# Genomics of Sorghum

Authors: Andrew H. Paterson
Journal: International Journal of Plant Genomics
 (2008-02-12)

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## Abstract

Sorghum (Sorghum bicolor (L.) Moench) is a subject of plant genomics research based on its importance as one of the world's leading cereal crops, a biofuels crop of high and growing importance, a progenitor of one of the world's most noxious weeds, and a botanical model for many tropical grasses with complex genomes.  A rich history of genome analysis, culminating in the recent complete sequencing of the genome of a leading inbred, provides a foundation for invigorating progress toward relating sorghum genes to their functions.  Further characterization of the genomes other than Saccharinae cereals may shed light on mechanisms, levels, and patterns of evolution of genome size and structure, laying the foundation for further study of sugarcane and other economically important members of the group.


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## Body

## 1. Why Sorghum?

As a food and feed crop,  sorghum is
an important “failsafe” in the global agroecosystem.  Worldwide, sorghum is the 5th most important
grain crop grown based on tonnage, after maize, wheat, rice, and barley
(www.fao.org). Sorghumis unusually
tolerant of low input levels, an essential trait for areas such as Northeast Africa and the US Southern Plains that receive
too little rainfall for most other grains. Increased demand for limited fresh
water supplies, increasing use of marginal farmland, and global climatic
trends, all suggest that dryland crops such as sorghum will be of growing
importance to feed the world’s expanding populations.Currently the 2nd source
of grain-based ethanol in the US (after maize), sorghum is a biofuel crop of
growing importance. The generally lower
water demands and market price for sorghum than maize, versus their equal per-bushel
ethanol yields, suggests that sorghum will be of growing importance in meeting
grain-based biofuels needs.
Cellulosic biofuel production offers
compelling advantages over seed-based production [1], but will require greater
utilization of marginal lands to make the low per-unit value of biomass
production economical, and will be heavily dependent upon the use of perennials
to be sustainable [2, 3].  A relatively advanced state of knowledge of
the genetic control of perenniality in sorghum [4, 5] and early progress in functional genomics of
perenniality  [6] add to its promise as a cellulosic biofuels crop. “Sweet sorghums” with high sugar content in stems, already
grown for forage and silage, may be especially promising.TheSorghum genus also offers the opportunity to gain new insights into
biology of weeds and invasives. Vegetative dispersal by rhizomes (underground
stems) and seed dispersal by disarticulation of the mature inflorescence (shattering)
cause “Johnsongrass" [Sorghum
halepense (L.) Pers, 2n=2x=40] to rank among the world's most noxious
weeds [7].  Johnsongrass is an interspecific hybrid of Sorghum bicolor and S. propinquum, the latter contributing rhizomatousness. Sorghum bicolor and S. propinquum are readily crossed, and their progeny provide a
system in which to dissect the genetic basis of rhizomatousness [4]. The same features that make
Johnsongrass such a troublesome weed are actually desirable in many forage, turf, and biomass crops which are
genetically complex.  Therefore, sorghum offers novel learning opportunities relevant to weed biology
as well as to improvement of a wide range of other forage, turf, and biomass
crops.The small
genome of sorghum has long been an attractive model for advancing understanding
of the structure, function, and evolution of cereal genomes. Sorghum is representative
of tropical grasses in that it has “C4” photosynthesis, using complex
biochemical and morphological specializations to improve carbon assimilation at
high temperatures.  By contrast, rice is
more representative of temperate grasses, using “C3” photosynthesis.  Its lower level of gene duplication than many
other tropical cereals makes sorghum, like
rice, an attractive model for functional genomics.  However, sorghum is much more closely related
than rice to many major cereal crops with complex genomes and high levels of
gene duplication.Sorghum and Zea (maize, the leading US crop
with a farm-gate value of $15–20 billion/y) diverged from a common
ancestor ~12 mya [8, 9] versus ~42 mya for rice and
the maize/sorghum lineage [10]. Saccharum (sugarcane), arguably the most important biofuels crop worldwide, valued at
~$30 billion including $1 billion/y in the US*, may have shared
ancestry with sorghum as little as 5 million years ago [11], retains similar gene order [12], and even produces viable
progeny in some intergeneric crosses [13]. Zea has undergone one whole-genome duplication since its divergence from Sorghum [14], and Saccharum has undergone at least two [12].


