Sorghum Genome Research Articles

Linkage group alignment of sorghum RFLP maps using a RIL mapping population

Several molecular maps have been constructed in sorghum (Sorghum bicolor L. Moench) using a variety of probes from different grass species such as sorghum, maize, sugarcane, rice, oat, and barley. In order to enhance the utility of the existing mapping information by the sorghum research community, alignment and integration of all major molecular maps is necessary. To achieve this objective, a genetic map of 214 loci with a total map distance of 1200 cM was constructed using 98 F7 sorghum recombinant inbred lines (RILs) from a cross between two inbred lines, B35 and Tx7000. Few cDNA clones of sorghum and maize related to photosynthesis and drought stress were mapped on this map for the first time. Five major restriction fragment length polymorphism (RFLP) maps independently developed in this species were used for alignment purpose. The distributions of previously mapped markers were compared with their respective sorghum maps to align each of the linkage groups. In general, consistent linear order among markers was maintained in all the linkage maps. The successful alignment of these RFLP maps will now allow selection of a large number of markers for any region of the sorghum genome with many potential applications ranging from fine mapping and marker-assisted selection to map-based cloning for the improvement of sorghum and related species.

Agrobacterium-mediated sorghum transformation.

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a 'Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.

Colinearity and its exceptions in orthologous adh regions of maize and sorghum.

Orthologous adh regions of the sorghum and maize genomes were sequenced and analyzed. Nine known or candidate genes, including adh1, were found in a 225-kilobase (kb) maize sequence. In a 78-kb space of sorghum, the nine homologues of the maize genes were identified in a colinear order, plus five additional genes. The major fraction of DNA in maize, occupying 166 kb (74%), is represented by 22 long terminal repeat (LTR) retrotransposons. About 6% of the sequence belongs to 33 miniature inverted-repeat transposable elements (MITEs), remnants of DNA transposons, 4 simple sequence repeats, and low-copy-number DNAs of unknown origin. In contrast, no LTR retroelements were detected in the orthologous sorghum region. The unconserved sorghum DNA is composed of 20 putative MITEs, transposon-like elements, 5 simple sequence repeats, and low-copy-number DNAs of unknown origin. No MITEs were discovered in the 166 kb of DNA occupied by the maize LTR retrotransposons. In both species, MITEs were found in the space between genes and inside introns, indicating specific insertion and/or retention for these elements. Two adjacent sorghum genes, including one gene missing in maize, had colinear homologues on Arabidopsis chromosome IV, suggesting two rearrangements in the sorghum and three in the maize genome in comparison to a four-gene region of Arabidopsis. Hence, multiple small rearrangements may be present even in largely colinear genomic regions. These studies revealed a much higher degree of diversity at a microstructural level than predicted by genetic mapping studies for closely related grass species, as well as for comparisons of monocots and dicots.

A Plastid Sigma Factor Sequence from the C4 Monocot Sorghum bicolor

Abstract: All plastomes of land plants and alga analyzed to date encode homologues of the core subunits of an eubacteria‐like RNA polymerase. However, the plastomes lack genes for sigma factors which are imperative for proper promoter recognition by this type of RNA polymerase and hence must be encoded by nuclear genes. By searching plant “expressed sequence tags” (EST5) for similarities to eubacterial sigma factors an Arabidopsis EST (155H23T7) was identified which was used for the isolation of a homologous full‐length cDNA from the C4 monocot, Sorghum bicolor. The cDNA sequence encodes a protein exhibiting the typical sequence motives of cs70‐type sigma factors and the corresponding gene was therefore designated Sigi. The SIG1 protein shows the greatest similarity to recently identified plastidial sigma factors of A. thaliana, O. sativa and S. alba. Like these proteins the Sorghum SIG1 protein contains an aminoterminal extension reminiscent of plastidial transit sequences indicating that the SIG1 protein functions as a plastidial sigma factor. Accordingly, Sigi transcripts are found preferentially in leaves and accumulate in a light‐dependent manner. Sigi transcript levels are similar in the mesophyll and bundle‐sheath cells suggesting that at least this sigma factor is not involved in the differential expression of plastid genes in the two cell types. Southern analyses indicate that the Sigl sequence is present as single copy gene in the Sorghum genome.

