Genetic variability of foxtail millet (Setaria italica P. Beauv.)

Summary The genetic diversity of a world collection of foxtail millet strains (Setaria italica) and some samples of wild populations (Setaria viridis) was studied by means of electrophoresis on five enzymes (10 loci) Est, Acph, Got, Mdh, Pgd. In spite of an overall limited polymorphism, the diversity appeared to be clearly regionalized. The wild populations collected in France and China introduced new genetic variability to the cultivated forms. However, the interregional diversity within both species was greater than the between species (S. viridis/S. italica) diversity.     

Outcrossing and hybridization in wild and cultivated foxtail millets: consequences for the release of transgenic crops

Summary Outcrossing rates within the wild green foxtail, Setaria viridis, and the cultivated foxtail millet, S. italica, are very low. However, spontaneous interspecific hybridizations in the experimental garden occurred in both directions at rates ranging from 0.002% to 0.6% according to plant density and distance between parents. Offtypes found in farmers' fields where foxtail millet is cultivated were shown to have originated from such interspecific crosses. Differences in the EcoR1 patterns of chloroplast DNA between cultivated and wild plants indicated that reciprocal crosses do occur in the field. These findings indicate that even a largely selfing cultivated species may exchange genetic information with wild relatives at rates that may cause problems if transgenic cultivars are released.     

Mapping of Quantitative Trait Locus (QTLs) that Contribute to Germination and Early Seedling Drought Tolerance in the Interspecific Cross Setaria italica×Setaria viridis

Drought tolerance is an important breeding target for enhancing the yields of grain crop species in arid and semi-arid regions of the world. Two species of Setaria, domesticated foxtail millet (S. italica) and its wild ancestor green foxtail (S. viridis) are becoming widely adopted as models for functional genomics studies in the Panicoid grasses. In this study, the genomic regions controlling germination and early seedling drought tolerance in Setaria were identified using 190 F₇ lines derived from a cross between Yugu1, a S. italica cultivar developed in China, and a wild S. viridis genotype collected from Uzbekistan. Quantitative trait loci were identified which contribute to a number of traits including promptness index, radical root length, coleoptile length and lateral root number at germinating stage and seedling survival rate was characterized by the ability of desiccated seedlings to revive after rehydration. A genetic map with 128 SSR markers which spans 1293.9 cM with an average of 14 markers per linkage group of the 9 linkage groups was constructed. A total of eighteen QTLs were detected which included nine that explained over 10% of the phenotypic variance for a given trait. Both the wild green foxtail genotype and the foxtail millet cultivar contributed the favorite alleles for traits detected in this trial, indicating that wild Setaria viridis populations may serve as a reservoir for novel stress tolerance alleles which could be employed in foxtail millet breeding.     

Discriminative flavonoid patterns within a preliminary collection of the Setaria italica specific complex

One hundred and eighty individuals of Setaria italica ssp. italica, reflecting the worldwide variability of cultivated foxtail millet, and 40 individuals of wild S. i. ssp. viridis, were analysed for their flavonoid content. S. i. spp. italica accumulates native or acylated O-glycosyl derivatives of 8-Cglycosylflavones and O-glycosylfiavones, showing a large qualitative as well as quantitative inter-varietal diversity. The subspecies is strongly organized into seven chemotypes clearly defined by the presence or absence of O-pentosylation and acylation, and by the balance between apigenin and luteolin derivatives. This organization only partly corroborates a previous classification based on morphological and electrophoretical data, chemotypes affording an additional discriminative criterion in most cases. Flavonoid patterns of S. i. ssp. viridis fall in the chemotypes defined from the cereal, and underlines the conspecific nature of the two taxa. Similarities between wild and cultivated foxtail millet from same geographical origin suggest independent domestication from already differentiated gene pools.     

