYA1 gene. Here we show a suppression of up to

B70% for the PHYA1 transcript, and compensatory overexpression of up to B20-fold in the remaining phytochromes in somatically regenerated PHYA1 RNAi cotton plants. Two independent transformants of three generations exhibited vigorous root and vegetative growth, early-owering, signicantly improved upper half mean bre length and an improvement in other major bre characteristics. Small decreases in lint traits were observed but seed cotton yield was increased an average 1017% compared with controls. RNAi-associated phenotypes were heritable and transferable via sexual hybridization. These results should aid in the development of early-maturing and productive Upland cultivars with superior bre quality.

1 Centre of Genomics and Bioinformatics, Academy of Sciences of Uzbekistan, Ministry of Agriculture & Water Resources of Uzbekistan, and Uzpakhtasanoat Association, University street-2, Kibray region, Tashkent 111215, Uzbekistan. 2 USDA-ARS, Crop Science Research Laboratory, Genetics and Precision Agriculture, P. O. Box 5367, 812 Highway 12E, Mississippi State, Mississippi 39762, USA. 3 Department of Biology, Texas A&M University, College Station, Texas 77843, USA. w Former address: Centre of Genomic Technologies, Institute of Genetics and Plant Experimental Biology, Academy of Sciences of

Uzbekistan, Yuqori Yuz, Kibray Region, Tashkent District 111226, Uzbekistan. Correspondence and requests for materials should be addressed to I.Y.A. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 1

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4062

Light is one of the most important environmental factors controlling plant development and physiology. It affects virtually all aspects of plant growth, from seed germination

to vegetative morphology, oral initiation, control of circadian rhythms, regulation of gene expression, gravitropism and photo-tropism13. Plants respond to light through several photoreceptor systems. The phytochrome red (R)/far-red (FR) photoreceptor gene family is best characterized in the model plant Arabidopsis thaliana, which has ve phytochrome apoprotein genes, designated PHYA, PHYB, PHYC, PHYD and PHYE1. In cultivated cottons, the phytochrome gene family has particular importance because there is evidence that the FR/R photon ratio inuences the length and diameter of developing bre. For example, bres from bolls that received a high FR/R photon ratio during development were longer than those that received normal photosynthetic light4.

Genetic improvement of bre quality and yield is a primary objective of cotton-breeding programmes worldwide5,6. To make cotton cultivation sustainable and competitive on the world market, it is imperative to develop early maturing, productive cotton cultivars producing ner (moderately lower micronaire (MIC)) and longer bres with improved strength6. Conventional breeders have faced serious challenges because of negative correlations between bre quality traits and yield components6,7, and between bre quality and maturity8. We hypothesized that phytochrome genes could play a signicant role in the regulation of cotton bre length9. For this reason, we characterized the phytochrome gene family and its evolution in the A and D diploid Gossypium spp., as well as AD allotetrapoloid cottons Gossypium hirsutum and Gossypium barbadense9,10. The AD tetraploid Gossypium spp. inherited homologues of each of the canonical phytochrome apoprotein subfamilies PHYA, PHYB, PHYC and PHYE from each of the A- and D-genome diploid ancestral species. Further, both diploid and tetraploid cotton species have two divergent PHYA subfamilies, designated PHYA1 and PHYA2, resulting from a Malvaceae-specic duplication that occurred B14 million years ago, before the divergence of the

A- and D-genome diploid genome groups of Gossypium10. The distribution of gene function between the two PHYA paralogues is unknown; however, previous efforts directed towards quantitative trait loci (QTL) mapping of bre quality traits in a bi-parental population found a signicant association of the PHYA1 gene with bre length9.

In this study, the bre quality QTL-associated PHYA1 gene fragment was used to create a specic RNAi construct11 to explore the biological roles of PHYA1 and (indirectly) other phytochrome genes in cotton. We somatically transformed the RNAi construct into the highly embryogenic but non-commercial Coker-312 variety, which is considered the genetic standard for cotton regeneration work12. When compared with control plants, we observed heritable improvement of major bre quality traits, early-owering, early-maturity, vigorous root and shoot development, and productivity traits through several generations of two independently derived RNAi cotton transformants. We further demonstrate that the RNAi-associated traits were transferable from somatically regenerated RNAi Coker-312 lines to other commercial Upland cultivars via conventional sexual hybridization. Our results provide the rst molecular evidence of the importance of phytochromes in cotton bre development, and show the usefulness of phytochromespecic RNAi in simultaneously improving several important agronomic and bre quality traitsa goal that has proven difcult to achieve via conventional breeding6. These results open a new avenue for cotton breeders to rapidly develop early-maturing and productive elite Upland cultivars with superior bre qualities.

ResultsTransformation and phenotypic evaluation in T0T3. We successfully obtained transgenic plant embryos bearing the cotton PHYA1 RNAi construct (Fig. 1a) that were resistant to kanamycin, the selectable marker. Compared with 15 somatically regenerated non-transformed controls, all 68 candidate PHYA1 RNAi cotton plants showed comparatively rapid and vigorous lateral and main root development and elongated petioles (Fig. 1b; Supplementary Figs 1 and 2). Fibres obtained from transgenic T0 RNAi plants were at least 5 mm (17%) longer than bres on control plants that were somatically regenerated using the same media and conditions (Fig. 1c). PCR analysis conrmed the presence of the RNAi construct in genomic DNAs of T0 and

T1 plants (Supplementary Fig. 3), and the stable Mendelian inheritance of the transgene in subsequent generations. Compared with the control genotypes lacking the RNA construct (non-transformed Coker-312 and null segregant progeny from transformed T1 lines), the RNAi-positive families had more vigorous vegetative growth (Figs 2ae and 3a) and 510 days earlier owering and earlier boll maturing phenotypes (Fig. 2ac; Supplementary Fig. 4), signicantly (analysis of variance (ANOVA), Pr0.01) longer hypocotyls (Supplementary Figs 5 and 6; Supplementary Table 1), higher root mass and higher germination rate (Fig. 4; Supplementary Fig. 5; Supplementary Table 1), and more owers and bolls (Supplementary Table 2) that opened earlier (Supplementary Table 3) at the time of phenotypic evaluation. We also observed elongated petioles (Fig. 2d), elongated boll peduncles and fruiting branches, and early senescence-associated anthocyanin pigmentation in leaves and stems (Supplementary Fig. 4) in both RNAi lines. Further, when compared with near-isogenic control plants, we observed a 58-mm increase in bre staple length in both PHYA1 RNAi lines (Figs 4a and 5). In subsequent generations, staple length was further evaluated using high volume instrumentation.

Analysis of major bre characteristics and cottonseed yield. On the basis of phenotypic evaluation of T2-generation plants in the eld in 2009, we selected two elite plant families, designated T2-1

and T2-31, for further characterization. These two independent RNAi transformants had directionally similar but quantitatively different phenotypic characteristics for traits such as boll maturation and hypocotyl length (Supplementary Tables 1 and 3), root development (Fig. 4c, d) and some of the bre quality traits, for example, microniare (Table 1).

