o r i g i n a l r es e a r c h
Ghd7 (Ma6) Represses Sorghum Flowering
in Long Days: Ghd7 Alleles Enhance Biomass
Accumulation and Grain Production
Rebecca L. Murphy, Daryl T. Morishige, Jeff A. Brady, William L. Rooney,
Shanshan Yang, Patricia E. Klein, and John E. Mullet*
Abstract
Sorghum Ma6, a strong repressor of flowering in long days (LDs),
was identified as the CONSTANS, CO-like, and TOC1 (CCT)domain protein encoded by SbGhd7. Sorghum Ghd7 increases
photoperiod sensitivity and delays flowering by inhibiting
expression of the floral activator SbEhd1 and genes encoding
FT. SbGhd7 expression is light-dependent and gated by the
circadian clock. In LDs when flowering is repressed, SbGhd7
mRNA abundance peaks in the morning and evening. In short
days (SDs) when floral initiation occurs, the evening phase of
SbGhd7 expression occurs in darkness, reducing SbGhd7
mRNA abundance. In energy sorghum hybrids, dominant alleles
of SbGhd7 and SbPRR37 act in additive fashion to delay floral
initiation for ~175 d until daylengths decrease below 12.3 h.
In contrast, photoperiod-insensitive grain sorghum genotypes
containing recessive alleles of SbGhd7 and SbPRR37 flower
in 60 to 80 d. Recessive alleles of SbGhd7 and SbPRR37 are
present in historically important sorghum germplasm introduced
into the United States in the 1800s that was used to produce
early flowering grain and sweet sorghum. Recessive alleles ghd71 and prr37-1 are present in maturity standards such as SM100,
and in BTx406, a genotype used in the Sorghum Conversion
Program to convert late-flowering photoperiod-sensitive sorghum
accessions into early flowering photoperiod-insensitive germplasm
useful for grain sorghum breeding. The results show that alleles
of SbGhd7 confer differences in photoperiod sensitivity and
flowering times that are critical for production of late-flowering
high-biomass energy sorghum and early flowering grain sorghum.
Published in The Plant Genome 7
doi: 10.3835/plantgenome2013.11.0040
© Crop Science Society of America
5585 Guilford Rd., Madison, WI 53711 USA
An open-access publication
All rights reserved. No part of this periodical may be reproduced or
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Permission for printing and for reprinting the material contained herein
has been obtained by the publisher.
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ptimal flowering time is of central importance to a
plant’s reproductive success. Early flowering is useful for grain production in regions where growing seasons
are short and in environments where terminal drought
or adverse temperatures for grain production occur frequently during the latter part of growing seasons. Longer
duration of vegetative growth in forage crops increases
leaf and stem biomass production (Rooney et al., 2007). In
sweet sorghum (Sorghum bicolor subsp. bicolor), delayed
flowering increases the size of stems and the potential for
sucrose accumulation (Burks et al., 2013). In energy sorghum, delayed flowering and long duration of vegetative
growth is a key trait associated with high biomass yield
and nitrogen use efficiency (Rooney et al., 2007; Olson et
al., 2012, 2013). In Olson et al., 2012, biomass yield was
reported as 25 to 40 dry Mg/ha (or 25 to 40 dry metric t/
ha). Late-flowering, high-biomass energy sorghum and
other C4 energy grasses with long vegetative growth
duration could help meet the U.S. Energy Independence
and Security Act of 2007 mandate to produce 21B gals of
cellulosic biofuels per year (U.S. House, 2007).
Flowering time is regulated in a complex manner by development (autonomous pathway), daylength
R.L. Murphy, Centenary College of Louisiana, Shreveport, LA 71104;
R.L. Murphy, D.T. Morishige, S. Yang, and J.E. Mullet, Dep. of
Biochemistry and Biophysics, Texas AM Univ., College Station, TX
77843. J.A. Brady, Texas Agrilife Research and Extension Center,
Stephenville, TX 76401. W.L. Rooney, Dep. of Soil and Crop
Sciences, Texas AM Univ., College Station, TX 77843. P.E. Klein,
Dep. of Horticultural Sciences and Institute for Plant Genomics
and Biotechnology, Texas AM Univ., College Station, TX 77843.
Received 28 Nov. 2013. *Corresponding author (jmullet@tamu.edu).
Abbreviations: CCT, CONSTANS, CO-like, and TOC1 domain;
Ct, cycle threshold; DTF, days to flowering; INDEL, insertion-deletion
polymorphism; LD, long day, 14-h light/10-h dark; LL, constant light
free-running conditions; QTL, quantitative trait loci; SD, short day,
10-h light/14-h dark; SNP, single nucleotide polymorphism; SSR,
simple sequence repeat.
