Development of 5123 Intron-Length Polymorphic Markers for Large-Scale Genotyping Applications in Foxtail Millet

Generating genomic resources in terms of molecular markers is imperative in molecular breeding for crop improvement. Though development and application of microsatellite markers in large-scale was reported in the model crop foxtail millet, no such large-scale study was conducted for intron-length polymorphic (ILP) markers. Considering this, we developed 5123 ILP markers, of which 4049 were physically mapped onto 9 chromosomes of foxtail millet. BLAST analysis of 5123 expressed sequence tags (ESTs) suggested the function for ∼71.5% ESTs and grouped them into 5 different functional categories. About 440 selected primer pairs representing the foxtail millet genome and the different functional groups showed high-level of cross-genera amplification at an average of ∼85% in eight millets and five non-millet species. The efficacy of the ILP markers for distinguishing the foxtail millet is demonstrated by observed heterozygosity (0.20) and Nei''s average gene diversity (0.22). In silico comparative mapping of physically mapped ILP markers demonstrated substantial percentage of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (∼50%), maize (∼46%), rice (∼21%) and Brachypodium (∼21%) chromosomes. Hence, for the first time, we developed large-scale ILP markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.     

Genetic and Genomic Resources of Small Millets

Small millets are very promising agricultural entity to ensure global food security. They gained remarkable importance in agriculture due to their resilience to climatic changes and increasing demand for nutritious food and feed. The genetic variability in the core and mini-core germplasm of small millets was characterized for nutritional composition and capacity to tolerate abiotic stresses that can be infused in breeding programs. Other than the foxtail millet, availability of genomic information in small millets is far below the mark for use in marker-assisted breeding and other genetic improvement programs. The genome sequence of foxtail millet has recently triggered a plethora of post-genomic analysis and envisaged foxtail millet as a model organism for the C₄ grasses and bioenergy research. Recent developments in the next-generation sequencing technologies enabled us, with the simultaneous discovery of high-throughput markers and multiplexed genotyping of germplasm, to speedup marker-assisted breeding. In this context, an in-depth analysis of the wealth of diverse germplasm resources and future perspectives of integrating genomics in genome-wide marker-trait association and breeding in small millets is worthy.     

Development and characterization of microsatellite markers from tropical forage Stylosanthes species and analysis of genetic variability and cross-species transferability

A limited number of functional molecular markers has slowed the desired genetic improvement of Stylosanthes species. Hence, in an attempt to develop simple sequence repeat (SSR) markers, genomic libraries from Stylosanthes seabrana B.L. Maass & 't Mannetje (2n=2x=20) using 5' anchored degenerate microsatellite primers were constructed. Of the 76 new microsatellites, 21 functional primer pairs were designed. Because of the small number of primer pairs designed, 428 expressed sequence tag (EST) sequences from seven Stylosanthes species were also examined for SSR detection. Approximately 10% of sequences delivered functional primer pairs, and after redundancy elimination, 57 microsatellite repeats were selected. Tetranucleotides followed by trinucleotides were the major repeated sequences in Stylosanthes ESTs. In total, a robust set of 21 genomic-SSR (gSSR) and 20 EST-SSR (eSSR) markers were developed. These markers were analyzed for intraspecific diversity within 20 S. seabrana accessions and for their cross-species transferability. Mean expected (He) and observed (Ho) heterozygosity values with gSSR markers were 0.64 and 0.372, respectively, whereas with eSSR markers these were 0.297 and 0.214, respectively. Dendrograms having moderate bootstrap value (23%-94%) were able to distinguish all accessions of S. seabrana with gSSR markers, whereas eSSR markers showed 100% similarities between few accessions. The set of 21 gSSRs, from S. seabrana, and 20 eSSRs, from selected Stylosanthes species, with their high cross-species transferability (45% with gSSRs, 86% with eSSRs) will facilitate genetic improvement of Stylosanthes species globally.     