In combined ANOVA (Supplementary Table 4) of the 2009 and 2010 small replicated plot eld evaluations, both of the RNAi families showed signicantly increased (5%; ANOVA, Pr0.01)

upper half mean (UHM) bre length (Table 1; Figs 4a and 5), and improved MIC (48%; MIC, representing the thickness of cotton bre), with no decrease in bre uniformity, in comparisons with sibling plants lacking the RNAi construct (that is, null segregants) as well as non-transformed Coker-312. The modestly improved bre strength (STR; 5%) and bre elongation (ELO; the elasticity (%) of matured bre) traits (210%) in RNAi families were statistically different (ANOVA, Pr0.01) from non-transformed

Coker-312, but not the null segregant control, suggesting possible effects of somaclonal variation and/or environmental interactions on these traits. However, trait differences between non-transformed Coker-312 and null segregant controls were not statistically signicant at Pr0.01 (ANOVA) in any of the traits tested, indicating that any effects of somaclonal variation or environmental differences were minor. MIC, ELO and bre colour ( B) traits statistically differed (ANOVA, Pr0.01)

between T-1 and T-31 RNAi families. Genotype by environment interactions for major bre quality traits evaluated in the two

2 NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4062 ARTICLE

PHYA

1 kb

Hinge region

RNAi fragment

Exon

Coding Non-coding

Xho I Xho I Xba I Xba I

PHYA1 PDK intron 1AYHP

RNAi (T0)

Control

Control

RNAi (T0)

Figure 1 | Effects of PHYA1 RNAi in cotton. (a) Schematic representation of PHYA gene, RNAi fragment position and pHellsgate-8::PHYA1 RNAi plasmid; (b) shoot and root development and (c) bre length characteristics of T0-generation PHYA1 RNAi and control cotton plants, somatically regenerated by tissue culture. PDK introna pyruvate dehydrogenase kinase intron; blue boxes represent att sites attached to the target gene fragment.

Coker-312 (control)

T1 RNAi

T0 RNAi

Coker-312 (control)

T3 RNAi

Coker-312 (control) Coker-312 (control)

Figure 2 | Phytochrome-associated developmental changes in PHYA1 RNAi plants. (a) Enhanced vegetative growth and early owering in T0 RNAi plants; early owering in T1 generation RNAi plant (b,c); (d) difference in petiole length (T0); and (e) difference in root development (T3).

T0 RNAi

NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 3

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4062

Coker-312 (control) T3-31 RNAi family

AN-boyovut-2 AN-boyovut-2 RNAi coker-312 (F2)

Figure 3 | Differences in vegetative growth between eld-grown T3 RNAi and control plants. Transferability of the phytochrome-associated RNAi effects from RNAi Coker-312 to Upland cultivar (AN-Boyovut-2) is shown. Enhanced plant development in the eld test during 2009 (a,b); and improvement of bre samples (c) in the original cultivar (left) and RNAi F2 hybrids (right) grown in the same environment.

subsequent seasons were not signicant at Pr0.01 in combined ANOVA except for ELO (ANOVA, P 0.0006) and bre colour

( B; ANOVA, P 0.0001) traits (Supplementary Table 4).

Selected T2:3 RNAi families, examined over two seasons, showed small but statistically signicant (ANOVA, Pr0.01)

decreases of up to 3% in lint percentage and up to a 1012% decrease in lint index. However, the average number of bres per seed (Table 1) was not signicantly decreased in PHYA1 RNAi lines (B10,492, B10,289) when compared with the

Coker-312 wild-type (B10,848) or the null segregant controls (B10,485). Overall, a statistically signicant 3% increase in seed weight was observed in RNAi plants. The average seed cotton weight per boll signicantly increased in RNAi genotypes (B6.376.41 g) compared with wild-type (B5.67 g) and null segregant (5.57 g) controls (Table 1). Comparative measurements of the raw seed cotton weight per unit area from small replicated plots of T3 and T4 generations of elite RNAi families in the two subsequent seasons showed an average 10% yield increase over non-RNAi control plants (Supplementary Table 5).

Copy number of integrated RNAi vectors. Vector-specic PCR amplication (see Supplementary Table 6 for primers) conrmed the insertion of the pHellsgate-8::PHYA1 RNAi construct into

the genomes of the selected T3 RNAi lines (Fig. 4b). Quantitative PCR (qPCR) results based on standard curve (Supplementary Table 7) showed that T3-1_7 plant have three copies while T3-31_10 plant contained two copies of pHellsgate-8::PHYA1 RNAi construct in their genomes. As an additional control, we included genomic DNA from Bt-cotton that has a known single copy of the cry I transgene inserted in its genome (Supplementary Table 8).

Relative expression of cotton phytochrome genes. Abundance of phytochrome transcripts in leaf tissues of non-transformed Coker-312 was highest in the PHYA subfamily (PHYA24

PHYA1), incrementally lower in PHYB and PHYC and lowest in PHYE (Fig. 6; Supplementary Tables 911). In somatically regenerated transformants carrying the pHellsgate-8::PHYA1 RNAi vector, PHYA1 transcript was suppressed by B70% in

T3-1_7 and B25% in T3-31_10. The RNAi construct did not appear to suppress the expression of PHYA2 or other phyto-chrome genes, with the possible exception of a slight (B10%)

downregulation of PHYB in T3-31_10. Unexpectedly, we detected 2- to 20-fold overexpression of PHYA2, PHYB (in T3-1_7), PHYC and PHYE genes in both RNAi lines. The overexpression of the non-target phytochrome genes was more evident in the T3-1_7 line where the deeper suppression of PHYA1 gene expression was observed compared with T3-31_10.

Except for ELO, Pearsons R statistics between bre traits described in Table 1 and relative quantity of cotton phytochrome transcripts (Fig. 6; Supplementary Table 11) revealed negative ( 0.6 to 1) or positive (0.540.98) correlations. For instance,

a Pearson correlation between UHM and PHYA1 expression was equal to 0.854, UHM and PHYA2 was 0.859, UHM and PHYB

was 0.611, UHM and PHYC was 0.68, and UHM and PHYE was 0.753.

Transferability of phytochrome-specic RNAi effects. We crossed T0-generation PHYA1 RNAi Coker-312 plants (including

T-1 and T-31 RNAi families described herein) with several popular Upland cotton cultivars that are grown commercially in Uzbekistan. The F1 and F2 generation hybrids from these crosses (for example, sexual hybrids between AN-Boyovut-2 and T-1_7) had more vigorous vegetative growth (Fig. 3b), elongated petioles, elongated boll peduncles, elongated fruiting branches, more owers and bolls, earlier owering (510 days) and earlier boll maturity, and senescence-associated anthocyanin pigmentation in leaves and stems compared with the non-RNAi commercial control cultivars and null hybrid (Uzbek cultivar crossed with Coker-312) grown side by side in the same plots (Supplementary Figs 7 and 8).

In the F2 and F3 generation hybrids, major bre quality traits were signicantly improved, compared with the original cultivated variety and the null hybrid (Fig. 3c; Supplementary Fig. 9; Supplementary Table 12). For example, in the PHYA1 RNAi hybrid AN-Boyvut-2 Coker-312_ T0-1, UHM was 1.29 inch, MIC

was 4.36 and STR was 31.75 g/tex. These values were statistically different (ANOVA, Pr0.05) from the original variety

AN-Boyvut-2 and null hybrid (1.19 inch for UHM, greater than B4.64 for MIC and less than 30.61 g/tex for STR, based on averages of two seasons of eld evaluations). Similar trends in bre trait improvement, as well as vigorous owering and root development, were observed in F2 and F3 generations of crosses to several other

Uzbek commercial cotton varieties (Supplementary Fig. 10).