## 2. Progress in Sorghum Genome Characterization

### 2.1. Genetic Mapping

Linkage
mapping in sorghum takes advantage of its straightforward diploid genetics,
amenability to inbreeding, high levels of DNA polymorphism betweenSorghum species, and manageable levels
of DNA polymorphism within S. bicolor.  High-density reference maps of one intraspecific S. bicolor [15–18] and one interspecific S. bicolor x S. propinquum [19, 20] cross provide about 2600
sequence-tagged-sites (based on low-copy probes that have been sequenced), 2454
AFLP, and ~1375 sequence-scanned (based on sequences of genetically anchored
BAC clones) loci. These two maps share one common parent (S. bicolor “BTx623") and are
essentially colinear [21].  Cytological
characterization of the individual sorghum chromosomes has provided a generally
adopted numbering system [22].More than 800
markers mapped in sorghum are derived from other taxa (hence serve as
comparative anchors) and additional sorghum markers have been mapped directly
in other taxa, or can be plotted based on sequence similarity.   Anchoring of
the sorghum maps to those of rice [10, 23], maize [20, 24], sugarcane [12, 25], millet [26], switchgrass [27], bermuda grass [28], and others provides for the
cross-utilization of results to simultaneously advance knowledge of many important
crops.


### 2.2. Physical Mapping

Sorghum
was the first angiosperm for which a BAC library was published [29].  Estimates of the physical size of the sorghum
genome range from 700 Mbp based on Cot analysis [30] to 772 Mbp based on flow
cytometry [31].  This makes the sorghum genome about 60%
larger than that of rice, but only about 1/4 the size of the genomes of maize
or human. DNA renaturation kinetic analysis [30] shows the sorghum genome to
be comprised of about 16% foldback DNA, 15% highly repetitive DNA (with
individual families occurring at an average of 5200 copies per genome), 41%
middle-repetitive DNA (average 72 copies) and 24% low-copy DNA.  About 4% of the DNA remained single-stranded
at very high Cot values and is assumed to have been damaged (thus the other percentages
are slight underestimates).High-coverage BAC
libraries are available for BTx623 (about 12X coverage from HindIII and 8X from BamHI), S. propinquum (13-14X coverage from EcoRI (~7X) and
HindIII (~7X) and IS3620C (~9X coverage from HindIII).  A total of 69 545
agarose-based fingerprints from BTx623 BACs are also anchored with 211,558
hybridization loci from 7292 probes (about 2000 of which are genetically
mapped).  In parallel, 40 957
agarose-based fingerprints from S.
propinquum are anchored with 189 735
hybridization loci from 7481 probes (2000 genetically mapped). Targeted HICF of
additional contig-terminal BACs has been used to fill gaps.  Each of these has been assembled into
WebFPC-accessible physical maps (http://www.stardaddy.uga.edu/fpc/WebAGCoL/bicolor/WebFPC and
http://www.stardaddy.uga.edu/fpc/WebAGCoL/propinquum/WebFPC),
for which earlier versions have been described in detail [32]. About
456 S. propinquum and 303 S. bicolor BAC contigs (41% of BACs, 80% of
single-copy loci) appear to be well-anchored to euchromatic regions, with the
percentage of the genome attributable to euchromatin likely to rise with
additional anchoring.  The finding that
41% of BACs are anchored to euchromatin while only 24% of the sorghum genomic
DNA is single- or low-copy [with an overall kinetic complexity of 1.64×108 [30]], suggests that sorghum
euchromatin includes a mixture of low-copy and repetitive DNA.