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(photoperiod), temperature, gibberellins, shading, and
other factors (Imaizumi and Kay, 2006; Andrés and
Coupland, 2012; Sanchez et al., 2011; Higgins et al.,
2010; Tsuji et al., 2011). Many of the pathways that control flowering time converge on the regulation of floral
integrator FT-like genes of the PEBP gene family that
encode peptides (florigens) that move from the leaf to
the shoot apex where they bind to FD and induce floral
meristem transition. In Arabidopsis, photoperiod regulation of flowering time is mediated by circadian clock
and light regulation of CONSTANS (CO) activity, an
activator of FT in LDs (Imaizumi and Kay, 2006). In rice
(Oryza sativa L.), sorghum, and other grasses, FT-like
genes (OsHd3a/OsRFT1, ZCN8) are also regulated by the
timing and extent of EARLY HEADING DATE 1 (Ehd1)
expression, an activator of FT expression unique to
grasses, and by a large number of other genes that modulate Ehd1 and FT expression (Doi et al., 2004; Itoh et al.,
2010; Tsuji et al., 2011).
Sorghum is a drought-tolerant C4 grass that is
cultivated widely as a source of grain, forage, sugar,
and biomass, especially in drought-prone regions of
the world (Rooney, 2004, 2007; Vermerris, 2011; Smith
and Frederiksen, 2000; Dahlberg et al., 2001). Optimal
flowering time of sorghum varies from ~50 to >200 d,
depending on growing location and crop type. Grain sorghum is generally selected for early flowering to enhance
grain yield and yield stability, whereas energy sorghum
is selected for late flowering to enhance biomass yield
(Rooney, 2004, 2007). The sorghum germplasm collection
(n = 40,000) varies extensively in flowering time and photoperiod sensitivity (Quinby and Karper, 1945; Quinby,
1966; Craufurd et al., 1999). For example, a survey of
sorghum accessions revealed critical photoperiods ranging from 10.2 to 17.5 h (Craufurd et al., 1999). Sorghum
flowering time is modified by temperature (Major et al.,
1990), gibberellins (Morgan et al., 1986), and photoperiod (Major et al., 1990; Childs et al., 1997) among other
factors. More than 40 QTL for flowering time have been
identified in sorghum (Mace et al., 2013). The degree of
photoperiod sensitivity in sorghum, a SD plant, depends
in part on alleles of the maturity loci Ma1 through Ma6
(Quinby and Karper, 1945; Quinby, 1966; Rooney and
Aydin, 1999). Light signaling through phytochrome B
(Ma3) is required for photoperiod sensitivity in sorghum
as in other plants (Childs et al., 1997). Ma1 encodes PRR37
an inhibitor of flowering in LDs that represses expression
of the floral activator Ehd1 and SbCN8 (FT; Murphy et al.,
2011). In this study, we report the identification of Ma6, a
locus that causes very late flowering in LDs (Rooney and
Aydin, 1999), as SbGhd7, a floral repressor regulated by
the circadian clock and light signaling.
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Materials and Methods
Plant Materials
The ATx623 ´ R.07007 BC1F1 population (n = 1821) was
grown under field conditions in the summer of 2003 in
College Station, Vega, and Plainview, TX. For flowering
date determinations, days to midanthesis (pollen shed)
were evaluated. BTx623 F2 plants (n = 124) were grown
under LDs in greenhouse conditions in 2008 and phenotyped for DTF (anthesis). Tissue was collected from
individuals for genotyping, and seed was collected for
planting. Following F2 family genotyping, selected F3
progeny (n = 255) from that population were grown under
the same LD greenhouse conditions in 2010, and scored
for flowering at anthesis. Tissue was collected from this
generation as well, and 185 combined progeny from both
populations were used in downstream QTL mapping.
Map-Based Cloning of Ghd7
Genomic DNA was isolated from each mapping line
using the FastDNA Spin Kit (MP Biomedicals, Solon,
OH). High throughput genotyping by 77 bp single-end
sequencing on an Illumina Genome Analyzer IIx was
used to identify novel SNP markers of parental lines and
F2/F3 offspring. Analysis of raw sequencing data was
performed according to Morishige et al. (2013). Genetic
map construction was performed using Mapmaker/EXP
3.0 (Lander et al., 1987), and the resulting map was used
in combination with flowering time phenotypic data as
input in QTL Cartographer. The log of odds threshold
was set using permutations of the data at 1000 iterations.
Quantitative trait loci above this threshold were considered statistically significant.
The Ma6 locus was refined further in the BTx623 F2/
F3 and ATx623 ´ R.07007 BC1F1 populations using SSR
markers (SSRIT, Temnykh et al., 2001), cleaved amplified
polymorphic sequence markers, SNPs, and insertiondeletion polymorphisms (INDELS). The SNP and INDEL
markers were discovered by de novo sequencing of
regions surrounding predicted open reading frames. Capillary sequencing of these markers was performed on the
ABI 3130xl Genetic Analyzer, and sequence assembly and
analysis was performed using Sequencher v. 4.8 (Gene
Codes Corporation, Ann Arbor, MI). All markers used
for fine mapping Ma6 are listed in Supplemental Table S3.
Ghd7 Allele Sequencing
Genomic DNA from each line in Table 1 was extracted
using the FastDNA Spin Kit (MP Biomedicals, Solon,
OH), and amplified using primers flanking the Ghd7
region. Ghd7 gene and coding sequences were amplified, and sequencing reactions were performed using the
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied
Biosystems, Foster City, CA). These reactions were sent
for capillary sequencing on the Applied Biosystems
3130xl Genetic Analyzer. The results were analyzed using
Sequencher v. 4.8.