Comparison of genomic SSR and EST-SSR markers for estimating genetic diversity in cucumber

Thirteen genomic microsatellite (gSSR) and sixteen expressed sequence tag (EST)-SSR (eSSR) markers were compared to estimate genetic diversity among 29 cucumber (Cucumis sativus L.) accessions. gSSR markers detected mean 4.46 alleles with a mean polymorphic information content (PIC) of 0.664, against eSSR markers with mean 3.38 alleles and a mean PIC of 0.397. gSSRs amplified more null alleles than eSSRs. Genetic diversity within the accession set was estimated by construction of dendrograms using gSSR or eSSR data. There was a clear consistency between gSSR and eSSR trees in terms of positioning of most cucumber germplasms. gSSR markers could separate various types of cucumber germplasms on the whole, although clustering of some accessions was not based on their geographical origins in eSSR tree. eSSR markers identified an independent sub-cluster containing five accessions resistant to downy mildew, suggesting a probable relationship between eSSRs and disease-resistance trait in cucumber. The Mantel test between gSSR and eSSR matrices revealed a good fit correlation (r = 0.836). The general dendrogram constructed using the combined data of gSSRs and eSSRs was similar to those obtained separately with each marker.     

Population structure and association mapping of yield contributing agronomic traits in foxtail millet

Key message

Association analyses accounting for population structure and relative kinship identified eight SSR markers ( p < 0.01) showing significant association ( R ²  = 18 %) with nine agronomic traits in foxtail millet.

Abstract

Association mapping is an efficient tool for identifying genes regulating complex traits. Although association mapping using genomic simple sequence repeat (SSR) markers has been successfully demonstrated in many agronomically important crops, very few reports are available on marker-trait association analysis in foxtail millet. In the present study, 184 foxtail millet accessions from diverse geographical locations were genotyped using 50 SSR markers representing the nine chromosomes of foxtail millet. The genetic diversity within these accessions was examined using a genetic distance-based and a general model-based clustering method. The model-based analysis using 50 SSR markers identified an underlying population structure comprising five sub-populations which corresponded well with distance-based groupings. The phenotyping of plants was carried out in the field for three consecutive years for 20 yield contributing agronomic traits. The linkage disequilibrium analysis considering population structure and relative kinship identified eight SSR markers (p < 0.01) on different chromosomes showing significant association (R ² = 18 %) with nine agronomic traits. Four of these markers were associated with multiple traits. The integration of genetic and physical map information of eight SSR markers with their functional annotation revealed strong association of two markers encoding for phospholipid acyltransferase and ubiquitin carboxyl-terminal hydrolase located on the same chromosome (5) with flag leaf width and grain yield, respectively. Our findings on association mapping is the first report on Indian foxtail millet germplasm and this could be effectively applied in foxtail millet breeding to further uncover marker-trait associations with a large number of markers.     

Sequence-based novel genomic microsatellite markers for robust genotyping purposes in foxtail millet [<Emphasis Type="Italic">Setaria italica</Emphasis> (L.) P. Beauv.]

The unavailability of microsatellite markers and saturated genetic linkage map has restricted the genetic improvement of foxtail millet [Setaria italica (L.) P. Beauv.], despite the fact that in recent times it has been documented as a new model species for biofuel grasses. With the objective to generate a good number of microsatellite markers in foxtail millet cultivar ‘Prasad’, 690 clones were sequenced which generated 112.95 kb high quality sequences obtained from three genomic libraries each enriched with different microsatellite repeat motifs. Microsatellites were identified in 512 (74.2%) of the 690 positive clones and 172 primer pairs (pp) were successfully designed from 249 (48.6%) unique SSR-containing clones. The efficacies of the microsatellite containing genomic sequences were established by superior primer designing ability (69%), PCR amplification efficiency (85.5%) and polymorphic potential (52%) in the parents of F₂ mapping population. Out of 172 pp, functional 147 markers showed high level of cross-species amplification (~74%) in six grass species. Higher polymorphism rate and broad range of genetic diversity (0.30–0.69 averaging 0.58) obtained in constructed phylogenetic tree using 52 microsatellite markers, demonstrated the utility of markers in germplasm characterizations. In silico comparative mapping of 147 foxtail millet microsatellite containing sequences against the mapping data of sorghum (~18%), maize (~16%) and rice (~5%) indicated the presence of orthologous sequences of the foxtail millet in the respective species. The result thus demonstrates the applicability of microsatellite markers in various genotyping applications, determining phylogenetic relationships and comparative mapping in several important grass species.     