### 2.3. Genome Sequence

The shotgun
sequencing of a leading US sorghum inbred, BTx623, is now complete, with ~10.5 million reads (~8X coverage) deposited in the NCBI Trace Archive. Early
analysis confirms that the sorghum genome sequence will be a suitable substrate
for a complete and high-quality annotation. In a preliminary assembly
(that is expected to further improve with ongoing analysis), more than 97% of
sorghum protein-coding genes (ESTs) were captured in the ~250 longest
scaffolds. The vast majority of these can be linked, ordered, and
oriented using the genetic and physical map to reconstruct complete
chromosomes. Alignments of the preliminary assembly to sorghum
methyl-filtered sequence; sorghum, maize, and sugarcane transcript assemblies;
and theArabidopsis and rice
proteomes confirms the base-level accuracy of the assembly and correct local
structure of protein-coding loci.Additional resources from
reduced-representation sequencing will contribute to the identification of
expressed portions of the genome sequence. The sorghum gene space is presently
represented by approximately 204 000
expressed sequence tags, many of which have been clustered into ~22 000
unigenes representing more than 20 diverse libraries from several genotypes [33].  About 500 000
methyl-filtered (MF) reads that provide an estimated 1x coverage of the
MF-estimated gene space [34] have been assembled into contigs
(SAMIs, http://magi.plantgenomics.iastate.edu).


## 2.1. Genetic Mapping

Linkage
mapping in sorghum takes advantage of its straightforward diploid genetics,
amenability to inbreeding, high levels of DNA polymorphism betweenSorghum species, and manageable levels
of DNA polymorphism within S. bicolor.  High-density reference maps of one intraspecific S. bicolor [15–18] and one interspecific S. bicolor x S. propinquum [19, 20] cross provide about 2600
sequence-tagged-sites (based on low-copy probes that have been sequenced), 2454
AFLP, and ~1375 sequence-scanned (based on sequences of genetically anchored
BAC clones) loci. These two maps share one common parent (S. bicolor “BTx623") and are
essentially colinear [21].  Cytological
characterization of the individual sorghum chromosomes has provided a generally
adopted numbering system [22].More than 800
markers mapped in sorghum are derived from other taxa (hence serve as
comparative anchors) and additional sorghum markers have been mapped directly
in other taxa, or can be plotted based on sequence similarity.   Anchoring of
the sorghum maps to those of rice [10, 23], maize [20, 24], sugarcane [12, 25], millet [26], switchgrass [27], bermuda grass [28], and others provides for the
cross-utilization of results to simultaneously advance knowledge of many important
crops.


## 2.2. Physical Mapping

Sorghum
was the first angiosperm for which a BAC library was published [29].  Estimates of the physical size of the sorghum
genome range from 700 Mbp based on Cot analysis [30] to 772 Mbp based on flow
cytometry [31].  This makes the sorghum genome about 60%
larger than that of rice, but only about 1/4 the size of the genomes of maize
or human. DNA renaturation kinetic analysis [30] shows the sorghum genome to
be comprised of about 16% foldback DNA, 15% highly repetitive DNA (with
individual families occurring at an average of 5200 copies per genome), 41%
middle-repetitive DNA (average 72 copies) and 24% low-copy DNA.  About 4% of the DNA remained single-stranded
at very high Cot values and is assumed to have been damaged (thus the other percentages
are slight underestimates).High-coverage BAC
libraries are available for BTx623 (about 12X coverage from HindIII and 8X from BamHI), S. propinquum (13-14X coverage from EcoRI (~7X) and
HindIII (~7X) and IS3620C (~9X coverage from HindIII).  A total of 69 545
agarose-based fingerprints from BTx623 BACs are also anchored with 211,558
hybridization loci from 7292 probes (about 2000 of which are genetically
mapped).  In parallel, 40 957
agarose-based fingerprints from S.
propinquum are anchored with 189 735
hybridization loci from 7481 probes (2000 genetically mapped). Targeted HICF of
additional contig-terminal BACs has been used to fill gaps.  Each of these has been assembled into
WebFPC-accessible physical maps (http://www.stardaddy.uga.edu/fpc/WebAGCoL/bicolor/WebFPC and
http://www.stardaddy.uga.edu/fpc/WebAGCoL/propinquum/WebFPC),
for which earlier versions have been described in detail [32]. About
456 S. propinquum and 303 S. bicolor BAC contigs (41% of BACs, 80% of
single-copy loci) appear to be well-anchored to euchromatic regions, with the
percentage of the genome attributable to euchromatin likely to rise with
additional anchoring.  The finding that
41% of BACs are anchored to euchromatin while only 24% of the sorghum genomic
DNA is single- or low-copy [with an overall kinetic complexity of 1.64×108 [30]], suggests that sorghum
euchromatin includes a mixture of low-copy and repetitive DNA.