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Table 1. Analysis of Ghd7 alleles in historically
important sorghum accessions.
Genotype
Energy sorghum
R.07019
R.07020
R.07018
Historical genotypes
Red Kafir
Tx Blackhull Kafir
Dwarf Yellow Milo
Double Dwarf Feterita
Hegari
Double Dwarf
Yellow Milo
Early White Milo
Standard Blackhull Kafir
Kalo
Caprock (Tx7000)
Combine 7078
CK60 (BTx3197)
Tx623
Rio
Conversion program
BTx406
IS12646
IS12646c
Maturity genotypes
F1 (Tx623 ´ R.07007)
100M
SM100M
Ma6
Ma1
DTF†
Description
Ghd7-2
Ghd7-5
Ghd7-6
PRR37
PRR37
PRR37
>175
>175
>175
very photoperiod sensitive
very photoperiod sensitive
very photoperiod sensitive
ghd7-2
ghd7-1
prr37-2
prr37-3
51
63
ghd7-1
PRR37
–
ghd7-2
ghd7-2
prr37-3
PRR37
65
140
ghd7-1
PRR37
–
ghd7-1
ghd7-1
ghd7-2
ghd7-1
ghd7-1
ghd7-1
ghd7-1
ghd7-2
prr37-1
prr37-2
prr37-2
prr37-2
prr37-1
prr37-3
prr37-3
prr37-2
59
68
60
58
57
64
71
85
grain sorghum (1876)
grain sorghum (1890)
grain sorghum (1905;
ma2ma3)
grain sorghum (1906)
grain sorghum (1908; ma4)
grain sorghum (1918;
ma2ma3)
grain sorghum (1920)
grain sorghum (1924)
grain sorghum (1933)
grain sorghum (1941)
grain sorghum (1944)
grain sorghum (1950)
grain sorghum (1960s)
sweet sorghum (1972)
ghd7-1
Ghd7-3
ghd7-1
prr37-1 60–70‡
PRR37 >175
prr37-1 60–70‡
Ghd7
ghd7-1
ghd7-1
PRR37
PRR37
prr37-1
>175
143
69
conversion program
exotic accession
converted accession
Ma1Ma2Ma3Ma4Ma5Ma6
Ma1Ma2Ma3Ma4Ma5ma6
ma1Ma2Ma3Ma4Ma5ma6
Days to flowering (DTF) were determined under long day (LD) summer greenhouse conditions. The
relative order of flowering times among the genotypes studied were similar in LD summer and fall
greenhouse conditions, although all plants flowered later under the higher temperature conditions in
the summer.
†
DTF data obtained from the USDA-ARS Germplasm Resource Information Network National Plant
Germplasm System.
‡
For the ghd7-2 allele, a protocol for flanking PCR
was used to delimit the border of a repetitive sequence
inserted into the intron (Siebert et al., 1995). Genomic
DNA (5 µg) from control genotypes (ghd7-1 and Ghd7),
as well as from a ghd7-2 line, was digested using blunt
end cutting restriction enzymes (New England BioLabs,
Inc.). Adapters (Supplemental Table S4) were then ligated
on to the end of the fragments with T4 DNA ligase (Promega), and PCR was performed and subjected to capillary
sequencing, followed by analysis with Sequencher v. 4.8.
Expression Analysis
The expression of Ghd7 was analyzed in leaf tissue collected from 39-d-old 100M and ATx623 ´ R07007 F1
plants treated with LD or SD light conditions at 3-h
intervals, as previously described (Murphy et al., 2011).
Raw cycle threshold (Ct) values were collected for each
gene, and calibrated to 18S ribosomal RNA to obtain
Figure 1. Phenotypic analysis of field-grown sorghum. The
ATx623 ´ R.07007 F1 (center) flowers later, is taller, and accumulates more biomass than ATx623 (left) and R.07007 (right)
parental lines used to generate the F1. Plants were planted on 16
Apr. 2010 in College Station, TX, and photographed in August.
∆Ct values. Relative expression was calculated using the
Comparative Ct (∆∆Ct) method (Bookout and Mangelsdorf, 2003) with the most highly expressed sample used
as the calibration sample between both LD and SD samples. Mean values are based on three technical replicates
and three biological replicates for both reference and target genes, ±SEM (Bookout and Mangelsdorf, 2003).
Accession Codes
Ghd7 allele sequences have been deposited at GenBank,
Accession No. JX448383, JX448384, JX448385, JX448386,
JX448387, JX448388, JX448389, JX448390.