The lint percentage (33.82%) and average number of bres per seed (B12,661) were signicantly decreased (ANOVA,

Pr0.05) in the RNAi commercial variety hybrids compared

with the null hybrid (34.04 lint% and B12,979 bres per seed),

4 NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4062 ARTICLE

T3-1_7 T3-31_10

Coker-312

M

1 2 3 4 5

1 kb 800 bp

700 bp 600 bp 500 bp 400 bp 300 bp

200 bp

100 bp

Staple length (cm) Root length (cm)

Coker-312

T3-1_7

T3-31_10

Coker-312

T3-1_7

Figure 4 | Fibre length and root development characteristics of selected T3-generation PHYA1 RNAi plant families. (a) Staple length of bre; (b) PCR verication using 35S-F/PDK-R primer pairs (Supplementary Table 6): M-100 bp ladder, 1T3-1_7; 2T3-31_10; 3Coker-312; 4pHellsgate-8::PHYA1 plasmid; 5no DNA template control. The same plants were used for copy-number identication and relative expression analyses using qRTPCR.

Coker-312 (wild)

Coker-312 (wild)

Coker-312 (wild)

pGFP line

Somatic clone

Null segregant

T 2-1_7 (RNAi)

T 2-31_10 (RNAi)

Figure 5 | Fibre staple length of control and PHYA1 RNAi cotton plants. pGFP plant was transformed in our Uzbekistan laboratory with green uorescent protein (GFP) construct (pBINmGFP5-ER ) used in cotton transformation12; somatically regenerated PCR-negative non-RNAi plant that escaped from PHYA1 RNAi vector transformation; pGFP, RNAi and somatic clones were regenerated from the same media, time and condition. Null segregant is non-RNAi plants segregated from T-1 and T-31 transformation events.

and the original cultivar (35.97 lint% and B13,351 bres per seed; Supplementary Table 12). However, the average seed cotton weight per boll was increased in RNAi hybrids (B6.58 g)

compared with the non-hybrid cultivar (B6.08 g) and null hybrid (B6.13 g) controls (Supplementary Table 12). We observed an average 17% increase in seed cotton yield (Supplementary Table 5) per unit area over original non-RNAi variety, as can be clearly seen in the RNAi hybrid versus control plant rows shown in Supplementary Figs 710.

DiscussionPhytochrome genes play key regulatory roles in a wide range of plant developmental processes13. As such, the manipulation of phytochrome gene function has long been considered as a

promising avenue for the modication of economically important traits in crops13,14. For example, overexpression of a transgenic PHYA gene in tobacco resulted in suppression of shade avoidance and greater allocation to leaf growth, resulting in an improved leaf harvest index15. Overexpression of the Arabidopsis PHYB gene in transgenic potato (Solanum tuberosum) resulted in increased tuber yields16. Previously, we observed a statistical association (Logarithm of odds (LOD) 4.13; the KruskalWallis

test, P 0.0001) between a PHYA1 single nucleotide

polymorphism marker and bre quality in an interspecic cross between Pima S-7 (G. barbadense) and Tamcot SP37(G. hirsutum) consisting of 96 F2 individuals9. We selected a unique PHYA1-specic fragment for the RNAi construct based on our previous sequencing of the phytochrome gene family in cotton10. The 213 bp PHYA1 fragment used in this construct

NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 5

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4062

Table 1 | Fibre quality seed and yield traits averaged from 2009 and 2010 growing environments.

Traits T-1 family (T23 RNAi) T-31 family (T23 RNAi) Coker-312 (wild type) Null segregant* (control) Major bre quality traits

UHM (s.e.) 1.29 (0.003)wzy 1.28 (0.002)wzy 1.23 (0.004)w 1.22 (0.006)w MIC (s.e.) 4.99 (0.07)wzy|| 5.21 (0.04)wzy 5.43 (0.08)w 5.40 (0.13)w

STR (s.e.) 31.20 (0.28)wz 31.25 (0.13)wz 29.71 (0.37)w 31.01 (0.31)w ELO (s.e.)z 9.82 (0.18)wzy 10.50 (0.09)wzy|| 9.58 (0.21)w 10.34 (0.38)w

UI (s.e.) 88.42 (0.46)w 88.06 (0.08)w 87.25 (0.25)w 87.13 (0.39)w RD (s.e.) 76.50 (0.26) 77.11 (0.16) 75.70 (0.28) 77.41 (0.99)

B (s.e.)z 8.97 (0.11)y 9.47 (0.08)y|| 9.51(0.22) 8.60 (0.18) No. samples/replications 46/6 175/9 20/5 7/2

Seed and lint percentage traitsWeight of 100 seeds, g (s.e.)z 12.61 (0.25) 12.90 (0.17) 12.65 (0.23) 12.34 (0.42) Lint% (s.e.)z 36.14 (0.56)wz 36.23 (0.43)wz 39.20 (0.33)w# 36.33 (0.46)w

Seed weight% (s.e.)z 63.86 (0.56)wz 63.77 (0.43)wz 60.80 (0.33)w# 63.67 (0.46)w Lint index (s.e.)z 7.15 (0.18)wz 7.33 (0.12)wz 8.14 (0.13)w# 7.03 (0.26)w

No. samples/replications 30/4 29/4 28/4 12/4

Yield traitsASCWPB, g (s.e.) 6.37 (0.27)wy 6.41 (0.08)wy 5.67 (0.25)w 5.57 (0.11)w No. samples/replications 9/3 9/3 6/2 12/4 Number of bre/seedSTD 10,492.0430.8 10,289.6282.6 10,848.4589.2 10,485.8618.3 No. samples/replications 3/3 3/3 3/3 6/6

ASCWPB, average seed cotton weight per boll; B, bre colour; ELO, elongation (or bre elasticity, %); MIC, micronaire; RD, reectance (%); RNAi, RNA interference; STR, bre strength (g/tex); UHM,

upper half mean (inches); UI, bre uniformity (%);

Lint percentages (weight of lint bres/weight of seed cotton) 100; lint index (Lint percentage weight of 100 seeds)/seed weight percentage. ASCWPB from 2009 evaluation. Average number of

bres per seed (non-signicant in ANOVA test) were counted according to Zhang et al.36*Only T -generation (2009) season bre quality data were presented for PCR-negative null segregant control (see also Supplementary Table 13).

wStatistical signicance (ANOVA, Pr0.01) between PCR-positive (T-1 and T-31 RNAi lines) and -negative groups (wild and null segregant controls).

zBetween wild Coker-312 and T-1/T-31 RNAi lines. yBetween T-1/T-31 RNAi lines and null segregant.

||Between T-1 and T-31 families.zGenotype by environment interactions between two years (2009 and 2010) of evaluations were shown.

#Between wild Coker-312 and null segregant.

Relative expression level (log 2)

5

4

3

2

1

0

PHYA1 PHYA2 PHYB PHYC PHYE

Coker-312

T3-31_10 T3-1_7

Figure 6 | Quantitative analysis of transcript abundance using phytochrome subfamily-specic qRTPCR. The mean abundance of PHYE in non-transformed Coker-312 was arbitrarily set at a value of 1. Relative expression of other transcripts is displayed on a log2 scale. Error bars indicated the s.e.m. based on at least three biological replications.

shared 87% nucleotide similarity with the cotton PHYA2 genes, 75% nucleotide similarity with Arabidopsis PHYA and only 5059% nucleotide similarity with cotton PHYB, PHYC and PHYE genes. Since effective RNAi suppression typically requires 80100% nucleotide identity17, we anticipated that our

construct would only target the PHYA1 genes and perhaps PHYA2 genes as well.