Nucleotide Sequence Diversity and Linkage Disequilibrium of Four Nuclear Loci in Foxtail Millet (Setaria italica)

Foxtail millet (Setaria italica (L.) Beauv) is one of the earliest domesticated grains, which has been cultivated in northern China by 8,700 years before present (YBP) and across Eurasia by 4,000 YBP. Owing to a small genome and diploid nature, foxtail millet is a tractable model crop for studying functional genomics of millets and bioenergy grasses. In this study, we examined nucleotide sequence diversity, geographic structure, and levels of linkage disequilibrium at four nuclear loci (ADH1, G3PDH, IGS1 and TPI1) in representative samples of 311 landrace accessions across its cultivated range. Higher levels of nucleotide sequence and haplotype diversity were observed in samples from China relative to other sampled regions. Genetic assignment analysis classified the accessions into seven clusters based on nucleotide sequence polymorphisms. Intralocus LD decayed rapidly to half the initial value within ~1.2 kb or less.     

A microsatellite-based, gene-rich linkage map reveals genome structure, function and evolution in Gossypium

The mapping of functional genes plays an important role in studies of genome structure, function, and evolution, as well as allowing gene cloning and marker-assisted selection to improve agriculturally important traits. Simple sequence repeats (SSRs) developed from expressed sequence tags (ESTs), EST-SSR (eSSR), can be employed as putative functional marker loci to easily tag corresponding functional genes. In this paper, 2218 eSSRs, 1554 from G. raimondii-derived and 754 from G. hirsutum-derived ESTs, were developed and used to screen polymorphisms to enhance our backbone genetic map in allotetraploid cotton. Of the 1554 G. raimondii-derived eSSRs, 744 eSSRs were able to successfully amplify polymorphisms between our two mapping parents, TM-1 and Hai7124, presenting a polymorphic rate of 47.9%. However, only a 23.9% (159/754) polymorphic rate was produced from G. hirsutum-derived eSSRs. No relationship was observed between the level of polymorphism, motif type, and tissue origin, but the polymorphism appeared to be correlated with repeat type. After integrating these new eSSRs, our enhanced genetic map consists of 1790 loci in 26 linkage groups and covers 3425.8 cM with an average intermarker distance of 1.91 cM. This microsatellite-based, gene-rich linkage map contains 71.96% functional marker loci, of which 87.11% are eSSR loci. There were 132 duplicated loci bridging 13 homeologous At/Dt chromosome pairs. Two reciprocal translocations after polyploidization between A2 and A3, and between A4 and A5, chromosomes were further confirmed. A functional analysis of 975 ESTs producing 1122 eSSR loci tagged in the map revealed that 60% had clear BLASTX hits (<1e(-10)) to the Uniprot database and that 475 were associated mainly with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function; many of the ESTs were associated with two or more category functions. The results presented here will provide new insights for future investigations of functional and evolutionary genomics, especially those associated with cotton fiber improvement.     

Construction of a foxtail millet linkage map and mapping of spikelet-tipped bristles 1(stb1) by using transposon display markers and simple sequence repeat markers with genome sequence information

We attempted genetic analysis and mapping of a gene responsible for the trait “spikelet-tipped bristles” (stb) in foxtail millet, Setaria italica (L.) P.Beauv., as the first step in positional cloning of the gene. This trait is important not only in grain yield such as grain number per panicle of this millet but also in the evolutionary development of the “bristle grass” clade including genera Setaria, Pennisetum and Cenchrus in subfamily Panicoideae. First of all, we confirmed that this trait is controlled by a single recessive gene, using two populations of F2 plants; one was a cross combination between two Taiwanese landraces and the other was a combination between a Taiwanese landrace and a Japanese landrace. Using the latter of the two F2 populations, with transposon display (TD) markers and simple sequence repeat (SSR) markers developed previously, we constructed a genetic map with 13 linkage groups and mapped the responsible gene (stb1) on chromosome 2. We also developed novel SSR markers by using foxtail millet genome sequence information, and we finally constructed nine linkage groups corresponding to nine chromosomes with a total length of 1287.5 cM, and mapped stb1 more precisely on chromosome 2. This work suggests that the foxtail millet genome sequences recently published are useful for developing genome-wide SSR markers for constructing linkage maps and mapping genes in this millet.     

Development and utilization of novel intron length polymorphic markers in foxtail millet (Setaria italica (L.) P. Beauv.)