Results
To analyze how various combinations of Ma1 and Ma6
alleles affect flowering time, late-flowering F1 hybrids
were produced by crossing Tx623 (ma1, Ma5, ma6; ~56 d
to flowering, DTF) to R.07007 (Ma1, ma5, Ma6; 74 DTF;
Fig. 1). When planted in College Station, TX, in early
April, F1 plants initiate flowering in September, after
~175 d of growth when daylengths decrease below 12.3
h, reaching anthesis in mid-to-late October (Rooney and
Aydin, 1999). The long duration of vegetative growth
of the F1 energy sorghum hybrids results in more than
twice as much biomass accumulation compared with
grain sorghum hybrids that flower in June (Olson et al.,
2012). To examine the role Ma6 plays in determining
flowering time, F1 plants were selfed to create F2 and F3
populations (n = 182) that were grown and phenotyped
for flowering time in a greenhouse under 14 h LD conditions during the fall. Genetic analysis revealed three
major quantitative trait loci (QTL) in this population that
modified flowering time; Ma5 (SBI-01), Ma6 (SBI-06, ~0.7
Mbp), and Ma1 (SbPRR37; SBI-06 at 40.28 Mbp; Murphy
et al., 2011; Rooney and Aydin, 1999; Fig. 2A). Under LD
mu rphy et al .: ghd 7 represses flowering in energy sorghum 3
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Figure 2. Quantitative trait loci (QTL) mapping of Ma6 (blue triangle). (A) Statistically significant QTL are those with log of odds
(LOD) scores that surpass the threshold value (horizontal red line),
determined by permutation test at 1000 iterations. Chromosome
numbers are given along the abscissa. Ma1 (purple triangle) and
Ma5 (green triangle) are noted above their respective QTL peaks.
(B) Fine mapping of Ma6 . The interval was refined to 267kb
between SNP1 and SSR5 on the end of chromosome 6. Close
up of the region. IND1 lies in the promoter of the strongest candidate gene, Grain number, plant height, and heading date (Ghd7;
Supplemental Table S3). Recombinants are given in parentheses.
(C) Ghd7 allele sequences. The dominant Ghd7-1 allele from
R.07007 contains a CONSTANS, CO-like, and TOC1 (CCT)
domain. The ghd7-1 allele from ATx623 contains a five base
insertion in the first exon. This causes a frameshift and a premature stop. The ghd7-2 allele contains a large insertion in the
intron (black triangle), which affects the integrity of the second
exon. Exons are shown as boxes, introns as solid lines. Golden
yellow color is the protein coding sequence; red indicates CCT
domain; pale yellow indicates extensive missense. 100-bp scale
is given. The pink area represents the portion of the DNA that
would code for the CCT domain if not for the insertion in ghd7-2.
greenhouse conditions, F3 plants recessive at ma1 and
ma6 (ma1ma1, ma6ma6) flowered in 65 d, plants dominant
for either Ma1 or Ma6 (ma1ma1, Ma6_; Ma1_, ma6ma6)
flowered in 96 to 100 d and plants dominant for both Ma1
and Ma6 (Ma1_, Ma6_) flowered in ~120 d. These results
indicate that Ma1 (PRR37) and Ma6 act in an additive
fashion to repress flowering under LD conditions.
Differences in flowering time in this population
conferred by inheritance of various Ma1 and Ma6 allelic
combinations were examined under field conditions in
2012. Selected genotypes were planted in College Station,
TX, in early April, and harvested in mid-September.
During this period, daylengths increase from 12.5 h at
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Figure 3. Flowering time in BTx623 ´ R.07007-derived lines
with various Ma1 and Ma6 allelic combinations. In the parental
lines Tx623 and R.07007, and in other lines recessive for both
ma1 and ma6 (lines 04, 07), 7 to 10 internodes were produced.
In lines recessive for either ma1 or ma6 (lines 09 and 10), 15 to
16 internodes were produced. In F1 plants dominant for both
Ma1 and Ma6 (as well as Ma5), 40 to 43 internodes were produced. Selected genotypes were planted under field conditions
in College Station, TX, in early April 2012 and harvested in late
September. The number of stem nodes was used as an indirect
measure of floral initiation.
emergence to 14.3 h in July, and then decrease to 12.3 h
in mid-September. All genotypes were harvested in late
September, and variation in the number of stem nodes
(equivalent to leaf number) was used as an indirect
measure of differences in days to floral initiation (Major
et al., 1990). The parental lines Tx623 and R.07007, and
progeny genotypes that were recessive for both Ma1
and Ma6 , produced 7 to 10 internodes (Fig. 3). Plants
producing this number of internodes initiate flowering
in late May or early June, and reach anthesis in late June
or early July. Lines with genotypes that were recessive
for either Ma1 or Ma6 in otherwise dominant maturity
allelic backgrounds produced 15 to 16 internodes (Fig. 3).
F1 plants that were dominant for both Ma1 and Ma6 had
40 to 43 internodes at the end of September, consistent
with the additive repressing action of Ma1 and Ma6 on
flowering time in LD field environments.
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Figure 4. Relative expression of Ghd7 in the 100M genotype for a full 72-hour time course. Plants were treated under 14-h light/10-h
dark (long day, LD, solid line) or 10-h light/14-h dark (short day, SD, red dashed line) conditions. The ordinate represents expression
normalized to 18S ribosomal RNA expression and relative to a calibrator sample and is based on three biological and three technical
replicates ± SEM (Bookout and Mangelsdorf, 2003). The black bar above the plot indicates the dark period for LD-treated plants; the
gray bar indicates subjective dark during constant light free-running (LL) conditions. Red bars indicate darkness for SD-treated plants;
pink indicates subjective dark during LL conditions. Open bars denote light periods. Light-gray shading within the plot area indicates
darkness for SD treated plants only; dark-gray shading indicates darkness for both LD- and SD-treated plants.