A Sorghum propinquum BAC library, suitable for cloning genes associated with loss-of-function mutations during crop domestication

Al arge insertSorghum propinquumBAC library has been constructed to analyze the physical organization of the sorghum genome and to facilitate positional cloning of sorghum genes and QTLs associated with the early stages of grain crop domestication. This library was established from 12 different ligations using high-molecular-weight DNA generated from either one cycle or two cycles of size selection. This library consists of 38 016 BAC clones with an estimated average insert size of 126 kb and coverage of 6.6 genome equivalents. The 6.6 genome-equivalent BAC library of S. propinquum provides a 99.7% probability of finding one or more BACs that contain genes of interest. Twenty mapped DNA probes, ten homologous and ten heterologous, were used to screen the library, and 121 positive clones were identified, 6.05 per locus or 6.37 per probe. Abbreviations:BAC, bacterial artificial chromosome; QTL, quantitative trait locus

Evidence that a Recent Increase in Maize Genome Size was Caused by the Massive Amplification of Intergene Retrotransposons

Estimates of the size and copy number of the retrotransposons in a 240 kb region flanking the adh1gene of maize suggest that 33–62% of the maize genome is composed of the high copy-number retrotransposons found in this region. An additional 16% of the maize genome is estimated to be composed of middle and low copy-number retrotransposons. The sorghum genome, which is more than three-fold smaller than the maize genome, does not have any detected copies of the maize retrotransposons in a region orthologous to that of maize adh1. Thus, it appears that retrotransposons have increased the size of the maize genome two- to five-fold since the divergence of maize and sorghum from a common ancestor about 16 million years ago.

Cloning and characterization of a centromere-specific repetitive DNA element from Sorghum bicolor

A 823-bp Sau3AI fragment (pSau3A10) was subcloned from a sorghum bacterial artificial chromosome (BAC) clone, 13I16, that contains DNA sequences specific to the centromeres of grass species. Sequence analysis showed that pSau3A10 consists of six copies of an approximately 137-bp monomer. The six monomers were organized into three dimers. The monomers within the dimers shared 62–72% homology and the dimers were 79–82% homologous with each other. Fluorescence in situ hybridization (FISH) analysis indicated that the Sau3A10 family is present only in the centromeres of sorghum chromosomes. Sequencing, Southern hybridization, and Fiber-FISH analyses indicated that the Sau3A10 family is tandemly arranged and is present in uninterrupted stretches of up to at least 81 kb of DNA. Slot-blot analysis estimated that the Sau3A10 family constitutes 1.6–1.9% of the sorghum genome. The long stretches of Sau3A10 sequences were interrupted by other centromeric DNA elements. Southern analysis indicated that the Sau3A10 sequence is one of the most abundant DNA families located in sorghum centromeres and is conserved only in closely related sorghum species. Methylation experiments indicated that the cytosine of the CG sites in sorghum centromeric regions is generally methylated. The structure and organization of the Sau3A10 family shared similarities with centromeric DNA repeats in other eukaryotic species. It is suggested that the Sau3A10 family is probably an important part of sorghum centromeres.

Grass genomes.

For the most part, studies of grass genome structure have been limited to the generation of whole-genome genetic maps or the fine structure and sequence analysis of single genes or gene clusters. We have investigated large contiguous segments of the genomes of maize, sorghum, and rice, primarily focusing on intergenic spaces. Our data indicate that much (>50%) of the maize genome is composed of interspersed repetitive DNAs, primarily nested retrotransposons that insert between genes. These retroelements are less abundant in smaller genome plants, including rice and sorghum. Although 5- to 200-kb blocks of methylated, presumably heterochromatic, retrotransposons flank most maize genes, rice and sorghum genes are often adjacent. Similar genes are commonly found in the same relative chromosomal locations and orientations in each of these three species, although there are numerous exceptions to this collinearity (i.e., rearrangements) that can be detected at the levels of both the recombinational map and cloned DNA. Evolutionarily conserved sequences are largely confined to genes and their regulatory elements. Our results indicate that a knowledge of grass genome structure will be a useful tool for gene discovery and isolation, but the general rules and biological significance of grass genome organization remain to be determined. Moreover, the nature and frequency of exceptions to the general patterns of grass genome structure and collinearity are still largely unknown and will require extensive further investigation.

Sequence Organization and Conservation in sh2/a1-Homologous Regions of Sorghum and Rice

Previously, we have demonstrated microcolinearity of gene composition and orientation in sh2/a1-homologous regions of the rice, sorghum, and maize genomes. However, the sh2 and a1 homologues are only about 20 kb apart in both rice and sorghum, while they are separated by about 140 kb in maize. In order to further define sequence organization and conservation in sh2/a1-homologous regions, we have completely sequenced a 42,446-bp segment of sorghum DNA. Four genes were identified: a homologue of sh2, two homologues of a1, and a putative transcriptional regulatory gene. A solo long terminal repeat of the retroelement Leriathan was detected between the two a1 homologues, and eight miniature inverted repeat transposable elements were found in this region. Comparison of the sorghum sequence with the sequence of the homologous segment from rice indicated that only the identified genes were evolutionarily conserved between these two species, which have evolved independently for over 50 million years. The introns of the a1 homologues have evolved faster than the introns of the sh2 homologue. The a1 tandem duplication appears to be an ancient event that may have preceded the ancestral divergence of maize, sorghum, and rice.