## 2.3. Genome Sequence

The shotgun
sequencing of a leading US sorghum inbred, BTx623, is now complete, with ~10.5 million reads (~8X coverage) deposited in the NCBI Trace Archive. Early
analysis confirms that the sorghum genome sequence will be a suitable substrate
for a complete and high-quality annotation. In a preliminary assembly
(that is expected to further improve with ongoing analysis), more than 97% of
sorghum protein-coding genes (ESTs) were captured in the ~250 longest
scaffolds. The vast majority of these can be linked, ordered, and
oriented using the genetic and physical map to reconstruct complete
chromosomes. Alignments of the preliminary assembly to sorghum
methyl-filtered sequence; sorghum, maize, and sugarcane transcript assemblies;
and theArabidopsis and rice
proteomes confirms the base-level accuracy of the assembly and correct local
structure of protein-coding loci.Additional resources from
reduced-representation sequencing will contribute to the identification of
expressed portions of the genome sequence. The sorghum gene space is presently
represented by approximately 204 000
expressed sequence tags, many of which have been clustered into ~22 000
unigenes representing more than 20 diverse libraries from several genotypes [33].  About 500 000
methyl-filtered (MF) reads that provide an estimated 1x coverage of the
MF-estimated gene space [34] have been assembled into contigs
(SAMIs, http://magi.plantgenomics.iastate.edu).


## 3. Postgenomics of Sorghum

With the genome sequence available,
one can anticipate renewed interest and accelerated progress in relating
sorghum genes to their functions.  Prior
efforts will benefit from the sequence as a means of integrating diverse data
types, providing for the formulation and testing of new hypotheses about roles
of specific genes in particular traits.
Existing data from QTL mapping, expression profiling, and early
association genetics studies are likely to figure prominently in this
merger.  To fully realize the fruits of
the sorghum sequence, additional functional genomics resources will be needed
that provide for identification and study of crippling mutations in specific
sorghum genes, in a manner that can be targeted to the subset of genes for
which sorghum is a preferred system over rice, maize, or other cereal
models.


### 3.1. QTL Mapping

Motivated by interest in a range of basic and applied questions, the
linkage maps of sorghum have been employed in the “tagging” (mapping) of genes
for a large number of traits.  The
interspecific population has been especially useful for characterization of
genes related to domestication, such as seed size, shattering [23], tillering, and
rhizomatousness [4]. Plant height and flowering
time [35, 36] have been a high priority.  Similarly, the importance of hybrid sorghum
motivated much research into the genetic control of fertility restoration [37–39].  Resistance genes have been tagged for
numerous diseases [40–47], key insect pests [48–51], and also the parasitic
weed, striga [40, 52].  Genes and QTLs have been identified that are
related to abiotic stresses including postreproductive stage drought tolerance
(stay-green) [53–56]; preharvest sprouting [57, 58], and aluminum tolerance [59]. Additional morphological
characteristics have also been mapped in interspecific and/or intraspecific
populations [21].


### 3.2. Expression Profiling

Progress in characterization of the transcriptome has been paralleled by
identification of differential gene expression in response to biotic and abiotic
factors, including greenbug feeding [60], dehydration,
high salinity and ABA [61], and methyl
jasmonate, salicylic acid, and aminocyclopropane carboxylic acid treatments [62].


### 3.3. Association Genetics

Much of the value of the sorghum sequence may be realized through better
understanding of the levels and patterns of diversity in extant germ plasm,
which can contribute both to functional analysis of specific sorghum genes and
to deterministic improvement of sorghum for specific needs and environments.
Sorghum is well suited to association mapping methods because of its
medium-range patterns of linkage disequilibrium [63] and its self-pollinating
mating system. Extensive ex situ sorghum
germplasm collections exist within the U.S. National Plant Germplasm
System and ICRISAT.  Early
characterization of complementary association genetics panels developed by a
group of US scientists [6], and by Subprogram 1 of the
Generation Challenge Program, is in progress. At present, more than 750 SSR
alleles and 1402 SNP alleles discovered in 3.3 Mb of sequence [63–66] are freely available from the Comparative
Grass Genomics Center relational database [67].  Extensive studies
of sequence variation in sorghum show that haplotype diversity is low, even
when nucleotide diversity is high: for regions of average length 671 bp
surveyed in 17 accessions, the median number of haplotypes was three and the
mode was two [63]. Common sequence variation can therefore be captured in a small sample
of accessions.