Ma1 was recently identified as PRR37 (Murphy
et al., 2011), but the molecular nature of Ma6 was not
known before this study. The gene underlying Ma6 was
identified using a BC1F1 population (n = 1821) created by
backcrossing F1 plants derived from Tx623 ´ R.07007
to the male sterile genotype ATx623 (ma1Ma5ma6).
This mapping population segregated for flowering time
caused by alleles of Ma1 (PRR37) and Ma6. Markers for
Ma1 alleles enabled variation in flowering time due to
this locus to be accounted for. The remaining variation
in flowering time and markers spanning the Ma6 locus
were used to delimit the interval encoding Ma6 to a
region between markers SNP1 and SSR5 (Fig. 2B; SNP =
single nucleotide polymorphism, SSR = simple sequence
repeat), covering 267 kb and 14 genes (Supplemental
Table S1; Phytozome v. 8.0, http://www.phytozome.net/,
verified 25 Mar. 2014). The best candidate for Ma6 was
Sb06 g000570, an ortholog of rice Ghd7 as determined
by sequence similarity and comparative analysis of Ghd7
in grass genomes (Yang et al., 2012). The functional
Ghd7 allele in R.07007 consists of a 741 bp coding-region
translating to a 246 amino acid product, which contains
a CCT domain spanning residues 187 to 231 (Fig. 2C).
The sequence of ghd7-1 from Tx623 (ma6) contained a
five base insertion, creating a frameshift and premature
termination of the protein (Fig. 2C; Supplemental Table
S2). No obvious functional polymorphisms were detected
in the other genes in the delimited Ma6 locus.
Sorghum PRR37 mRNA abundance peaks in the
morning and evening in LDs, but only in the morning
in SDs, through coordinate regulation by light and the
circadian clock (Murphy et al., 2011). SbPRR37 and
SbGhd7 both encode repressors that inhibit flowering in
LD, suggesting that expression of these genes might be
regulated in a similar way. To examine this possibility, the
expression of SbGhd7 was analyzed by qRT-PCR in leaves
of plants entrained in LD or SD, followed by constant
light free-running conditions (LL; Murphy et al., 2011;
Itoh et al., 2010; Samach and Coupland, 2000; Millar
and Kay, 1991). In 100M (Ma1Ma5ma6) plants grown in
LD conditions, Ghd7 mRNA abundance peaks 3 h (Fig.
4; arrow) and 15 h (Fig. 4, arrowhead) after initial light
exposure in the morning, similar to SbPRR37 (Murphy et
al., 2011). By contrast, in SD, the evening peak of SbGhd7
expression is reduced relative to LD (Fig. 4, red dashed
line), indicating differential expression in LD and SD
photoperiods. Regulation of Ghd7 expression was further
examined by quantifying expression in 100M under
constant illumination (Fig. 4, 24 to 72 h LL). The peaks in
SbGhd7 mRNA abundance observed in the morning and
evening in LD plants continued for at least 2 d under LL
conditions, indicating that SbGhd7 is controlled by the
circadian clock. Furthermore, when SD-treated plants
that show only a morning peak of Ghd7 RNA abundance
are allowed to free run in LL, the evening peak of Ghd7
expression reappears (Fig. 4, 39 and 63 h). Taken together,
these data indicate that differential Ghd7 expression
in LD and SD photoperiods depends on exposure to
light and gating by the circadian clock, consistent with
prior observations on PRR37 in sorghum (Murphy et
al., 2011). To further examine the light dependence of
SbGhd7 expression, leaf tissue was collected from F1
plants (Ma1Ma5Ma6) grown in LD and then released
into constant light (Fig. 5A) or dark (DD; Fig. 5B). Ghd7
mRNA abundance varied as observed in 100M during
the 24 h LD cycle, (Fig. 5A/B; arrow and arrowhead), but
abundance was reduced during DD, indicating that light is
required for high levels of Ghd7 expression (Fig. 5B).
In rice, Ghd7 represses flowering by inhibiting
expression of Ehd1, an activator of the FT-genes Hd3a and
RFT1 (Xue et al., 2008). In sorghum grown in LD, PRR37
was found to reduce the expression of SbEhd1 and to act
mu rphy et al .: ghd 7 represses flowering in energy sorghum 5
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Figure 5. Ghd7 expression is clock- and light-dependent. Plants
were treated under14-h light/10-h dark (long day, LD, solid line)
or 10-h light/14-h dark (short day, SD, red dashed line) conditions. Relative expression of Ghd7 was analyzed at 3-h intervals
by quantitative RT-PCR. (A) ATx623 ´ R.07007 F1 plants Ghd7
expression increased in the morning (arrow) and evening (arrowhead) of LDs. (B) The expression of Ghd7 is diminished in ATx623
´ R.07007 F1 plants grown in 14-h/10-h LD and then released
into DD at time 24 h. The ordinate represents expression normalized to 18S ribosomal RNA expression and relative to a calibrator sample and is based on three biological and three technical
replicates ± SEM (Bookout and Mangelsdorf, 2003). The black
bar above the plot indicates the dark period for LD-treated plants;
the gray bar indicates subjective dark during constant light freerunning (LL) conditions. Red bars indicate darkness for SD-treated
plants; pink indicates subjective dark during LL conditions. Open
bars denote light periods. Light-gray shading within the plot area
indicates darkness for SD treated plants only; dark-gray shading
indicates darkness for both LD- and SD-treated plants.
together with Ma6 to inhibit flowering (Murphy et al., 2011;
Rooney and Aydin, 1999). To determine if SbGhd7 inhibits
expression of the floral activator SbEhd1, the expression of
this gene in leaves of F1 plants (Ma1, Ma6) and 100M (Ma1,
ma6) plants grown in LDs was quantified and compared.