Comparative mapping of Andropogoneae: Saccharum L. (sugarcane) and its relation to sorghum and maize.

Comparative genetic maps of Papuan Saccharum officinarum L. (2n = 80) and S. robustum (2n = 80) were constructed by using single-dose DNA markers (SDMs). SDM-framework maps of S. officinarum and S. robustum were compared with genetic maps of sorghum and maize by way of anchor restriction fragment length polymorphism probes. The resulting comparisons showed striking colinearity between the sorghum and Saccharum genomes. There were no differences in marker order between S. officinarum and sorghum. Furthermore, there were no alterations in SDM order between S. officinarum and S. robustum. The S. officinarum and S. robustum maps also were compared with the map of the polysomic octoploid S. spontaneum 'SES 208' (2n = 64, x = 8), thus permitting relations to homology groups ("chromosomes") of S. spontaneum to be studied. Investigation of transmission genetics in S. officinarum and S. robustum confirmed preliminary results that showed incomplete polysomy in these species. Because of incomplete polysomy, multiple-dose markers could not be mapped for lack of a genetic model for their segregation. To coalesce S. officinarum and S. robustum linkage groups into homology groups (composed of homologous pairing partners), they were compared with sorghum (2n = 20), which functioned as a synthetic diploid. Groupings suggested by comparative mapping were found to be highly concordant with groupings based on highly polymorphic restriction fragment length polymorphism probes detecting multiple SDMs. The resulting comparative maps serve as bridges to allow information from one Andropogoneae to be used by another, for breeding, ecology, evolution, and molecular biology.

Microcolinearity in sh2-homologous regions of the maize, rice, and sorghum genomes.

Large regions of genomic colinearity have been demonstrated among grass species by recombinational mapping, but the degree of chromosomal conservation at the sub-centimorgan level has not been extensively investigated. We cloned the rice and sorghum genes homologous to the sh2 locus of maize on bacterial artificial chromosomes (BACs), and observed that a homologue of the maize a1 gene was also present on each of these BACs. In sorghum, we found a direct duplication of a1 homologues separated by about 10 kb. In maize, sh2 and a1 are approximately 140 kb apart and transcribed in the same direction, with sh2 upstream of a1. In rice and sorghum, this arrangement is fully conserved. However, the sh2 and a1 homologues are separated by about 19 kb in both rice and sorghum. We found low-copy-number and repetitive DNAs between the sh2 and a1 homologues of sorghum and rice. The sh2 and a1 homologues cross-hybridized, but the repetitive DNA and most low-copy-number sequences between these genes did not. These results indicate that maize, sorghum, and rice have conserved gene order and composition in the sh2-a1 region, but have acquired extensive qualitative and quantitative differences in the sequences between these genes.

Construction of a composite sorghum genome map and comparison with sugarcane, a related complex polyploid

A sorghum composite linkage map was constructed with two recombinant inbred line populations using heterologous probes already mapped on maize and sugarcane. This map includes 199 loci revealed by 188 probes and distributed on 13 linkage groups. A comparison based on 84 common probes was performed between the sorghum composite map and a map of a sugarcane (Saccharum spp.) cultivar being developed and presently comprising 10 tentative linkage groups. A straight synteny was observed for 2 pairs of linkage groups; in two cases, 1 sorghum linkage group corresponded to 2 or 3 sugarcane linkage groups, respectively; in two cases 1 sugarcane link- age group corresponded to 2 separate sorghum linkage groups; for 2 sorghum linkage groups, no complete correspondance was found in the sugarcane genome. In most cases loci appeared to be colinear between homoeologous chromosomal segments in sorghum and sugarcane. These results are discussed in relation to published data on sorghum genomic maps, with specific reference to the genetic organization of sugarcane cultivars, and they, illustrate how investigations on relatively simple diploid genomes as sorghum will facilitate the mapping of related polyploid species such as sugarcane.

A 120 kilobase resolution contig map of the rice genome.