Our results demonstrated that the suppression level of PHYA1 modied the expression of other phytochrome genes. Cross-regulation among the phytochrome genes has been observed in Arabidopsis and other plants. For example, Arabidopsis loss-of-function phyB mutants show reduced expression of PHYC in dark-grown tissues, and reduced PHYC, PHYD and PHYE in light-grown tissues18. In light-grown tissues from a sorghum phyB mutant, PHYA expression was increased in all light conditions tested19. These cross-regulatory effects may be the manifestations of a compensatory regulatory network2022. In cotton, we observed a substantial increase in PHYB transcript in the strongest of our T3-1_7 RNAi line (Fig. 6). This sharply contrasts with ndings from Arabidopsis in which loss-of-function phyA mutations showed no increase in PHYB expression23. Similarly, both of the PHYA1 RNAi lines showed increased transcript levels for PHYA2, PHYC and PHYE. These observations also contrast with Arabidopsis18,23, and indicate that the phytochrome regulatory network of cotton may have a fundamentally different dynamic architecture than that of Arabidopsis.

While earlier observations suggested an effect of increased FR/R light ratio on bre length and diameter4, and identied a preliminary association of PHYA1 with a bre length QTL in cotton9, our results provide solid evidence of a molecular role for phytochromes in regulating cotton bre traits. As cotton lacks the sophisticated molecular and genomic tool kit of the model plant Arabidopsis, including facile Agrobacterium transformation24 and comprehensive collections of knockout mutants25, it is not possible at this point to ascribe the bre phenotypes directly to the suppression of PHYA1 or, alternatively, to the indirect effects of the increased levels of PHYA2, PHYC and PHYE. In either

6 NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4062 ARTICLE

case, enhancement of bre length could occur because of a change in phytochrome-mediated plant hormone signalling1,26,27. Auxin (indole-3-acetic acid, IAA), abscisic acid, gibberellic acid, brassinosteroids, ethylene and cytokinin are known factors associated with bre development6. For instance, recent spatiotemporal manipulation of auxin biosynthesis in developing cotton ovules showed enhancement of bre yield and quality parameters6. There is well-supported evidence of molecular crosstalk between auxin and light signalling. Interactions of auxin with PHYA through phytochrome-dependent phosphorylation of auxin-related proteins, increases in auxin biosynthesis or regulation of auxin distribution26 could explain the bre length improvement in our PHYA RNAi plants6. Further, we previously observed that photomorphogenesis-related factors (for example, PHYC, SPA1, FAR1, COP1, COP9, CIP7, CIP8 and RTP2) were targeted by putative ovule-derived short interfering RNAs during the initiation and elongation phases of bre development28, thus indirectly supporting a role for light signalling in bre traits.

Our data suggested that some bre traits (for example, ELO and STR) might be minimally affected by somaclonal variation. However, the major phenotypic variations (for example, bre length, owering, plant architecture, shoot and root developments, maturity and so on; Figs 15) observed among plants from different transformation events were correlated with the level of PHYA1 RNAi suppression, which could in turn be due to differences in RNAi copy number (Supplementary Table 8), the positions of genome insertions or the arrangement of the transgene locus in the cotton genome. In our study, the T3-1_7 line had the most dramatic phenotypic effects, and also had the greater suppression of PHYA1 gene and the higher estimated copy number of the RNAi construct.

Somaclonal variation resulting from extended tissue culture may have also contributed to the decreased lint percentage and lint index, as well as a non-signicant decrease in the average number of bres per seed in RNAi genotypes and in the null segregant. Further lint percentage decreases in RNAi plants might be directly associated with the average number of bres per seed, or it could be inuenced by the increased neness of RNAi plant bres. Improvements in bre quality characteristics such as microniare, STR, uniformity index and others could also be the result of modied owering time that made the plants better adapted to local environmental conditions. We also speculate that an early senescence of vegetative organs (in particular leaves) in the RNAi cotton lines may lead to an earlier mobilization of leaf cell nutrient resources, and transfer to the developing fruits, fostering simultaneous maturation of all cotton plant bolls, thus improving bre quality.

Moreover, increased yield may be due to some combination of more vigorous shoot growth (including greater numbers of fruiting branches, owers and bolls, as well as improved seed cotton weight per boll) and root development (potentially improving water and nutrient assimilation) in RNAi plants. The multiple-fold overexpression of cotton PHYE (as well as PHYB in line T3-1_7) in PHYA1 RNAi plants might result in increased yield potential, as has been seen in tobacco15 and potato16 since PHYE is most closely related to PHYB, both functionally and phylogenetically13. This hypothesis is supported by the observation that both the potato PHYB-overexpressing lines16 and our cotton PHYA1 RNAi suppression lines showed markedly increased anthocyanin production, particularly close to the time of senescence. The increased yield could also be associated with enhanced root development of RNAi lines, thus mediating better assimilation of soil nutrients or water.

Early maturity and owering of cotton also minimizes late season risks of insects/pathogen attacks and aids in proper

timing of crop rotation (for example, wheat-cotton-wheat), potentially increasing farm incomes by reducing the input costs for fertilizers, water, insecticides and pesticides29. In particular, unfavourable late season weather conditions signicantly decrease bre quality parameters29. Early maturity of cotton cultivars is especially vital for cotton-growing plantations of northern latitudes with short cotton seasons such as Uzbekistanthe northernmost cotton growing country. Through manipulation of PHYA1 expression using RNAi, we have achieved simultaneous improvement of several important traits, such as bre length and early maturity, without negatively affecting other key parameters such as yielda goal that has largely eluded conventional breeding. For instance, introgression of superior bre quality traits from Pima cotton (G. barbadense) into Upland (G. hirsutum) cultivars using interspecies genetic hybridization is challenging owing to segregation distortion and linkage drag that often results in introgressed progenies that are late maturing or have poor agronomic quality30. Application of RNAi has allowed us to effectively solve these fundamental problems, and rapidly improve maturity, owering, yield and major bre quality traits, thus transforming commercial Upland varieties into more long-stapled, productive and early-maturing cultivars.

Coker-312 is the only G. hirsutum line that is easily amenable to tissue culture regeneration12,31,32; however, it is not a commercially competitive bre producer. While our RNAi construct was initially introduced and tested in Coker-312, a critical challenge was to show genetic stability and transferability of this construct and its resulting suite of traits to elite commercial cultivars. Using conventional sexual crosses, we successfully transferred the RNAi into the elite Uzbek commercial line AN-Boyovut-2 (which is well adapted for local growing conditions and cultivation practices). We succeeded in the efcient and stable transfer of the RNAi effects that were observed in original RNAi Coker-312 line, while maintaining the adaptive characteristics of the original cultivar, AN-Boyovut-2, thus developing a new superior cotton cultivar. While some small effects on bre traits (for example, STR) may have been inuenced by factors such as hybrid vigour (heterosis), environmental conditions and varietal background (Supplementary Table 12), the dramatic improvements observed in RNAi hybrids (for example, UHM, MIC and STR) were not seen in variety Coker-312 null-hybrid plants (that is, without the RNAi

construct), and are therefore probably due to action of the RNAi transgene itself. Similar trend of key agronomic and bre quality trait improvements in several other Uzbek Upland cultivar backgrounds (Supplementary Fig. 10) further demonstrated the usefulness of PHYA1 RNAi for conventional cotton breeding. With the intent of future commercialization, later generations (F4:5) of RNAi families with improved traits are being tested in larger eld plots in Uzbekistan in 20122015.

