Phylogenetic and population genomic analysis of foxtail millet (Setaria italica) landraces via ddRAD-seq, with emphasis on Japanese germplasm.

Foxtail millet shows variation in positive phenol color reaction (Phr) and negative Phr in grains, but predominant accessions of this crop are negative reaction type, and the molecular genetic basis of the Phr reaction remains unresolved. In this article, we isolated polyphenol oxidase (PPO) gene responsible for Phr using genome sequence information and investigated molecular genetic basis of negative Phr and crop evolution of foxtail millet. First of all, we searched for PPO gene homologs in a foxtail millet genome database using a rice PPO gene as a query and successfully found three copies of the PPO gene. One of the PPO gene homologs on chromosome 7 showed the highest similarity with PPO genes expressed in hulls (grains) of other cereal species including rice, wheat, and barley and was designated as Si7PPO. Phr phenotypes and Si7PPO genotypes completely co-segregated in a segregating population. We also analyzed the genetic variation conferring negative Phr reaction. Of 480 accessions of the landraces investigated, 87 (18.1 %) showed positive Phr and 393 (81.9 %) showed negative Phr. In the 393 Phr negative accessions, three types of loss-of-function Si7PPO gene were predominant and independently found in various locations. One of them has an SNP in exon 1 resulting in a premature stop codon and was designated as stop codon type, another has an insertion of a transposon (Si7PPO-TE1) in intron 2 and was designated as TE1-insertion type, and the other has a 6-bp duplication in exon 3 resulting in the duplication of 2 amino acids and was designated as 6-bp duplication type. As a rare variant of the stop codon type, one accession additionally has an insertion of a transposon, Si7PPO-TE2, in intron 2 and was designated as "stop codon +TE2 insertion type". The geographical distribution of accessions with positive Phr and those with three major types of negative Phr was also investigated. Accessions with positive Phr were found in subtropical and tropical regions at frequencies of ca. 25-67 % and those with negative Phr were broadly found in Europe and Asia. The stop codon type was found in 285 accessions and was broadly distributed in Europe and Asia, whereas the TE-1 insertion type was found in 99 accessions from Europe and Asia but was not found in India. The 6-bp duplication type was found in only 8 accessions from Nansei Islands (Okinawa Prefecture) of Japan. We also analyzed Phr in the wild ancestor and concluded that the negative Phr type was likely to have originated after domestication of foxtail millet. It was also implied that negative Phr of foxtail millet arose by multiple independent loss of function of PPO gene through dispersal because of some advantages under some environmental conditions and human selection as in rice and barley.

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.

The author examined fossilized deer bones with forks at one or both ends which were excavated from the Gohezu cave of Ie-jima, Okinawa Prefecture, and whichhave been considered them as the result of shaping by hominids. He decided that they were not artificial, but in a much more advanced stage of bone damage due to chewing by deer himself as a result of osteophagia. So, at present, any archaeological evidences in palaeolithic age are not recognized inOkinawa Prefecture. Recently an amorphous flake industry with wedge shaped tools has been found in the layer, contained AT-tephra dated more than 20, 000 years B.P., at the Yaaya and Kisikawa, Amamioshima Island, Kagoshima Prefecture. The undated same industry, too, is reported from the Amangusuku, Tokunoshima Island, Kagoshima Prefecture. The author asserts that the flake industry in final Upper Pleistocene should been widely distributed throughout the Nansei (Southeastern) Islands of Okinawa and Kagoshima Prefecture. Further, the same flake industry with wedgeshaped tools exists at the Baxian-dong caves (5, 000-15, 000ys. B.P.) in Eastern Taiwan, Lang Rongrien rockshelter (27, 000-37, 000 ys. B.P.) in Southwestern Thailand, and Devil's Lair cave (12, 000-31, 000 ys. B.P.) in Southwestern Australia. The author thinks that the prehistoric people with the flake industry had adapted themselves to coastal environment somewhere around Southeast Asia about 30, 000 years ago, because of findingsof fish bone sand fishing tools from the sites. They had known about the technology how to sail across the sea, and dispersed on each islands northward along the Nansei Islands from the cradle of flake industry in Southeast Asia until 20, 000 years ago.