92% of the rice genome (4.3 x 10(8) bp, 2n = 24) was covered by 631 contigs of various length, which were generated by fingerprinting from a representative and genetically stable bacterial artificial chromosome (BAC) library of the Guang Lu Ai 4 (a O. Sativa variety) genome with the average insert of 120 kb in length. To form the contig map, 565 molecular markers of RFLP, STS, cDNA and anchor set derived from two O. Sativa varieties were by colony hybridization mapped to the contigs, which were then assigned to and ordered along the particular chromosomes according to the marker colinearity. Being highly conserved DNA sequences shared among the genomes of rice, barley, wheat, oat, maize, sorghum and sugar cane, 89 anchor markers mapped help to identify the rice genes through the information provided by the maps of relative genomes, and vice versa. Numerous repeated DNA sequences of various length were identified and mapped to the chromosomes. Physical distances have been determined for hundreds pairs of adjacent markers, which would facilitate the identification by map-based cloning the rice genes of interest. The accuracy of clone overlaps in contigs was further confirmed by the existence in contigs of well fit stacks of marker-lodged clones independently identified by hybridization. Large scale DNA sequencing of individual chromosomes could now be initiated simply by selecting and sequencing the minimally overlapped BAC clones of the contigs.

Comparative genetic mapping between duplicated segments on maize chromosomes 3 and 8 and homoeologous regions in sorghum and sugarcane

Comparative mapping within maize, sorghum and sugarcane has previously revealed the existence of syntenic regions between the crops. In the present study, mapping on the sorghum genome of a set of probes previously located on the maize and sugarcane maps allow a detailed analysis of the relationship between maize chromosomes 3 and 8 and sorghum and sugarcane homoeologous regions. Of 49 loci revealed by 46 (4 sugarcane and 42 maize) polymorphic probes in sorghum, 42 were linked and were assigned to linkage groups G (28), E (10) and I (4). On the basis of common probes, a complete co-linearity is observed between sorghum linkage group G and the two sugarcane linkage groups II and III. The comparison between the consensus sorghum/sugarcane map (G/II/III) and the maps of maize chromosomes 3 and 8 reveals a series of linkage blocks within which gene orders are conserved. These blocks are interspersed with non-homoeologous regions corresponding to the central part of the two maize chromosomes and have been reshuffled, resulting in several inversions in maize compared to sorghum and sugarcane. The results emphasize the fact that duplication will considerably complicate precise comparative mapping at the whole genome scale between maize and other Poaceae.

Comparative analysis of QTLs affecting plant height and maturity across the Poaceae, in reference to an interspecific sorghum population.

Correspondence among QTLs affecting height and/or flowering was investigated across the five races of sorghum, an interspecific sorghum F2 population, and 32 previously published sorghum, maize, rice, wheat, and barley populations revealing 185 QTLs or discrete mutants. Among nine QTLs mapped in the interspecific sorghum population (six affecting height and three affecting flowering), at least seven (78%) are associated with "conversion," backcross-introgression of alleles imparting reduced height or earlier flowering from cultivated sorghums into one or more exotic Sorghum bicolor races. One chromosomal region was "converted" in all S. bicolar races--in the interspecific F2, this region explained 54.8% of height variation (putatively the Dw2 gene) and 85.7% of flowering time variation (putatively Ma1). Comparative data suggest that Ma1 and Dw2 orthologs influence height and flowering of other Poaceae taxa and support classical dogma that the sorghum phenotypes attributed to Ma1 and Dw2 (respectively) are due to different genetic loci. Other sorghum QTLs also showed correspondence with those in other Poaceae, more frequently than would be expected by chance. Possible homoeologous QTLs were found within both the maize and sorghum genomes. Comparative QTL mapping provides a means to unify, and thereby simplify, molecular analysis of complex phenotypes.

Comparative genome mapping of sugar cane with other species within the Andropogoneae tribe

Comparative mapping within the tribe Andropogoneae has recently progressed with the development of mapped maize genomic probes that can be used for sorghum and sugar cane genomes. In the present study, data from previous reports were used to locate various linkage groups of sugar cane and sorghum on the genomic map of maize. Syntenic genome regions in the three plants were determined according to existing bridge-loci. The distribution of these synteny clusters closely matched the duplication pattern in maize. In several cases, the two arms of a single maize chromosome corresponded to at least two synteny clusters. There seem to be common chromosome rearrangements between maize and sugar cane and between maize and sorghum. In this respect, sugar cane and sorghum appear to be more closely related than either one with maize. A more detailed analysis of two synteny clusters was undertaken using recent sugar cane data to compare gene orders and recombination rates of the three plants. The three genomes showed colinearity in these regions. Distances between genes were similar in maize and sorghum, whereas sugar cane tended to display less recombination, at least in the varietal progeny investigated.

Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor.

The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.