In SDs, SbEhd1 mRNA abundance peaked twice during
the night in both genotypes (Fig. 6A, red line). In the F1,
expression of SbEhd1 was greatly repressed in LD relative
to SD and at times was undetectable (Fig. 6A, black line).
SbEhd1 expression was also repressed in 100M in LD
compared with SD (Murphy et al., 2011). Comparison of
SbEhd1 RNA levels in 100M and F1 plants grown in LD
showed that Ehd1 RNA abundance was >10-fold higher in
leaves of 100M (Ma1, ma6) compared with F1 (Ma1, Ma6)
plants (Supplemental Fig. S1A; 100M, green line vs. F1,
orange line). In rice, Ehd1 up-regulates the expression of
Hd3a (FT) and RFT1, members of the PEBP-gene family
that act as florigens in this species (Doi et al., 2004).
Sorghum lacks RFT1 but possesses a gene that is collinear
with Hd3a (SbCN15), as well as the FT-like gene SbCN8, a
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Figure 6. Ehd1 and SbCN8 expression is repressed in LD in Ghd7
plants. Plants were treated under14-h light/10-h dark (long day,
LD, solid line) or 10-h light/14-h dark (short day, SD, red dashed
line) conditions. Relative expression of Ehd1 and SbCN8 was
analyzed at 3-h intervals by quantitative RT-PCR. In the ATx623
´ R.07007 F1, (A) Ehd1 and (B) SbCN8 are strongly repressed
in LDs, but not in SDs. The ordinate represents expression normalized to 18S ribosomal RNA expression and relative to a calibrator sample and is based on three biological and three technical
replicates ± SEM (Bookout and Mangelsdorf, 2003). The black
bar above the plot indicates the dark period for LD-treated plants;
gray bars indicate subjective dark during constant light freerunning (LL) conditions. Red bars indicate darkness for SD-treated
plants; pink indicates subjective dark during LL conditions. Open
bars denote light periods. Light-gray shading within the plot area
indicates darkness for SD treated plants only; dark-gray shading
indicates darkness for both LD- and SD-treated plants.
predicted ortholog of ZCN8, the florigen in maize (Meng
et al., 2011; Lazakis et al., 2011; Supplemental Table S3). In
F1 plants (Ma1, Ma6), SbCN8 is repressed in LD relative to
SD (Ma1, ma6; Murphy et al., 2011; Fig. 6B) and repression
occurs to a greater extent in F1 plants compared with 100M
(Supplemental Fig. S1B). Analysis of SbCN12, SbCN15, and
CO expression in the F1 and 100M indicated that SbGhd7
in Ma1 genetic backgrounds modifies expression of these
genes to a more limited extent (Supplemental Fig. S2),
and the expression patterns of SbCN8 and SbCN12 are
similar (each has two peaks) but not identical (Fig. 6B and
Supplemental Fig. S2A; Murphy et al., 2011).
Five additional functional alleles of SbGhd7 were
identified in highly photoperiod-sensitive sorghum
genotypes (Supplemental Table S2). Sorghum accessions
encoding functional alleles of Ghd7 and PRR37 (Ma1)
flowered in >175 d in LD summer greenhouse conditions
(Table 1). Many of the genotypes that flowered earlier in
LDs contained the ghd7-1 null allele, including historically
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important progenitors of the U.S. grain sorghum-breeding
program, such as Texas Blackhull Kafir (circa 1890, Table
1). A second recessive allele, ghd7-2, was identified in
grain sorghum genotypes such as Red Kafir (circa 1876),
Double Dwarf Feterita (1906), Hegari (1908), and the
sweet sorghum genotype Rio (1972; Table 1, Supplemental
Table S2, Supplemental Fig. S3). The ghd7-2 allele is
characterized by insertion of a large repetitive DNA
at position 268 bp within Ghd7’s only intron (Fig. 2C,
Supplemental Fig. S4). The size of this insertion was too
large to characterize precisely; however, BLAST analysis
of sequences from the insertion revealed related repetitive
sequences throughout the sorghum genome. Populations
derived from BTx623 ´ Rio segregated for Ma1 but not for
Ma6, indicating that the recessive ghd7-2 allele in Rio is
null or very weak (Murray et al., 2008).