Sixteen RFLP loci in 62 landraces were assayed to study geneticdifferentiation in foxtail millet, Setariaitalica (L.) P. Beauv. Among 52 bands, 47 werepolymorphic among foxtail millet landraces. A dendrogram constructedbased on RFLPs was divided into five major clusters (clusterI–V). Clusters I and II contained strains mainly from EastAsia. Cluster III consisted of strains from subtropical and tropicalregions in Asia such as Nansei Islands of Japan, Taiwan and thePhilippines and India and cluster IV consisted of some strains fromEast Asia, a strain from Nepal and a strain from Myanmar. Cluster Vcontained strains from central and western regions of Eurasia such asAfghanstan, Central Asia and Europe. Chinese landraces wereclassified into four clusters. These results indicate that foxtailmillet landraces have differentiated genetically between differentregions and that Chinese landraces were highly variable.

Influence of natural and artificial selection on the yield differences among progeny derived from crossing between subspecies in cultivated rice

As an ancient cereal of great importance for dryland agriculture even today, foxtail millet (Setaria italica) is fast becoming a new plant genomic model crop. A genotypic analysis of 250 foxtail millet landraces, which represent 1% of foxtail millet germplasm kept in the Chinese National Gene Bank (CNGB), was conducted with 77 SSRs covering the foxtail millet genome. A high degree of molecular diversity among the landraces was found, with an average of 20.9 alleles per locus detected. STRUCTURE, neighbor-jointing, and principal components analyses classify the accessions into three clusters (topmost hierarchy) and, ultimately, four conservative subgroups (substructuring within the topmost clusters) in total, which are in good accordance with eco-geographical distribution in China. The highest subpopulation diversity was identified in the accessions of Pop3 from the middle regions of the Yellow River, followed by accessions in Pop1 from the downstream regions of the Yellow River, suggesting that foxtail millet was domesticated in the Yellow River drainage area first and then spread to other parts of the country. Linkage disequilibrium (LD) decay of less than 20 cM of genetic distance in the foxtail millet landrace genome was observed, which suggests that it could be possible to achieve resolution down to the 20 cM level for association mapping.

Foxtail millet (Setaria italica (L.) P. Beauv.) is highly valued for nutritional traits, stress tolerance and sustainability in resource-poor dryland agriculture. However, the low productivity of this crop in semi-arid regions of Southern India, is further threatened by climate stress. Landraces are valuable genetic resources, regionally adapted in form of novel alleles that are responsible for cope up the adverse conditions used by local farmers. In recent years, there is an erosion of genetic diversity. We have hypothesized that plant genetic resources collected from the semi-arid climatic zone would serve as a source of novel alleles for the development of climate resilience foxtail millet lines with enhanced yield. Keeping in view, there is an urgent need for conservation of genetic resources. To explore the genetic diversity, to identify superior genotypes and novel alleles, we collected a heterogeneous mixture of foxtail millet landraces from farmer fields. In an extensive multi-year study, we developed twenty genetically fixed foxtail millet landraces by single seed descent method. These landraces characterized along with four released cultivars with agro-morphological, physiological, yield and yield-related traits assessed genetic diversity and population structure. The landraces showed significant diversity in all the studied traits. We identified landraces S3G5, Red, Black and S1C1 that showed outstanding grain yield with earlier flowering, and maturity as compared to released cultivars. Diversity analysis using 67 simple sequence repeat microsatellite and other markers detected 127 alleles including 11 rare alleles, averaging 1.89 alleles per locus, expected heterozygosity of 0.26 and an average polymorphism information content of 0.23, collectively indicating a moderate genetic diversity in the landrace populations. Euclidean Ward’s clustering, based on the molecular markers, principal coordinate analysis and structure analysis concordantly distinguished the genotypes into two to three sub-populations. A significant phenotypic and genotypic diversity observed in the landraces indicates a diverse gene pool that can be utilized for sustainable foxtail millet crop improvement.