Grasses as a single genetic system: genome composition, collinearity and compatibility

The best-known grasses are the cereals, of which three speciesrice, wheat and maizeaccount for about half of total world food production. These and other domesticated grasses (including barley, millet, oats, rye, sorghum and sugar-cane) are among the 10-11 thousand species that comprise the grass (Gramineae) family of monocotyledonous flowering plantsL Because of their unrivaled economic importance and relative ease of genetic manipulation, the cereals and forage grasses have been major targets for basic and applied plant science. Each grass species has unique agronomic properties, particularly with respect to environmental preferences and tolerances, and this variation has led to an array of independent research programs for the study and improvement of each grass. Although many individual species have been investigated intensively, the information and materials acquired have not often been used in the full range of grass species. Recent results from genome mapping and intergeneric sexual hybridization experiments suggest that we can now overcome the barriers that have traditionally isolated these systems. These studies show that the various grasses can best be viewed as different manifestations of one tractable genome, and that research priorities should be set to reflect this rich biological potential. Phylogenies based on morphology, molecular sequences and fossils argue that there is a relatively close evolutionary relationship among grass species. The first grass fossils appear in paleocene-eocene deposits some 50-60 million years old z, at a time when modem orders of mammals are known to have existed, and crops were domesticated from their wild progenitors only a few thousand years ago. Grasses are morphologically welldifferentiated from other families and have a single (monophyletic) origin3. Given the close relatedness of the grasses, it is not surprising that many cross-species sexual hybrids have been documented. Moreover, the movement of chromosome segments from one grass species to another has been a powerful tool in both basic and applied plant research. Very wide-ranging crosses between grasses have recently been made, producing transient allodiploidy or chromosomal addition line#. These interspecies and intergeneric hybrids can now be used in traditional mendelian analyses of segregants to identify the genes, mechanisms and designs that underlie the morphological and physiological differences that have evolved in the Gramineae5. Until recently the quality of genetic maps for grasses, including the cereal crops, ranged from fair to non-existent. This general deficiency was rapidly overcome with the advent of maps based on DNA markers. Now, maps have been constructed for all the major cereals, with restriction fragment length polymorphisms (RFLP) and/or randomly amplified polymorphic DNA (RAPD) probes providing anywhere from hundreds to thousands of markers. The first extensive use of DNA markers for the grasses was in studies of maize, and these programs generated large libraries of mapped probes 6-8. The fwst detailed genetic map of sorghum was generated using probes from maize9: when maize probes were hybridized to sorghum DNA, single-o,!?y sequences hybridized well (>95°/6 detected similarly low copy number bands in sorghum under standard high-stringency conditions), while most repetitive sequences hybridized poorly, or not at all. This suggests that the discrepancy in size between the maize and sorghum genomes l°.l~ (the maize genome is 3.5 times larger than that of sorghum) is due, not to differences in the number or types of genes, but rather to differences in the amount of repetitive DNA. Most interestingly,

RFLP linkage map and genome analysis of Saccharum spontaneum

An RFLP linkage map of the wild sugarcane species Saccharum spontaneum L. (2n = 8x = 40-128) was constructed, comprising 216 loci, detected by 116 DNA probes, and distributed over 44 linkage groups. At a density of at least one marker every 25-cM interval, the coverage of the genome was estimated as 86%. For the generation of RFLP markers, probes were surveyed from seven DNA libraries: three sugarcane cDNA, one oat cDNA, one rice cDNA, and one barley cDNA, as well as one sugarcane genomic. Sixty-two maize genomic clones that were previously mapped on maize were used to initiate a comparative map between the sugarcane, sorghum, and maize genomes. Based on the RFLP segregation data, we conclude that this species is an autopolyploid, with an estimated genome size of 2107 cM.

Structure and evolution of the genomes ofsorghum bicolor andZea mays

Cloned maize genes and random maize genomic fragments were used to construct a genetic map of sorghum and to compare the structure of the maize and sorghum genomes. Most (266/280) of the maize DNA fragments hybridized to sorghum DNA and 145 of them detected polymorphisms. The segregation of 111 markers was analyzed in 55 F2 progeny. A genetic map was generated with 96 loci arranged in 15 linkage groups spanning 709 map units. Comparative genetic mapping of sorghum and maize is complicated by the fact that many loci are duplicated, often making the identification of orthologous sequences ambiguous. Relative map positions of probes which detect only a single locus in both species indicated that multiple rearrangements have occurred since their divergence, but that many chromosomal segments have conserved synteny. Some sorghum linkage groups were found to be composed of sequences that detect loci on two different maize chromosomes. The two maize chromosomes to which these loci mapped were generally those which commonly share duplicated sequences. Evolutionary models and implications are discussed.