### 3.4. Need for Mutants and their Characterization

A collection of
~400S. bicolor mutants, now
under the curation of C. Franks (USDA-ARS, Lubbock TX), provides a start toward
testing hypotheses about the functions of individual genes, but a much broader
set is needed, ideally providing for the identification of multiple
loss-of-function mutants in each gene. Sorghum offers an opportunity to
complement more extensive reverse genetics resources in for Oryza and Zea, providing for the study of genes/gene families that are less
tractable in maize or rice (e.g., which remain duplicated in both taxa,
but are single copy in sorghum), and also for targeting functional analyses to
specific sorghum genes implicated in key traits by association genetics or
other approaches.To accelerate
identification in a targeted manner of mutants useful to relateSorghum genes to their functions, 1600 M3 annotated individually pedigreed mutagenized lines using ethyl methane
sulfonate have been generated for sorghum genotype BTx623 and their preliminary
characterization is in progress [68]. To date, every M3 row
inspected closely has been distinguishable from the original stock, and many
have multiple mutant phenotypes (Z. Xin, personal communication). More effort
in this area is desirable.Transposon
tagging warrants further exploration as a means to obtain additional mutants in
sorghum.Cs1 is the first active
transposable element isolated from sorghum, and offers several advantages as an
insertion mutagen. Cs1-homologous
sequences are present in low copy number in
sorghum and other grasses, including sudangrass, maize, rice, teosinte, and
sugarcane [69].  The low copy number and high transposition
frequency of Cs1 implies that this
transposon could prove to be an efficient gene isolation tool. Preliminary
studies of Cs1 as a mutagen (S.
Chopra, personal communication) indicate the feasibility of using this
transposon as a tagging tool.


## 3.1. QTL Mapping

Motivated by interest in a range of basic and applied questions, the
linkage maps of sorghum have been employed in the “tagging” (mapping) of genes
for a large number of traits.  The
interspecific population has been especially useful for characterization of
genes related to domestication, such as seed size, shattering [23], tillering, and
rhizomatousness [4]. Plant height and flowering
time [35, 36] have been a high priority.  Similarly, the importance of hybrid sorghum
motivated much research into the genetic control of fertility restoration [37–39].  Resistance genes have been tagged for
numerous diseases [40–47], key insect pests [48–51], and also the parasitic
weed, striga [40, 52].  Genes and QTLs have been identified that are
related to abiotic stresses including postreproductive stage drought tolerance
(stay-green) [53–56]; preharvest sprouting [57, 58], and aluminum tolerance [59]. Additional morphological
characteristics have also been mapped in interspecific and/or intraspecific
populations [21].


## 3.2. Expression Profiling

Progress in characterization of the transcriptome has been paralleled by
identification of differential gene expression in response to biotic and abiotic
factors, including greenbug feeding [60], dehydration,
high salinity and ABA [61], and methyl
jasmonate, salicylic acid, and aminocyclopropane carboxylic acid treatments [62].


## 3.3. Association Genetics

Much of the value of the sorghum sequence may be realized through better
understanding of the levels and patterns of diversity in extant germ plasm,
which can contribute both to functional analysis of specific sorghum genes and
to deterministic improvement of sorghum for specific needs and environments.
Sorghum is well suited to association mapping methods because of its
medium-range patterns of linkage disequilibrium [63] and its self-pollinating
mating system. Extensive ex situ sorghum
germplasm collections exist within the U.S. National Plant Germplasm
System and ICRISAT.  Early
characterization of complementary association genetics panels developed by a
group of US scientists [6], and by Subprogram 1 of the
Generation Challenge Program, is in progress. At present, more than 750 SSR
alleles and 1402 SNP alleles discovered in 3.3 Mb of sequence [63–66] are freely available from the Comparative
Grass Genomics Center relational database [67].  Extensive studies
of sequence variation in sorghum show that haplotype diversity is low, even
when nucleotide diversity is high: for regions of average length 671 bp
surveyed in 17 accessions, the median number of haplotypes was three and the
mode was two [63]. Common sequence variation can therefore be captured in a small sample
of accessions.