Ribosomal DNA (rDNA) spacer length polymorphism was studied in foxtail millet (Setaria italica ssp. italica) landraces from Pakistan and Afghanistan and in its wild ancestor (S. italica ssp. viridis) from Pakistan by PCR-based methods. Sequence polymorphism was also investigated for accessions selected based on the observed length polymorphism. The PCR-based length polymorphism and sequence polymorphism of rDNA intergenic spacer (IGS) clearly demonstrated genetic differentiation between cultivated and wild forms in the region. Genetic differentiation was observed between different areas to some extent in the cultivated form, and between different regions in the wild form of northern Pakistan. Based on the results, we discuss the genetic differentiation of foxtail millet and wild ancestor in this region and possible utility of rDNA markers to trace the dispersal of this crop in the region.

Assessing nutritional and genetic variations within foxtail millet (Setaria italica) landraces collected from indigenous communities across the Philippines

In order to understand how divergence may appear within a gene pool without limitations on gene flow, experiments were performed to investigate whether the genetic structure of a population may be effective in controlling the response to natural selection. Starting from plateaued populations of Drosophila melanogaster selected for wing length on the two sexes separately, the response to natural selection was studied after artificial selection had been discontinued. The results show that populations derived from artificial selection applied to the different sexes respond to subsequent natural selection in dissimilar ways, so as to suggest that the relationships between fitness and wing length have been changed. A second experiment comparing the response to natural and to artificial reversed selection suggests that different genes or gene complexes may be involved in wing length determination: these different genes show a dissimilar response in the different sexes and to natural and artificial selection. This variety of responses suggests that the interactions of different genes or gene complexes with artificial and natural selection could possibly lead to differentiation within the population.

The infection by Haemonchus contortus is a significant challenge to sheep production in tropical regions, particularly in developing countries. Although several genomic studies have been conducted on this topic, there is still a lack of research combining evolutionary information on resistance and resilience to nematode infection. The aim of this study was to provide evidence of different types of selection and their effects on traits associated with infection levels and animal productivity, using pedigree, phenotypic, and genomic data. It was hypothesized that these patterns would reflect indirect artificial selection and relaxed natural selection. Phenotypic data were collected for Faecal Egg Count (FEC), Eye Color Chart (ECC), Packed Cell Volume (PCV), Total Plasma Protein (TPP), Body Weight (BW), and Body Condition Score (BCS) from 1,283 Santa Inês sheep. A total of 638 animals were genotyped using the Ovine SNP50 BeadChip. After estimating breeding values using the BLUPF90 software, statistical models were employed to assess differences in the intensities of natural and artificial selection and to identify the type of selection acting on each trait, in comparison with classic studies of sexual selection. Selection signatures were investigated using Wright's fixation index, in addition to analyses of runs of homozygosity. The gene content of the identified regions and their associated pathways were examined using the Ensembl BioMart tool and the Panther Classification System, respectively, along with alignments of quantitative trait loci (QTL). BCS was found to be the best indirect trait correlated with parasitological traits, and selection intensity analysis showed that natural selection contributed 76%, compared to 24% from artificial selection. Traits such as BW, PCV, and TPP exhibited directional selection, while FEC and ECC varied according to the challenge level applied. A total of 15 selection signatures were identified (11 for natural selection and 4 for artificial selection), with 9 overlapping with islands of homozygosity, encompassing 131 genes and 49 QTL. A critical analysis revealed that both types of selection contribute to the phenomena of resistance and resilience. However, evidence of directional selection, hard sweeps, and functional enrichment of innate immunity was found for artificial selection, while natural selection exhibited evidence of stabilizing selection, soft sweeps, and functional enrichment of adaptive immunity.

We sequenced ribosomal DNA intergenic spacer subrepeats and their flanking regions of foxtail millet landraces from various regions in Europe and Asia and its wild ancestor to elucidate phylogenetic differentiation within each of types I-III found in our previous work and to elucidate relationships among these three types. Type I was classified into seven subtypes designated as Ia-Ig based on subrepeat sequences; C repeats downstream of those subrepeats are also polymorphic. Of these, subtypes Ia-Id and Ig were found in foxtail millet landraces. Subtypes Ia and Ib were distributed broadly throughout Asia and Europe. Subtype Ic was distributed in China, Korea and Japan. Subtype Id has a 20-bp deletion in subrepeat 3 and has a unique C repeat sequence. This subtype was found in a morphologically primitive landrace group from Afghanistan and northwestern Pakistan and differed greatly from other type I subtypes, implying that these landraces were domesticated independently. Subtypes Ig was found in a landrace from Pakistan and Ia and Ie-Ig were in six wild ancestor accessions. Type II was also highly polymorphic and four subtypes were found and designated as subtypes IIa-IId, but sequence analyses indicated type III as monomorphic. The present work indicates that type III should be classified as a subtype of type II (subtype IIe). Sequence polymorphism of subrepeats of types I-III indicated that subrepeats of subtype IIa are greatly divergent from others. Relationships among types I-III are much more complicated than anticipated based on previous RFLP work.