In the 1940s, sorghum maturity standards that
varied in alleles of Ma1 through Ma4 were constructed
from ‘Double Dwarf Yellow Milo’ and ‘Early White
Milo’ (Quinby, 1967). 100M and SM100, two of the
maturity standards that differ in Ma1 (SbPRR37) alleles,
were used previously to determine how PRR37 modifies
the expression of genes in the flowering time pathway
(Murphy et al., 2011). Sequence analysis revealed that
the maturity standards 100M and SM100 and their
progenitors possess ghd7-1 (Table 1). Rooney and Aydin
(1999) showed that 100M and SM100 are dominant for
Ma5. When grown in 14-h daylengths in a greenhouse in
summer conditions, the relative flowering times of the
F1 (Tx623 ´ R.07007, >175 d, Ma1ma1, Ma6ma6), 100M
(~140 d, Ma1Ma1, ma6ma6), and SM100 (~69 d, ma1ma1,
ma6ma6) were consistent with the additive repressing
action of Ma1 and Ma6 on floral induction.
In 1963, the USDA established the Sorghum
Conversion Program to convert late-flowering exotic
sorghum accessions into early flowering genotypes useful
for grain production (Dahlberg, 2000). The genotype
BTx406 was developed as a source of recessive ma1
(prr37-1) and dw2 for use in the conversion program
(Stephens et al., 1967). This genotype was crossed to tall
late-flowering exotic sorghum accessions to substitute
these alleles for Ma1 and Dw2 in exotic germplasm,
converting them to early flowering, short genotypes,
respectively (Supplemental Fig. S3). Sequence analysis
showed that BTx406 contains a null allele of Ma6 (ghd71) in addition to the null allele of Ma1 (prr37-1, Table 1).
Conversion of the late-flowering line IS12646 to early
flowering IS2646c involved substituting ghd7-1 and
prr37-1 from BTx406 for the corresponding dominant
alleles present in IS12646 (Table 1). These results indicate
that the molecular basis for the success of the Sorghum
Conversion Program based on BTx406 is that recessive
alleles for ma6 , ma1, and dw2 are all present in this
genotype at one end of LG-06, resulting in the efficient
conversion of tall (Dw2), photoperiod-sensitive exotic
genotypes that contained either Ma1ma6 , ma1Ma6,
or Ma1Ma6 into short, early flowering lines (Klein et
al., 2008).
Discussion
Ma6 , a repressor of flowering in sorghum, was identified in this study as SbGhd7. Ghd7 also functions as a
repressor of flowering in rice (Xue et al., 2008), wheat
(Triticum aestivum L.; Yan et al., 2004), barley (Hordeum
vulgare L.; Trevaskis et al., 2006), and maize (Zea mays
L.; Ducrocq et al., 2009; Hung et al., 2012). In sorghum,
Ghd7 confers photoperiod sensitivity and delays flowering in LDs, even in genotypes that are null for PRR37
(Ma1), another repressor of flowering in LDs (Murphy et
al., 2011). Therefore, as observed in rice (Koo et al., 2013),
Ghd7 can repress flowering in sorghum through a pathway independent of PRR37. In sorghum genotypes that
contain functional PRR37 alleles, Ghd7 acts in an additive fashion with PRR37 to enhance photoperiod sensitivity and delay flowering until daylengths are <12.3 h
under field conditions (Fig. 7). In rice, Ghd7 and PRR37
were also reported to repress flowering in an additive
fashion (Koo et al., 2013). In LDs, SbGhd7 and SbPRR37
both reduce expression of the floral activator SbEhd1
and SbCN8 (ortholog of ZCN8, a source of florigen in
maize) and flowering (Murphy et al., 2011; this study).
SbGhd7 further reduced the expression of the floral activator SbEhd1 in Ma1 (PRR37) genotypes consistent with
the additive repressing action of Ma1 and Ma6 on floral
induction. The molecular basis of the additive repressing
action of Ghd7 and PRR37 on Ehd1 expression in sorghum remains to be elucidated. However, studies of the
interaction of wheat ZCCTs (Ghd7-like factors), CO, and
NF-Y transcription factors that modulate flowering time
gene expression may provide insight into the biochemical
basis of repression (Li et al., 2011).
In sorghum, Ghd7 and PRR37 decrease Ehd1 expression in LDs but not SDs, consistent with their ability to
modulate flowering in response to photoperiod. Expression of both genes peaks in the morning and evening
in LDs, and evening phase expression is attenuated in
SDs coincident with floral induction. Expression of
Ghd7 and PRR37 is regulated by the circadian clock and
light, suggesting common upstream regulation of the
two genes. Regulation of Ghd7 expression by photoperiod is observed in rice (Xue et al., 2008), wheat (Yan et
al., 2004; Distelfeld and Dubcovsky, 2010), and barley
(Trevaskis et al., 2006). In rice, high Ghd7 expression
requires the activity of GIGANTEA (GI), a component
of the circadian clock (Itoh et al., 2010). The two peaks
of Ghd7 RNA observed in sorghum during LDs have not
been reported in rice. In barley, Ghd7 (VRN2) genes show
two to three peaks of expression in LDs (Trevaskis et
al., 2006). The significance of these differences is not yet
clear; however, in sorghum, variation in the amplitude
of the evening peak of Ghd7 expression in LDs vs. SDs is
highly correlated with differences in flowering time.