## 3.4. Need for Mutants and their Characterization

A collection of
~400S. bicolor mutants, now
under the curation of C. Franks (USDA-ARS, Lubbock TX), provides a start toward
testing hypotheses about the functions of individual genes, but a much broader
set is needed, ideally providing for the identification of multiple
loss-of-function mutants in each gene. Sorghum offers an opportunity to
complement more extensive reverse genetics resources in for Oryza and Zea, providing for the study of genes/gene families that are less
tractable in maize or rice (e.g., which remain duplicated in both taxa,
but are single copy in sorghum), and also for targeting functional analyses to
specific sorghum genes implicated in key traits by association genetics or
other approaches.To accelerate
identification in a targeted manner of mutants useful to relateSorghum genes to their functions, 1600 M3 annotated individually pedigreed mutagenized lines using ethyl methane
sulfonate have been generated for sorghum genotype BTx623 and their preliminary
characterization is in progress [68]. To date, every M3 row
inspected closely has been distinguishable from the original stock, and many
have multiple mutant phenotypes (Z. Xin, personal communication). More effort
in this area is desirable.Transposon
tagging warrants further exploration as a means to obtain additional mutants in
sorghum.Cs1 is the first active
transposable element isolated from sorghum, and offers several advantages as an
insertion mutagen. Cs1-homologous
sequences are present in low copy number in
sorghum and other grasses, including sudangrass, maize, rice, teosinte, and
sugarcane [69].  The low copy number and high transposition
frequency of Cs1 implies that this
transposon could prove to be an efficient gene isolation tool. Preliminary
studies of Cs1 as a mutagen (S.
Chopra, personal communication) indicate the feasibility of using this
transposon as a tagging tool.


## 4. Beyond Sorghum-Broader Characterization of the Saccharineae

Sorghum sprung from the loins of the
Saccharinae group of cereals, which
also includes cultivated sugarcane and weedy/invasive Johnsongrass and Microstegium.  This curious group shows a 6-fold variation in
genome size among closely related species with the same chromosome number (S, bicolor and propinquum versus nitidum)
[70]; an apparent reduction in
chromosome number from the ancestral 20 to 10 in most parasorghums [71]; at least two chromosome
doublings in Saccharum since its divergence from the remainder of
the group [12]; and both natural (Sorghum halepense: [4]) and human-mediated
polyploidization (Saccharum cultivars: [12]). Knowledge of the
mechanisms, levels, and patterns of evolution of genome size and structure in this
curious group will help to reveal the path by which
the sorghum genome has arrived at its present state, also laying the foundation
for further study of sugarcane and other economically important members of the
group.Of singular importance is the role that sorghum may play in
clarifying the fates and consequences of genes duplicated in recent
whole-genome duplications inSaccharum,
and Zea (albeit not in the Saccharinae). Zea is the less complicated of these
opportunities—a
genomewide (or largely so) duplication in the Zea lineage shortly followed the Sorghum-Zea divergence [14, 72], making Sorghum an excellent outgroup for deducing the ancestral state at duplicated loci with
regard to location, sequence, regulatory and other features.  This opportunity is less complicated in that Zea is relatively advanced in
restoration of the diploid state with regard to chromosome pairing, behaving
for practical purposes as a diploid. Saccharum offers insight into an earlier
stage following polyploid formation, behaving largely as an autopolyploid
although with varying degrees of preferential pairing in different taxa and
crosses [12, 73, 74]. Sorghum halepense, although far less
well studied than either Zea or Saccharum, appears to be even closer to
polyploid formation, in that its formation postdates the divergence of S. bicolor and S. propinquum which we roughly estimate to be 1-2 million years ago
(based on ~1.2% divergence of coding nucleotides).  While it is very possible that these three
polyploidizations differed in the degree of pairing specificity that was
possible at the outset of polyploid evolution, insight into the relative
degrees of duplicate gene loss, and/or silencing would be a valuable resource
toward clarifying recent hypotheses about adaptation of genomes to the
polyploid state [75].



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*Source: 362451-2008-04-23.xml*