Green foxtail (Setaria viridis) is a new model plant for the genomic investigation of C4 photosynthesis biology. As the ancestor of foxtail millet (Setaria italica), an ancient cereal of great importance in arid regions of the world, green foxtail is crucial for the study of domestication and evolution of this ancient crop. In the present study, 288 green foxtail accessions, which were collected from all geographical regions of China, were analysed using 77 simple sequence repeats (SSRs) that cover the whole genome. A high degree of molecular diversity was detected in these accessions, with an average of 33.5 alleles per locus. Two clusters, which were inconsistent with the distribution of eco-geographical regions in China, were inferred from STRUCTURE, Neighbor–Joining, and principal component analysis, indicating a partially mixed distribution of Chinese green foxtails. The higher subpopulation diversity was from accessions mainly collected from North China. A low level of linkage disequilibrium was observed in the green foxtail genome. Furthermore, a combined analysis of green foxtail and foxtail millet landraces was conducted, and the origin and domestication of foxtail millet was inferred in North China.

Although the origin and domestication process of foxtail millet (Setaria italica subsp. italica (L.) P. Beauv.) has been studied by several groups, the issue is still ambiguous. It is essential to resolve this issue by studying a large number of accessions with sufficient markers covering the entire genome. Genetic structures were analyzed by transposon display (TD) using 425 accessions of foxtail millet and 12 of the wild ancestor green foxtail (Setaria italica subsp. viridis (L.) P. Beauv.). We used three recently active transposons (TSI-1, TSI-7, and TSI-10) as genome-wide markers and succeeded in demonstrating geographical structures of the foxtail millet. A neighbor-joining dendrogram based on TD grouped the foxtail millet accessions into eight major clusters, each of which consisted of accessions collected from adjacent geographical areas. Eleven out of 12 green foxtail accessions were grouped separately from the clusters of foxtail millet. These results indicated strong regional differentiations and a long history of cultivation in each region. Furthermore, we discuss the relationship between foxtail millet and green foxtail and suggest a monophyletic origin of foxtail millet domestication.

The effectiveness of artificial vs. natural selection for improving lint yield and other traits in American Pima cotton (Gossypium barbadense L.) when grown in predictable environments was evaluated at low and high elevations. A cross was made between ‘Pima S‐3,’ adapted to the less fertile soils at high elevations, and ‘Pima S‐4,’ adapted to low elevations and to the more fertile soils at high elevations. F2 through F8 populations from this cross were grown at one low‐elevation and two high‐elevation locations. These populations were subjected to natural selection by bulking all plants to form the next generation, or to artificial selection by bulking only selected plants to form the next generation. Phenotypic differences for lint yield, plant height, and fruiting height among plants within the succeeding generations were greatest at Phoenix, next greatest at Safford, and least at El Paso. Therefore, selection of genotypicaliy productive plants was most effective at Phoenix, somewhat less effective at Safford, and the least effective at El Paso.Artificial‐ vs. natural‐selection cycles 0 (F2), 3, and 6 from each of the three locations were evaluated at all locations. The populations developed by artificial selection at a given location tended to yield more than naturally selected populations at that location, with the greatest advantage at Phoenix, next greatest at Safford, and no advantage at El Paso. Likewise, among locations, natural selection had the most influence on yield potential at Phoenix. In some years, the artificially selected populations from one location tended to yield well at the other locations, but no population yielded well at all‐three locations in both years. Selection for improvement of traits such as lint yield in the environment where the selected population will be utilized was effective. However, if the Pima improvement program were confined to one location, Phoenix appears to be the best of the three locations. At Phoenix, the magnified expression of productivity, and fruiting‐ and plant‐height differences would permit the selection of genotypes that would be productive at each elevation of the American Pima Belt.