Thus, modication of cotton through modulation of phyto-chrome photoreceptor activity using RNAi not only provides a solution to a longstanding problem for conventional cotton breeders (bre quality versus yield and early maturity), but may also lead to signicantly increased income for cotton producers worldwide, thus opening a new paradigm in Upland cotton improvement. The demands for longer, ner and stronger cotton lint bre, as well as early maturity, early owering and improved seed cotton yield potential are key economic incentives in the global cotton market, and highlight the importance of our ndings.

Methods

Plant materials. Plant materials used in this study were the somatically regenerable cotton genotype G. hirsutum line Coker-312 and its transgenic derivative lines, transformed with pHellsgate-8::PHYA1 vector. To verify the transferability of

NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 7

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4062

the observed RNAi effects, we used several commercially important Uzbek cotton cultivars, for example, G. hirsutum cv. AN-Boyovut-2, C-6524, Namangan-77 and/or Toshkent-6 for the conventional genetic hybridization experiments with RNAi Coker-312 plants.

RNAi vector construction. For RNAi vector construction, cotton PHYA1 gene-specic primers with the attB sites (Gos_PHYA1attB1-F and Gos_PHYA1attB2-R) were designed (Supplementary Table 6) and purchased from Integrated DNA Technologies Inc., (IA, USA). The specic cotton PHYA1 fragment (213 bp) was amplied from G. hirsutum (cv. TM-1) genomic DNA with KODHiFi high delity proofreading DNA polymerase (Novagen, USA) using non-attB gene-specic primers (PHYA1-F: 50-GTGCTCGGAGTTAGTCCCATCAC-30; PHYA1-R:50- GTCCGTATGATTGTTGATTGTCG-30). The PCR was conducted according to the manufacturers instructions and protocol. The rst round gene-specic PCR product sizes were veried using agarose gel electrophoresis; then, attB1 and attB2 sites were attached to the PCR products in a second round of PCR with attB-anked gene-specic primers (Supplementary Table 6). PCR products were puried with polyethylene glycol solution (containing 26% polyethylene glycol 8,000, 6.5 mM MgCl2 and 0.6 mM sodium acetate pH 5.2) to remove remaining attB primers.

Site-specic recombination between attB site-anked gene fragment and the vector pDONOR221 (Invitrogen, USA) was conducted as described by Helliwell et al.11 in a total reaction volume of 10 ml, with 2 ml BP clonase buffer (Invitrogen), 2 ml attB site-anked PCR product, 150 ng plasmid vector and 2 ml BP clonase (Invitrogen). The reaction mix was incubated at 25 C overnight and 2 ml of this recombination mix was transformed into chemically competent DH5-alpha Escherichia coli cells (Invitrogen). Transformed cells were grown in lysogeny broth (LB) agar plates containing 50 mg l 1 spectinomycin and colonies were subjected to plasmid isolation by the NaOH/SDS lysis method33 and analysis by the PCR of the insertion with vector-specic M13 primers.

Recombinations reaction between the pDONOR221-attB-PHYA1 and the pHellsgate-8 vector with 35S promoter11 were conducted in a total volume of 10 ml, with 2 ml LR clonase buffer (Invitrogen), 2 ml recombinant pDONOR221-attB-PHYA1 (150 ng), 300 ng pHellsgate-8 and 2 ml LR clonase (Invitrogen). Reactions were incubated for overnight at room temperature, treated with proteinase K and a 2-ml aliquot was transformed into DH5-cells (Invitrogen). Cells were grown in LB plate containing selective antibiotic (spectinomycin) and colonies picked for verication of correct recombination with attB sites by restriction analyses with XhoI (for sense orientation site) and Xba (anti-sense orientation site). Positive clones were selected for further RNAi vector preparation as described by Helliwell et al.11 RNAi vectors were transformed into Agrobacterium tumefaciens strain LBA4404 for plant transformation experiments.

Cotton transformation and somatic embryogenesis. We used a combination of available methodologies for cotton tissue culture12,31,32. Hypocotyl sections (57 mm thickness) from 1-week-old seedlings of Coker-312 were wounded with a laboratory razor and placed on P1 medium (4.31 g l 1 Murashige and Skoog basal salt mixture (MS salt), 0.4 mg l 1 thiamine HCl, 100 mg l 1 myoinositol,0.75 mg l 1 MgCl2, 3% glucose, 0.2% phytagel, 5 mg l 1 N6-(2-isopentenyl) adenine, 0.1 mg l 1 1-naphthaleneacetic acid, pH 5.8). On the top of these wounded hypocotyl sections, 5 ml of a suspension of A. tumefaciens LBA4404 bearing the pHellsgate-8::PHYA1 RNAi vector was applied, followed by an incubation at 22 C for 72 h in the dark. The LBA4404 suspension bearing pHellsgate-8::PHYA1 RNAi vector was grown in YEP medium (10 g l 1 Bacto peptone, 5 g l 1 NaCl, 10 g l 1 Bacto yeast extract, pH 7.0) containing rifampicin (10 m l 1) and spectinomycin (50 mg l 1) antibiotics. Bacterial cultures were grown in culture tubes for 36 h at 26 C with shaking at 200 r.p.m. Cells from ve tubes were pooled, harvested by centrifugation at 3,200 g for 10 min (Eppendorf 5810R, Germany) and resuspended in 10 ml of pre-induction medium (10 g l 1 glucose, 14.62 g l 1 2-(N-morpholino)ethanesulfonic acid, 20 ml l 1 sodium phosphate buffer pH 5.6 and 50 ml l 1 20 AB salt stock34 containing 100 mM

acetosyringone). For controls, 5 ml sterile water was applied instead of bacterial suspension.

After 72 h, infected and control hypocotyl sections were transferred to fresh P1 medium, containing kanamycin (50 mg l 1) and cephabol (500 mg l 1; analogue of clavamox), and cultures were grown under a 16-h photoperiod (10 mmol m 2 s 1). After 3 weeks, 3 mm callus tissues were transferred into new P7 medium(4.31 g l 1 MS salts, 0.4 mg l 1 thiamine HCL, 100 mg l 1 myoinositol,0.75 mg l 1 MgCl2, 3% glucose, 0.2% phytagel, 0.1 mg l 1 (2-isopentenyl) adenine, and 5 mg l 1 1-naphthaleneacetic acid, pH 5.8) and sub-cultured onto fresh media monthly. Callus tissues less than 3 mm were kept in P1 medium for another 3 weeks and subsequently transferred to the P7 medium. After 16 weeks, to induce the somatic embryogenesis callus tissues grown on selective P7 medium were transferred to new modied medium R5 (4.31 g l 1 MS salt, 1 ml l 1 vitamins

Gamborg solution, 1.9 g l 1 KNO3, 0.75 mg l 1 MgCl2, 3% maltose and 0.2% phytagel) in which somatic embryos arose after 1216 weeks. Somatic embryos 6

7 mm in size were transferred into modied SH1 (10 ml l 1 100 micronutrients,

50 ml l 1 50 macronutrients, 1 ml l 1 vitamin B5, 5 g l 1 sucrose, 15 g l 1

bactoagar and 2 g l 1 phytagel) medium and incubated in the dark for 10 days. After dehydration32 and root initiation, embryos were transferred to new SH-2

medium (10 ml l 1 100 micronutrients, 50 ml l 1 50 macronutrients,

1 ml l 1 vitamin B5, 20 g l 1 sucrose, 1 g l 1 phytagel and 5 g l 1 agar) and grown for 10 days under a 16-h photoperiod (10 mmol m 2 s 1). After the development of initial roots and leaves, embryo plantlets were transferred to SH-3 medium (10 ml l 1 100 micronutrients, 50 ml l 1 50 macronutrients,

1 ml l 1 vitamin B5, 20 g l 1 sucrose, 1 g l 1 phytagel and 2.25 g l 1 agar) and grown with increased light (70 mmol m 2 s 1) for full development of roots and leaves. After 10 days, fully developed embryo plantlets were transferred into plastic containers with SH-3 medium and grown until the development of four to ve leaves and additional roots. All reagents for cotton tissue culture were purchased from Phytotechnology Laboratories, USA.