A Ser–Gly substitution in plastid-encoded photosystem II D1 protein is responsible for atrazine resistance in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

A foxtail millet (Setaria italica L. Beauv.) line resistant to atrazine was obtained through interspecific hybridization between wild S. viridis L. Beauv. and cultivated S. italica. The resistance was proved to be controlled by a chloroplast-inherited gene and it has further been utilized in foxtail millet production. However, the sequence information of the putative atrazine resistance gene, psbA in foxtail millet’s chloroplast genome encoding photosystem II D1 protein (32 kDa thylakoid membrane protein) (photosystem Q_B protein) and the mutation site responsible for the resistance are not known. In this paper the psbA sequences of six atrazine susceptible/resistant foxtail millet varieties were obtained and compared. The results indicated that there was only one amino acid difference between susceptible and resistance gene, resulting from a single base substitution. It was concluded that a mutant allele of photosystem II protein D1 encoding a Gly residue instead of a Ser residue at position 264 is a major gene of resistance to atrazine. Moreover, the phylogenetic tree based on the psbA coding region of thirty-five plant species was carried out. The phylogenetic relationship between S. italica and other plants and the related evolutionary issues were discussed and it was suggested that psbA sequences could be used in phylogenetic studies in plants. Xiaoping Jia and Jincheng Yuan have equal contribution.     

Infraspecific variation and systematics of cultivatedSetaria italica,foxtail millet (Poaceae)

Foxtail millet (Setaria italica,) is grown as a cereal in southern Europe and in temperate, subtropical, and tropical Asia. Its closest wild relative isS. italica ssp.viridis (green foxtail). Green foxtail is native to temperate Eurasia, but was introduced and became widely established as a weed in temperate and warmer parts of the Americas. Spontaneous and cultivatedS. italica cross to produce fertile hybrids. Derivatives of such crosses, resembling foxtail millet in some inflorescence traits but with efficient natural seed dispersal, accompany the cereal across its range of cultivation. Giant green foxtail of Europe and the American corn belt is a weed of hybrid origin. Foxtail millet was domesticated in the highlands of central China; remains of cultivated foxtail millet are known from the Yang-shao culture period dating back some 5,000 yrs. Comparative morphology suggests that foxtail millet spread to Europe and India as a cereal soon after its domestication. Three cultivated races are recognized. Moharia, from Europe and southwestern Asia, includes cultivars with 5–52 culms, each bearing several, small, more or less erect inflorescences. Cultivars in race maxima are characterized by plants with 1–8 usually unbranched culms that bear large inflorescences; they occur in Transcaucasian Russia and the Far East. Race indica is intermediate in culm number (ave. 6.6) and inflorescence size between races moharia and maxima, and is cultivated in southern Asia.     

Isolation of gametes and zygotes from Setaria viridis

Setaria viridis, the wild ancestor of foxtail millet (Setaria italica), is an effective model plant for larger C₄ crops because S. viridis has several desirable traits, such as short generation time, prolific seed production and a small genome size. These advantages are well suited for investigating molecular mechanisms in angiosperms, especially C₄ crop species. Here, we report a procedure for isolating gametes and zygotes from S. viridis flowers. To isolate egg cells, ovaries were harvested from unpollinated mature flowers and cut transversely, which allowed direct access to the embryo sac. Thereafter, an egg cell was released from the cut end of the basal portion of the dissected ovary. To isolate sperm cells, pollen grains released from anthers were immersed in a mannitol solution, resulting in pollen-grain bursting, which released sperm cells. Additionally, S. viridis zygotes were successfully isolated from freshly pollinated flowers. Isolated zygotes cultured in a liquid medium developed into globular-like embryos and cell masses. Thus, isolated S. viridis gametes, zygotes and embryos are attainable for detailed observations and investigations of fertilization and developmental events in angiosperms.

     

Development and genetic mapping of SSR markers in foxtail millet [Setaria italica (L.) P. Beauv.]

SSR markers are desirable markers in analysis of genetic diversity, quantitative trait loci mapping and gene locating. In this study, SSR markers were developed from two genomic libraries enriched for (GA)n and (CA)n of foxtail millet [Setaria italica (L.) P. Beauv.], a crop of historical importance in China. A total of 100 SSR markers among the 193 primer pairs detected polymorphism between two mapping parents of an F₂ population, i.e. “B100” of cultivated S. italica and “A10” of wild S. viridis. Excluding 14 markers with unclear amplifications, and five markers unlinked with any linkage group, a foxtail millet SSR linkage map was constructed by integrating 81 new developed SSR markers with 20 RFLP anchored markers. The 81 SSRs covered nine chromosomes of foxtail millet. The length of the map was 1,654 cM, with an average interval distance between markers of 16.4 cM. The 81 SSR markers were not evenly distributed throughout the nine chromosomes, with Ch.8 harbouring the least (3 markers) and Ch.9 harbouring the most (18 markers). To verify the usefulness of the SSR markers developed, 37 SSR markers were randomly chosen to analyze genetic diversity of 40 foxtail millet accessions. Totally 228 alleles were detected, with an average 6.16 alleles per locus. Polymorphism information content (PIC) value for each locus ranged from 0.413 to 0.847, with an average of 0.697. A positive correlation between PIC and number of alleles and between PIC and number of repeat unit were found [0.802 and 0.429, respectively (P < 0.01)]. UPGMA analysis revealed that the 40 foxtail millet cultivars could be grouped into five clusters in which the landraces’ grouping was largely consistent with ecotypes while the breeding varieties from different provinces in China tended to be grouped together. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The occurrence and identification of <Emphasis Type="Italic">Setaria italica</Emphasis> (L.) P. Beauv. (foxtail millet) grains from the Chengtoushan site (ca. 5800 cal B.P.) in central China,with reference to the domestication centre in Asia