Sorghum genotypes that are dominant for both
Ma6 and Ma1 are highly photoperiod sensitive and
exhibit strong repression of Ehd1 and SbCN8 (FT-like
gene in maize and sorghum) in LDs resulting in very
mu rphy et al .: ghd 7 represses flowering in energy sorghum 7
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10
Figure 7. A simplified model depicting the co-regulation of
flowering by SbPRR37 (Ma1) and SbGhd7 (Ma6 ). Both light and
circadian clock contribute to the regulation of floral repressors
SbGhd7 and SbPRR37, which in turn downregulate expression of
SbCN genes in the leaves, thereby delaying the floral transition
in the shoot meristem.
late flowering under field conditions in College Station,
TX (~200 d). Knowledge of the action of these floral
repressors and the presence of alleles of Ma1 and Ma6 in
sorghum germplasm has aided the construction of early
flowering pollen and seed parent inbreds that can be
used to generate energy sorghum hybrid seed in locations
also used to produce grain sorghum hybrids (Rooney et
al., 2007). The resulting energy sorghum hybrids have
enhanced photoperiod sensitivity resulting in long duration of vegetative growth when grown in most locations
in the United States, greatly increased yield of biomass
for biofuels production (Olson et al., 2012), and enhanced
nitrogen use efficiency (Olson et al., 2013). The enhanced
biomass yield of energy sorghum hybrids and other C4
grasses with long duration of vegetative growth would
reduce the acreage needed for biomass production for
biofuels by up to 50% compared with biomass generated
by a grain sorghum crop (Olson et al., 2012; Somerville et
al., 2010; Carroll and Somerville, 2009; Byrt et al., 2011).
8
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Historically important sorghum accessions used for
development of grain sorghum were found to encode
recessive alleles of Ghd7 and, in most cases, recessive
alleles of PRR37. The genotypes from Africa with recessive ghd7 and prr37 alleles came from South Africa
(Blackhull Kafir, Red Kafir) and Egypt (Feterita, Hegari),
latitudes where daylengths may be too long during the
growing season to be compatible with grain genotypes
that have high photoperiod sensitivity. In contrast,
highly photoperiod-sensitive genotypes often originate
within 20 degrees of the equator where increased photoperiod sensitivity could be useful to delay flowering
when daylengths differ to only a small extent. Craufurd
et al. (1999) identified 16 sorghum accessions originating
within 20 degrees of the equator, with critical photoperiods <12.5h (10.2 to 12.5 h) similar to the F1 energy sorghum hybrids (Ma1, Ma6) described in this study. In contrast, the photoperiod insensitive sorghum accessions,
with one exception, were adapted to higher latitudes (20
to 45° N or S). Grain sorghum grown in the United States
is largely photoperiod insensitive and selected to flower
in ~60 to 80 d to minimize exposure to drought, insect
pressure, or cold temperature during the reproductive
phase depending on location. The finding that grain sorghum used in the United States contains recessive alleles
of Ma6 and Ma1 is consistent with the need for early
flowering at the latitudes (26 to 45° N) and daylengths
(>12.3 h) during the season where most sorghum grain
production occurs in the United States. Similarly, alleles
of Ghd7 and PRR37 have been found to be important for
growing rice at different latitudes (Xue et al., 2008). The
USDA Sorghum Conversion Program was established
in the 1960s to convert late-flowering exotic germplasm
into photoperiod insensitive early flowering sorghum
genotypes by introgressing recessive alleles of both Ma1
(prr37-1) and Ma6 (ghd7-1) from BTx406 into these accessions. The discovery that Ma6 is closely linked to Ma1 and
Dw2 on SBI-06 suggests that this linkage aided the success of the USDA Sorghum Conversion Program before
DNA marker-based selection. This program converted
~800 late-flowering accessions into short early flowering
lines useful for grain sorghum breeding, greatly increasing the genetic diversity of sorghum germplasm and
grain sorghum hybrids used in the United States and
elsewhere. Knowledge of the flowering time regulatory
pathway and alleles of Ghd7 and PRR37 is now enabling
marker-assisted selection of germplasm for production of
early flowering grain sorghum hybrids and late-flowering
energy sorghum hybrids (Rooney et al., 2007). In summary, alleles of Ghd7 have had an important impact on
sorghum, wheat, barley, maize, and rice breeding programs, demonstrating the special significance of this
gene family for cereal production by enabling optimized
flowering time for grain and energy crops.
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Supplemental Information Available
Supplemental information is included with this article,
see file Supplementary Information.
Acknowledgments
This research was supported by the Office of Science (Biological and
Environmental Research), U. S. Department of Energy, Grant No.
DE-FG02-06ER64306, the Perry Adkisson Chair (J.E. Mullet), and Ceres
Inc. The authors do not declare a conflict of interest. Author contributions: J.E. Mullet was the principal investigator who conceived the study.
R.L.Murphy, D.T. Morishige, J.A. Brady, S. Yang, and P.E. Klein carried
out QTL mapping and fine mapping studies. R.L. Murphy and D.T. Morishige conducted allele sequencing and expression analysis. P.E. Klein
performed bioinformatic analysis. W.L. Rooney created the populations
used and directed field studies. R.L. Murphy and J.E. Mullet were primarily responsible for writing the paper.
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