Identication of transformed plants using PCR. Genomic DNAs were isolated from frozen leaf tissues using the method of Dellaporta et al.35 Amplication reactions were performed in 50 ml volumes containing 4.5 ml 10 PCR buffer with

1.5 mM MgCl2, 1 ml BSA, 0.5 ml 25 mM of a dATP, dGTP, dTTP and dCTP mix,2.5 ml 50 ng ml 1 of each RNAi vector-specic 35S-F/PDK-R or PDK-F/OST-R primers pairs (Supplementary Table 6), 1 ml 50 ng ml 1 template DNA and 0.5 U

Taq DNA polymerase (Sigma, USA). Amplications were carried out with an initial denaturation at 94 C for 3 min followed by 45 cycles of 94 C for 1 min, 55 C for 1 min and 72 C for 2 min. A nal 5 min extension at 72 C was then performed. For determining PCR product sizes, 2% agarose (Sigma) gel electrophoresis was carried out in 0.5 TBE buffer. Gels were stained with

ethidium bromide.

RNAi plant evaluations and statistical analyses. PCR-positive transformants and control plants were transferred to soil and grown in the greenhouse environment to produce self-pollinated T1 seeds. PCR-positive PHYA1 RNAi embryonic plants were sexually hybridized with several popular Uzbekistan Upland cotton cultivars and F1 hybrid plant seeds were harvested. Subsequently, 1520 T1 or F1 seeds were germinated in small paper-soil pots in the greenhouse environment, and plants were checked to verify the presence of the transgene using PCR with RNAi vector-specic primers. PCR-positive T1 and F1 plants were transferred into larger pots and evaluated for owering time, boll maturation and bre staple length characteristics compared with non-transgenic Coker-312 plants grown in the same greenhouse environment. Flowering characteristics were determined from owering time (the rst ower opening date from planting) and number of opened owers. Maturity was determined based on number of opened bolls (above 50%) per plant at the time of harvesting the bolls. Fibre length was measured manually in comparison with control Coker-312 genotype bres or original non-RNAi Uzbek cultivar.

Further, 4045 T2 or F2 plants, derived from each superior quality T1 or F1 plant (single seed decent) of different transformation events, were grown as a family along side of 25 non-transgenic control Coker-312 plants in standard replicated eld plot design in a two-row (60 30 cm2 row-spacing) layout.

Evaluations were conducted at the controlled eld station (with no access of cross-pollinating insects) of the Institute of Genetics and Plant Experimental Biology, Tashkent, Uzbekistan in 2009. Plots were 6.3 1.2 m2. The average

indices for hypocotyl length, number of opened owers and opened bolls of each eld-grown T2 or F2 RNAi family and control plants were recorded. First, owers opened were tagged with date. All plants were self-pollinated to produce pure T3 or F3 generation seeds (by wrapping the petals with cotton threads before oral opening).

In 2010, self-pollinated T3 or F3 generation seeds from two superior RNAi families (T2-1_7 and T2-31_10) and control plants were grown in the eld station, Tashkent, Uzbekistan, with each family in 10 rows, 10 m long and 9 m wide plot, with 90 20 cm2 row-spacing layout. Evaluations were performed with at least

three replications for RNAi, RNAi hybrid and control plants. We measured yield potential by weighing the raw seed cotton yield from T3 or F3 RNAi cotton families and controls grown in the same eld and environmental conditions. Lint percentage, the weight of 100 seeds (seed index) and lint index were measured manually from randomly chosen individual plants of each selected T2:3 or F2:3

RNAi family and control plants, taking seed cottons from the six fully matured bolls per plant.

Root length of RNAi and control plants was measured 25 days after seed germination. Plastic pots were cut and roots were carefully washed. In addition, randomly chosen individual T2:3 or F2:3 plants from the eld were dug at the owering and boll maturation stages, roots were washed and root lengths were compared with control plants. Germination rates and hypocotyl lengths of T2:4

generation RNAi plants and controls were measured in triplicate under light (16 h photoperiod, 100 mmol m 2 s 1) and dark conditions in the laboratory.

Fibres of the second- and third-generation plants from replicated plots were ginned in small laboratory roller gin, and the main bre quality traits of the same eld-grown RNAi and control plants from 2009 and 2010, including UHM, MIC, STR, ELO, bre uniformity, Rd and B were measured using high volume

instrumentation at the bre-testing Centre SIFAT, Tashkent Uzbekistan and STRAR LAB at Knoxville, TN, USA.

The number of bres per seed was determined according to the methodology by Zhang et al.36 in a triplicate experiment, averaged from the independently repeated counting performed by three different individuals using microscopy. The statistical

8 NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4062 ARTICLE

signicance of trait differences between RNAi families and control plants were tested in combined ANOVA using main effects and two-way interactions of Visual Statistics software (ViSta37).

Copy-number identication for the integrated RNAi sequence. We followed the real-time qPCR (qRTPCR) methodology38 and calculations39 to estimate the copy number of the PHYA1 RNAi vector integrated into the transformed cotton genomes. We cloned the GhUBC1 fragment into the plasmid vector pCR4 TOPO-TA following the manufacturers protocol and instructions (Invitrogen), which contains nptII gene as a selectable marker. This way, we obtained a reference plasmid vector containing both nptII and GhUBC1 gene for constructing a standard curve based on absolute copies of the plasmid vector (Supplementary Table 7). We made 10-fold serial dilutions of plasmid vector and amplied target genes using qRTPCR primer pairs and probes (Supplementary Table 6). qRTPCR was carried out in an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster City, USA). PCR reactions were performed in a 12.5-ml volume with the following standard programme recommended by the manufacturer: 95 C for 10 min, followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. Each 12.5 ml reaction mixture contained 6.25 ml Master Mix (2 ), 0.25 ml (200 nM)

of each primer (10 mM), 1 ml (40 nM) of probe (0.5 mM), 4 ml of template DNA sample (0.2 pg20 ng) and 0.75 ml sterile deionized water. A 2 PCR Master Mix

contained No AmpErase UNG, AmpliTaq Gold DNA polymerase, deoxynucleo-side triphosphates with dUTP and Taq Man reaction buffer with magnesium chloride (Applied Biosystems).

Average Ct values were plotted against log of absolute copy numbers to obtain standard curves. Six replicates were conducted to construct standard curves for each target gene. Efciencies of amplication were calculated based on slopes of standard curves with the following formula: E 10( 1/slpoe) 1 (refs 38,40). The

copy-number calculation was performed as described by Weng et al.39 using the equation X0/R0 10[(Ct, X IX)/SX)] [(Ct, R IR)/SR], where X is nptII, R is UBC1, I is

intercept of the standard curve, S is slope of the standard curve for target (X) and reference (R) genes39. X0/R0 values were used directly (that is, without doubling)

for copy-number estimation since our samples were T3-generation plants, and not T0, as was the case in Weng et al.39 Coefcient of variation in copy-number estimates was calculated from coefcient of variation estimates for GhUBC1 (endogenous control) and nptII (target gene).