Archaeobotanical remains of Setaria grains and chaff were found at the Chengtoushan site in south-central China (ca. 5800 cal b.p.). Grain shape was determined, using length to breadth ratios, and morphological variation in the upper lemma of modern domesticated and wild Setaria species were examined using scanning electron microscopy as a basis for identifying archaeobotanical remains. Grains of S. viridis, S. yunnanensis, and S.×pycnocoma are slender, whereas S. italica, S. italica var. germinica, S. lutescenes, S. faberi, S. glauca, S. pallidefusca and S. intermedia are round in shape. The papillae distributed on the upper lemma of S. italica are small (8–15 μm) with a non-ridged base, while other Setaria species have large papillae (15–20 μm) with a widely ridged base. The remains of the Setaria from the Neolithic layers at Chengtoushan included S. italica, based on these identification characters. These new finds of foxtail millet are the earliest discoveries from the Yangtze River basin of southern China and are also the earliest evidence for co-cultivation of foxtail millet with rice. The implications of these findings for understanding foxtail millet domestication centres are discussed.     

Population genetics of Setaria viridis,a new model system

An extensive survey of the standing genetic variation in natural populations is among the priority steps in developing a species into a model system. In recent years, green foxtail (Setaria viridis), along with its domesticated form foxtail millet (S. italica), has rapidly become a promising new model system for C₄ grasses and bioenergy crops, due to its rapid life cycle, large amount of seed production and small diploid genome, among other characters. However, remarkably little is known about the genetic diversity in natural populations of this species. In this study, we survey the genetic diversity of a worldwide sample of more than 200 S. viridis accessions, using the genotyping‐by‐sequencing technique. Two distinct genetic groups in S. viridis and a third group resembling S. italica were identified, with considerable admixture among the three groups. We find the genetic variation of North American S. viridis correlates with both geography and climate and is representative of the total genetic diversity in this species. This pattern may reflect several introduction/dispersal events of S. viridis into North America. We also modelled demographic history and show signal of recent population decline in one subgroup. Finally, we show linkage disequilibrium decay is rapid (<45 kb) in our total sample and slow in genetic subgroups. These results together provide an in‐depth understanding of the pattern of genetic diversity of this new model species on a broad geographic scale. They also provide key guidelines for on‐going and future work including germplasm preservation, local adaptation, crossing designs and genomewide association studies.     

WEEDY ADAPTATION IN SETARIA SPP. I. ISOZYME ANALYSIS OF GENETIC DIVERSITY AND POPULATION GENETIC STRUCTURE IN SETARIA VIRIDIS

Setaria viridis is an important self-pollinating, cosmopolitan weed of temperate regions worldwide. Allozyme markers were used to investigate genetic diversity and structure in 168 accessions (including four S. italica) collected mainly from North America and Eurasia. Genetic diversity in green foxtail, and its population genetic structure, provided important clues about this weed's evolutionary history. Genetic diversity was low, with marked population differentiation: the percentage of polymorphic loci was 25% (0.95 criterion); mean number of alleles per locus was 1.86; mean panmictic heterozygosity was 0.07; and the coefficient of population genetic differentiation was 0.65. A common genotype occurred in 25 accessions distributed in six countries from both the Old World and New World, in a wide variety of ecological situations. Relatively little genetic divergence occurred between Eurasia and North America, with Nei's unbiased genetic identity between the two regions equaling 1.0. Populations from these two continents also had equivalent genetic diversity. Within North America, regional differentiation was indicated by northern and southern groups separated at 43.5° N latitude. No geographic pattern in genetic diversity was found within Eurasia. The size of the geographic range from which populations were sampled was not an accurate indicator of the extent of genetic diversity found among populations from that region. These results suggest that present patterning among green foxtail populations in North America is the consequence of multiple introductions into the New World followed by local adaptation and regional differentiation. Finally, S. italica and several green foxtail varieties did not differ isozymatically from typical forms of green foxtail. This supports the view that S. italica and S. viridis are conspecific, that the former (foxtail millet) is a domesticated form of the latter, and also questions the taxonomic validity of formally recognizing morphological varieties within green foxtail.     