Introns are noncoding sequences in a gene that are transcribed to precursor mRNA but spliced out during mRNA maturation and are abundant in eukaryotic genomes. The availability of codominant molecular markers and saturated genetic linkage maps have been limited in foxtail millet (Setaria italica (L.) P. Beauv.). Here, we describe the development of 98 novel intron length polymorphic (ILP) markers in foxtail millet using sequence information of the model plant rice. A total of 575 nonredundant expressed sequence tag (EST) sequences were obtained, of which 327 and 248 unique sequences were from dehydration- and salinity-stressed suppression subtractive hybridization libraries, respectively. The BLAST analysis of 98 EST sequences suggests a nearly defined function for about 64% of them, and they were grouped into 11 different functional categories. All 98 ILP primer pairs showed a high level of cross-species amplification in two millets and two nonmillets species ranging from 90% to 100%, with a mean of ～97%. The mean observed heterozygosity and Nei's average gene diversity 0.016 and 0.171, respectively, established the efficiency of the ILP markers for distinguishing the foxtail millet accessions. Based on 26 ILP markers, a reasonable dendrogram of 45 foxtail millet accessions was constructed, demonstrating the utility of ILP markers in germplasm characterizations and genomic relationships in millets and nonmillets species.     

Exploiting the Transcriptome of Euphrates Poplar,Populus euphratica (Salicaceae) to Develop and Characterize New EST-SSR Markers and Construct an EST-SSR Database

Background

Microsatellite markers or Simple Sequence Repeats (SSRs) are the most popular markers in population/conservation genetics. However, the development of novel microsatellite markers has been impeded by high costs, a lack of available sequence data and technical difficulties. New species-specific microsatellite markers were required to investigate the evolutionary history of the Euphratica tree, Populus euphratica, the only tree species found in the desert regions of Western China and adjacent Central Asian countries.

Methodology/Principal Findings

A total of 94,090 non-redundant Expressed Sequence Tags (ESTs) from P. euphratica comprising around 63 Mb of sequence data were searched for SSRs. 4,202 SSRs were found in 3,839 ESTs, with 311 ESTs containing multiple SSRs. The most common motif types were trinucleotides (37%) and hexanucleotides (33%) repeats. We developed primer pairs for all of the identified EST-SSRs (eSSRs) and selected 673 of these pairs at random for further validation. 575 pairs (85%) gave successful amplification, of which, 464 (80.7%) were polymorphic in six to 24 individuals from natural populations across Northern China. We also tested the transferability of the polymorphic eSSRs to nine other Populus species. In addition, to facilitate the use of these new eSSR markers by other researchers, we mapped them onto Populus trichocarpa scaffolds in silico and compiled our data into a web-based database (http://202.205.131.253:8080/poplar/resources/static_page/index.html).

Conclusions

The large set of validated eSSRs identified in this work will have many potential applications in studies on P. euphratica and other poplar species, in fields such as population genetics, comparative genomics, linkage mapping, QTL, and marker-assisted breeding. Their use will be facilitated by their incorporation into a user-friendly web-based database.     

Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I–V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses.     

Development, characterization, genetic diversity and cross-species/genera transferability of ILP markers in rubber tree (Hevea brasiliensis)

To construct a linkage map enriched with tapping panel dryness (TPD)-related markers, we firstly utilized rubber tree ESTs associated with TPD to develop intron length polymorphism (ILP) markers. In this study, 52 new ILP markers were further developed. Together with the ILP markers previously reported, 102 ILP markers developed from TPD-related ESTs were analyzed within 39 Hevea germplasm in detail. The PCR success rate and polymorphism rate of ILP markers was 97.06 and 61.62 %, respectively. The results based on PCR amplification and sequence analyses provided the evidences on cross-species/genera transferability of rubber tree ILP markers. The average polymorphic information content (PIC) values of 39 Hevea germplasm were about 0.1719, indicating that the genetic base of Hevea germplasm selected in this study was very narrow. Among 39 Hevea germplasm, the PIC value of wild rubber tree accessions was the highest, followed by Hevea species and cultivated rubber tree clones. Based on the similarity coefficient of ILP markers, 39 Hevea germplasm were divided into three groups including cultivated clones, wild accessions and Hevea species, suggesting that the classification was generally related to the characterization of Hevea germplasm. The ILP markers developed in this study further enriches the number of molecular marker in rubber tree, and the ILP markers will have a wide application in DNA fingerprinting, genetic diversity, marker-assisted selection and genetic mapping, etc. Moreover, the ILP markers transferred cross-euphorbiaceae family might be utilized in cassava, castor bean and physic nut.     

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.     

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.