RNA isolation and quantitative real-time PCR. Total RNAs were isolated from leaf tissues using combination of protocols described by Suzuki et al.41 and Wu et al.42 RNAs were isolated from the same control and RNAi plants that were used for copy-number identication and detailed phenotypic evaluations. Briey,100 mg of fresh leaf tissues was frozen in liquid N2 and powdered using a pestle and mortar, followed by addition of 2 ml extraction buffer of Wu et al.42, heated to 80 C and with 10 mM (1.54 mg ml 1) dithiothreitol freshly added. Then, 60 ml of 25 mg ml 1 proteinase K was added to the homogenate and ground further to mix.

The homogenate was then transferred to 2 ml plastic tubes, incubated at room temperature for 15 min, and was then centrifuged (Eppendorf 5415R, Germany) at top speed for 20 min at 4 C. The supernatant was transferred to fresh 2 ml tubes and RNA was isolated with a conventional water-saturated phenol:chloroform-isoamylacohol RNA precipitation procedure41,43. From this point, all steps were exactly as described and optimized by Suzuki et al.41 The resulting RNA pellet was washed with 75% (v/v) ethanol, air-dried and dissolved in sterile diethylpyrocarbonate-treated water.

The integrity of RNA was checked and judged by the presence and intensity of ribosomal RNA bands on a 1% agarose gel containing 2.2 M of formaldehyde in the presence of ethidium bromide44. RNA samples were treated with RNAase-free DNAase I (Ambion, USA) according to the manufacturers protocol and repuried with additional purication steps using acid phenol:chloroform (5:1; Ambion) and ethanol precipitation. The concentrations of total RNA samples were quantied using spectrophotometer (GENESYS 10UV, Thermo Scientic, USA). The rst-strand complementary DNAs were synthesized from B2 mg total RNAs using an

Avian RT cDNA kit (Sigma) with random nonamer primers according to the manufacturers protocol. The rst-strand cDNAs were diluted 1:15 with sterile water and used in expression analyses. RTPCR reactions were carried out with intron-specic primer pairs45 (A1341F/R; Supplementary Table S6) to check for DNA contamination, and with primers for catalytic subunit of protein phosphatase 2A gene of cotton (GhPP2A1)46 to check the quality of the cDNAs synthesized. RTPCR reactions were carried out using an RTPCR kit (Sigma) according to the manufacturers protocol. Samples were subjected to qRTPCR only if they failed to amplify intronic primer pairs, but successfully amplied the endogenous (GhPP2A1) reference gene.

To identify RNAi inuence in the expression of cotton phytochrome genes, we utilized a qRTPCR method using SYBR green-based amplicon detection and primer pairs shown in Supplementary Table 6. qRTPCR was carried out in an Applied Biosystems 7500 real-time PCR system (Applied Biosystems). PCR reactions were performed in a 25-ml volume with the following cycling conditions: 95 C for 10 min, followed by 40 cycles of 95 C for 15 s and 65 C for 45 s. Each 25 ml reaction mixture contained 12.5 ml SYBR Green Master Mix (2 ), 0.35 ml

(140 nM) of each primer (10 mM), 6 ml of template 1:15 times diluted cDNA template and 5.8 ml sterile deionized water. A 2 SYBR Green PCR master mix

contained No AmpErase UNG, AmpliTaq Gold DNA polymerase, deoxynucleo-side triphosphates with dUTP and SYBR Green reaction buffer with magnesium chloride (Applied Biosystems). Post real-time PCR dissociation curves were constructed for each primer pairs and used to rule out primer-dimers, genomic DNA contamination and mis-annealing. Analysis of qRTPCR amplications was conducted using 7500 System SDS v1.4 Software (Applied Biosystems).

For creation of standard curves for each gene used in qRTPCR analysis, twofold dilution series (1 , 2 up to 128 ) of 1:15 diluted cDNA from control

Coker-312 was created. Average Ct values from, at least, two replicates were plotted against log of starting amount to obtain standard curves. Efciencies of amplication were calculated based on slopes of standard curves with the following formula: E 10( 1/slope) 1 (refs 38,40). Slope and intercept indices from the

standard curves obtained for each gene analysed were used to calculate log input amount (Log input amount (Average Ct

target gene

target gene)/

Slope target gene); then, the input amount was calculated using 10log input amount.

Input amounts obtained for phytochrome genes were divided by the input amount of GhPP2A1. The relative quantity of the target genes was then calculated by dividing the normalized quantity of target gene expression in RNAi plants by the normalized quantity of the same gene expression in a control plant, chosenas a calibrator. Coefcient of variation was calculated from coefcient of variation estimates for GhPP2A1 and each of the phytochromes.

Intercept

References

1. Fankhauser, C. & Chory, J. Light control of plant development. Ann. Rev. Cell Dev. Biol. 13, 203229 (1997).

2. Bagnall, D. J. et al. Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh. Plant Physiol. 108, 14951503 (1995).

3. Lin, C. Photoreceptors and regulation of owering time. Plant Physiol. 123, 950 (2000).

4. Kasperbauer, M. J. Cotton bre length is affected by far-red light impinging on developing bolls. Crop. Sci. 40, 16731678 (2000).

5. El-Mogahzy, Y. E. & Chewning, Jr C. H. Cotton Fibre to Yarn Manufacturing Technology (Cotton Incorporated, 2001).

6. Zhang, M. et al. Spatiotemporal manipulation of auxin biosynthesis in cotton ovule epidermal cells enhances bre yield and quality. Nat. Biotechnol. 29, 453458 (2011).

7. Miller, P. A. & Rawlings, J. O. Selection for increased lint yield and correlated responses in Upland cotton, Gossypium hirsutum L. Crop Sci. 7, 637640 (1967).

8. Iqbal, M. et al. Correlation and path coefcient analysis of earliness and agronomic characters of upland cotton in Multan. Pak. J. Agron 2, 160168 (2003).

9. Abdurakhmonov, I. Y. Molecular Cloning and Characterization of Genomic Sequence Tags (GSTS) from the PHYA, PHYB and HY5 Gene Families of Cotton (Gossypium species). Thesis, (Texas A&M Univ., USA, 2001).

10. Abdurakhmonov, I. Y., Buriev, Z. T., Logan-Young, C. J., Abdukarimov, A. & Pepper, A. E. Duplication, divergence and persistence in the phytochrome photoreceptor gene family of cottons (Gossypium spp.). BMC Plant Biol. 10, 119 (2010).

11. Helliwell, C. A., Wesley, A. V., Wielopolska, A. J. & Waterhouse, P. M. High-throughput vectors for efcient gene silencing in plants. Funct. Plant. Biol. 29, 12171225 (2002).

12. Sunilkumar, G. & Rathore, K. S. Transgenic cotton: factors inuencing Agrobacterium-mediated transformation and regeneration. Mol. Breed. 8, 3752 (2001).

13. Smith, H. Phytochrome transgenics; functional, ecological and biotechnological applications. Semin. Cell Biol. 5, 315325 (1994).

14. Robson, P. R. H. & Smith, H. Fundamental and biotechnological applications of phytochrome transgenes. Plant Cell Environ. 20, 831839 (1997).

15. Robson, P. R. H., McCormac, A. C., Irvine, A. S. & Smith, H. Genetic engineering of harvest index in tobacco through overexpression of a phytochrome gene. Nat. Biotechnol. 14, 995998 (1996).

16. Thiele, A., Herold, M., Lenk, I., Quail, P. H. & Gatz, C. Heterologous expression of Arabidopsis phytochrome B in transgenic potato inuences photosynthetic performance and tuber development. Plant Physiol. 120, 7382 (1999).