Assessment of genetic relationships between Setaria italica and its wild relative S. viridis using AFLP markers

AFLP markers were used to assess genetic diversity and patterns of geographic variation among 39 accessions of foxtail millet (Setaria italica) and 22 accessions of green foxtail millet (S. viridis), its putative wild progenitor. A high level of polymorphism was revealed. Dendrograms based on Nei and Li distances from a neighbour joining procedure were constructed using 160 polymorphic bands. Bootstrap values revealed that no specific geographic structure can be extracted from these data. The high level of diversity among Chinese accessions was consistent with the hypothesis of a centre of domestication in China. The results also showed that accessions from Eastern Europe and Africa form two distinct clusters. The narrow genetic basis of these two gene pools may be the result of local-adaptation. Received: 1 June 1999 / Accepted: 16 September 1999     

Inheritance of some marker genes in Setaria italica (L.) P. Beauv.

Summary A study on a series of genetic markers was run on five hybrids of foxtail millet, Setaria italica, and on one interspecific hybrid S. viridisxS. italica (S. viridis is the wild relative of S. italica). Seven enzymatic systems were investigated using starch gel electrophoresis (esterase, alcohol dehydrogenase, glutamate oxaloacetate transaminase, acid phosphatase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, cathodic peroxidase). This genetic analysis of the 6 F2 has allowed us to define 12 polymorphic loci: Est-1, -2 and -3, Adh-1, Got-1 and -2, Acph-1, Mdh-1 and -2, Pgd-1 and -2, and Pox-1. All of them behaved like dimers, except Est-1 and Est-2 which showed monomeric structures. Two other markers were examined: waxy endosperm, which appeared to be controlled by one locus, and anthocyanic pigmentation of the collar, for which at least two loci are responsible. Studies of linkage carried out on three F2 showed two linkage groups: Mdh-1, Pox-1, Wx, Est-3, and a locus for collar colour, and Est-2, and one or two other loci of colouring.     

Foxtail millet: a model crop for genetic and genomic studies in bioenergy grasses

Foxtail millet is one of the oldest domesticated diploid C₄ Panicoid crops having a comparatively small genome size of approximately 515?Mb, short life cycle, and inbreeding nature. Its two species, Setaria italica (domesticated) and Setaria viridis (wild progenitor), have characteristics that classify them as excellent model systems to examine several aspects of architectural, evolutionary, and physiological importance in Panicoid grasses especially the biofuel crops such as switchgrass and napiergrass. Foxtail millet is a staple crop used extensively for food and fodder in parts of Asia and Africa. In its long history of cultivation, it has been adapted to arid and semi-arid areas of Asia, North Africa, South and North America. Foxtail millet has one of the largest collections of cultivated as well as wild-type germplasm rich with phenotypic variations and hence provides prospects for association mapping and allele-mining of elite and novel variants to be incorporated in crop improvement programs. Most of the foxtail millet accessions can be primarily abiotic stress tolerant particularly to drought and salinity, and therefore exploiting these agronomic traits can enhance its efficacy in marker-aided breeding as well as in genetic engineering for abiotic stress tolerance. In addition, the release of draft genome sequence of foxtail millet would be useful to the researchers worldwide in not only discerning the molecular basis of biomass production in biofuel crops and the methods to improve it, but also for the introgression of beneficial agronomically important characteristics in foxtail millet as well as in related Panicoid bioenergy grasses.     

Construction of RFLP-based maps of foxtail millet, Setaria italica (L.) P. Beauv.