17. Holzberg, S., Brosio, P., Gross, C. & Pogue, G. P. Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J. 30, 315327 (2002).

18. Hirschfeld, M., Tepperman, J. M., Clack, T., Quail, P. H. & Sharrock, R. A. Coordination of phytochrome levels in phyB mutants of Arabidopsis as revealed by apoprotein-specic monoclonal antibodies. Genetics 149, 523535 (1998).

19. Finlayson, S. A., Mullet, J. E. & Morgan, P. W. Phytochrome B and shade signals regulate phytochrome A expression. Physiol. Plantarum 127, 326338 (2006).

20. Hummler, E. et al. Targeted mutation of the CREB gene: compensation within the CREB/ATF family of transcription factors. Proc. Natl Acad. Sci. USA 91, 56475651 (1994).

NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 9

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4062

21. Tieman, D. M., Taylor, M. G., Ciardi, J. A. & Klee, H. J. The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family. Proc. Natl Acad. Sci. USA 97, 56635668 (2000).

22. Cheng, N. H., Pittman, J. K., Barkla, B. J., Shigaki, T. & Hirschi, K. D. The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters. Plant Cell 15, 4764 (2003).

23. Schepens, I., Duek, P. & Fankhauser, C. Phytochrome-mediated light signalling in Arabidopsis. Curr. Opin. Plant Biol. 7, 564569 (2004).

24. Clough, J. & Bent, A. F. Floral dip: a simplied method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735743 (1998).

25. Alonso, J. M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653657 (2003).

26. Colon-Carmona, A., Chen, D. L., Yeh, K. C. & Abel, S. Aux/IAA proteins are phosphorylated by phytochrome in vitro. Plant Physiol. 124, 17281738 (2000).

27. Stamm, P. & Kumar, P. P. The phytohormone signal network regulating elongation growth during shade avoidance. J. Exp. Bot. 61, 28892903 (2010).

28. Abdurakhmonov, I. Y. et al. Small RNA regulation of ovule development in the cotton plant, G. hirsutum L. BMC Plant Biol. 8, 93 (2008).

29. Panhwar, G. N. et al. Predicting earliness in cotton during crop development stage -11. Asian J. Plant Sci. 1, 3738 (2002).

30. Endrizzi, J., Turcotte, E. & Kohel, R. Genetics, cytology and evolution of Gossypium. Adv. Genet. 23, 271375 (1985).

31. Firoozabady, E. et al. Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Mol. Biol. 10, 105116 (1987).

32. Stewart, J. M. & Hsu, C. L. In ovulo embryo culture and seedling development of cotton (Gossypium hirsutum L.). Planta 137, 113117 (1977).

33. Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd edn (Cold Spring Harbor Laboratory Press, 1989).

34. Chilton, M. D. et al. Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc. Natl Acad. Sci. USA 71, 36723676 (1974).

35. Dellaporta, S. L., Wood, J. & Hicks, J. P. A plant DNA minipreparation: VersionII. Plant Mol. Biol. Rep. 1, 1921 (1983).36. Zhang, M., Zeng, Q., Hou, L., Li, D. & Pei, Y. A method for counting cotton mature bres per seed. Protoc. Exchange doi:http://dx.doi.org/10.1038/protex.2011.220

Web End =10.1038/protex.2011.220 (2011).

37. Young, F. W., Valero-Mora, P. M. & Friendly, M. Wiley Series in Probability and Statistics (Wiley &Sons, 2006).

38. Yi, C. X., Zhang, J., Chan, K. M., Liu, X. K. & Hong, Y. Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum). Anal. Biochem. 375, 150152 (2008).

39. Weng, H. et al. Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Mol. Biol. Rep. 22, 289300 (2004).

40. Ginzinger, D. G. Gene quantication using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503512 (2002).

41. Suzuki, Y., Makino, A. & Mae, T. An efcient method for extraction of RNA from rice leaves at different ages using benzyl chloride. J. Exp. Bot. 52, 15751579 (2001).

42. Wu, Y., Llewellyn, D. J. & Dennis, E. S. A quick and easy method for isolating good quality RNA from cotton (Gossypium hirsutum L.) tissues. Plant Mol. Biol. Rep. 20, 213218 (2002).

43. Chomczynski, P. & Sacchi, N. Single step method of RNA isolation by acid guanidinium thiocyanatephenolchloroform extraction. Anal. Biochem. 162, 156159 (1987).

44. Maniatis, T., Fritsch, E. F. & Sambrook, J. Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982).

45. Cronn, R. C., Small, R. L., Haselkorn, T. & Wendel, J. F. Rapid diversication of the cotton genus (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast genes. Am. J. Bot. 89, 707725 (2002).

46. Artico, S., Nardeli, S. M., Brilhante, O., Grossi-de-Sa, M. F. & Alves-Ferreira, M. Identication and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data. BMC. Plant. Biol. 21, 49 (2010).

Acknowledgements

This project was funded by the Ofce of International Research Programs of the United States Department of Agriculture (USDA) in the frame of USDAFormer Soviet Union cooperation programmes with the research Grant of P121/P121B to I.Y.A., S.S. and A.E.P. We acknowledge the Science and Technology Centre of Ukraine for project coordination, and the technical assistance of project participants of P121/P121B. The ISTEDOD (formerly UMID) Presidential Fund of the Government of Uzbekistan provided support for the I.Y.A. to conduct research at Texas A&M University. We also thank Cabinet of Ministries of Uzbekistan, Academy of Sciences of Uzbekistan, Ministry of Agriculture and Water Resources of Uzbekistan, Uzpakhtasanoat association, association of Oil-Fat& Food Industry, Ministry of Foreign Economic Relations, Investments and Trade of the Republic of Uzbekistan, bre quality testing Centre SIFAT of Uzbekistan and holding company )Uzvinprom Holding* for their continual in-house funding of this research. We are indebted to P. Waterhouse and C. Helliwell, CSIRO, Australia, for providing pHellsgate vector systems for our study. We greatly acknowledge the Uzbekistan and USA partner laboratories staff for the assistance with molecular experiments and eld evaluations described in this work.

Author contributions

I.Y.A and Z.T.B. performed vector construction, plant transformation, greenhouse and eld evaluations and molecular characterizations of RNAi plants; I.Y.A., A.A., S.S., J.N.J. and A.E.P. designed the study, performed data analyses and interpretation of the results, and edited the manuscript; I.Y.A. and A.E.P. drafted the manuscript.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Web End =http://www.nature.com/ http://www.nature.com/naturecommunications

Web End =naturecommunications

Competing nancial interests: A patent application for this work has been ledin Uzbekistan (IAP 20120069), the US Patent Ofce (USPTO:13/445696) and internationally (PCT/US13/27801). I.Y.A., Z.T.B., A.A., S.S., J.N.J. and A.E.P. are listed as co-inventors on the patent application entitled Cotton PHYA1 RNAi improves bre quality, root elongation, owering, maturity and yield potential in Gossypium hirsutum L.

Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

Web End =http://npg.nature.com/ http://npg.nature.com/reprintsandpermissions/

Web End =reprintsandpermissions/

How to cite this article: Abdurakhmonov, I. Y. et al. Phytochrome RNAi enhances major bre quality and agronomic traits of the cotton Gossypium hirsutum L.

Nat. Commun. 5:3062 doi: 10.1038/ncomms4062 (2014).

10 NATURE COMMUNICATIONS | 5:3062 | DOI: 10.1038/ncomms4062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.