 An RFLP-based map consisting of 160 loci was constructed in an intervarietal cross of foxtail millet [Setaria italica (L.) P. Beauv.], Longgu 25×Pagoda Flower Green. The map comprises nine linkage groups, which were aligned with the nine foxtail millet chromosomes using trisomic lines, and spans 964 cM. The intraspecific map was compared to an interspecific map, constructed in a S. italica×S. viridis cross. Both the order of the markers and the genetic distances between the loci were highly conserved. Deviations from the expected 1 : 2 : 1 Mendelian segregation ratios were observed in both the intra- and inter-specific populations. The segregation data indicate that chromosome VIII in the Longgu 25×Pagoda Flower Green cross carries a gene that strongly affects gamete fertility. Received: 18 December 1996 / Accepted: 4 August 1997     

Setaria genome sequencing: an overview

The genus Setaria includes two important C₄ Panicoid grass species, namely S. italica (cultivated) and S. viridis (weed; wild ancestor), which together represent an appropriate model system for architectural, physiological, evolutionary, and genomic studies in related grasses. It is a diploid, inbreeder, self-fertile annual cereal grass having short life cycle and minimal growth requirements. There close relatedness to biofuel crops like switch grass and napier grass further signifies their importance. Further, foxtail millet is an important food and fodder grain crop grown in arid and semi-arid regions in many parts of the world. Therefore, an increasing interest in these species has led to a gradual accumulation and development of genomic data and genetic resources. Setaria genome sequencing is an outcome of such endeavors. These sequencing efforts uncovered several distinctive attributes of Setaria genome that may help in understanding its physiology, evolution and adaptation. This will not only aid in comparative genomics studies of Setaria and related crops including bioenergy grasses but also help in rapid advancements of genomics information for developing varieties with superior traits either through marker-assisted selection (MAS) or using transgenic approaches in these crops.     

Methods for Performing Crosses in Setaria viridis,a New Model System for the Grasses

Setaria viridis is an emerging model system for C₄ grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced ^1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production ³ and developed systems for both transient and stable transformation ⁴. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 °C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.     

Development and Genetic Control of Plant Architecture and Biomass in the Panicoid Grass,Setaria

The architecture of a plant affects its ability to compete for light and to respond to environmental stresses, thus affecting overall fitness and productivity. Two components of architecture, branching and height, were studied in 182 F₇ recombinant inbred lines (RILs) at the vegetative, flowering and mature developmental stages in the panicoid C₄ model grass system, Setaria. The RIL population was derived from a cross between domesticated S. italica (foxtail millet) and its wild relative S. viridis (green foxtail). In both field and greenhouse trials the wild parent was taller initially, started branching earlier, and flowered earlier, while the domesticated parent was shorter initially, but flowered later, producing a robust tall plant architecture with more nodes and leaves on the main culm and few or no branches. Biomass was highly correlated with height of the plant and number of nodes on the main culm, and generally showed a negative relationship with branch number. However, several of the RILs with the highest biomass in both trials were significantly more branched than the domesticated parent of the cross. Quantitative trait loci (QTL) analyses indicate that both height and branching are controlled by multiple genetic regions, often with QTL for both traits colocalizing in the same genomic regions. Genomic positions of several QTL colocalize with QTL in syntenic regions in other species and contain genes known to control branching and height in sorghum, maize, and switchgrass. Included in these is the ortholog of the rice SD-1 semi-dwarfing gene, which underlies one of the major Setaria height QTL. Understanding the relationships between height and branching patterns in Setaria, and their genetic control, is an important step to gaining a comprehensive knowledge of the development and genetic regulation of panicoid grass architecture.     

Geographical distribution and genetic analysis of phenol color reaction in foxtail millet,Setaria italica (L.) P. Beauv.

Summary Phenol color reaction was examined in a total of 376 strains of foxtail millet, Setaria italica (L.) P. Beauv., collected from different areas throughout Eurasia. Positive and negative phenotypes, and no intermediate type could be recognized by the phenol color reaction. Of 376 strains examined, 50 were positive, 319 were negative, five were mixtures of both phenotypes, and the coloration in two strains with blackish lemmata and paleae could not be distinguished. The strains that showed the positive phenotype of phenol color reaction were found in rather limited regions, while those with the negative phenotype occurred in almost all the regions. The positive phenotype occurred more frequently in the lower latitudinal regions of Asia. Genetic analysis of the F₁ and F₂ generations between the two phenotypes showed that the phenol color reaction is controlled by a single gene, and that the positive phenotype is dominant.Contribution No. 28 from the Plant Germ-plasm Institute, Faculty of Agriculture, Kyoto University, Kyoto (Japan)