Genetic Diversity and Classification of Chinese Elite Foxtail Millet [ Setaria italica (L.) P. Beauv.] Revealed by Acid-PAGE Prolamin

Arid and semi-arid regions of China account for more than half of the coun-try. Because of drought resistance and high nutritive value, elite foxtail millet (Setaria Italica (L.) P. Beauv.) is one of the most important cereal crops in China. Evaluation of germplasm and genetic diversity of foxtail millet is still in its infancy, but prolamin could play an important role as a protein marker. To investigate the genetic diversity and population structure of foxtail millet from different ecological zones of China, 90 accessions of foxtail millet were collected from three major ecological areas: North, Northwest, and Northeast China. The prolamin contents were examined by acid polyacrylamide gel electrophoresis (acid-PAGE). Five to twenty-two prolamin bands appeared in tested varieties, of which were polymorphic, so prolamin patterns of foxtail millet varieties can be used in variety identification and evaluation. Structure analysis identified six groups, which matches their pedigree information but not their geographic origins. This indicated a high degree (87.78%) of consistency with a phylogenetic classification based on SSR. The results showed prolamin banding patterns were an effective method for analyzing foxtail millet genetic variability.


Introduction
Insufficient water supply is a major issue in the world for crop production. Chi-other types of preferred varieties [6] have accumulated in long-term cultivation and domestication processes, yet basic research on foxtail millet lags major cereal crops such as wheat. Evaluation of germplasm and genetic diversity of foxtail millet is still in its infancy. The genetic diversity analysis of some foxtail millet pedigrees suggests potential new cultivars based on agronomic performance [7].
In agricultural studies have focused on morphology [7] [8], cytology, physiology, and biochemistry [9]. Morphological characters are affected by the environment, which leads to challenges in genetic identification. Biochemical markers, such as isozymes and proteins, are the product of gene expression, not all show the codominant inheritance, polymorphism of produce is limited, the close genetic relationship and genetic basis of complex material are difficult to identify [9]. With the development of molecular biology and genomics, the research and application of DNA molecular markers have rapidly developed. Molecular markers, such as amplified fragment length polymorphism (AFLP) [10], random amplified polymorphic DNA (RAPD) markers [11], and simple sequence repeat (SSR) markers [12] [13] have been used in genetic diversity research in foxtail millet.
However, these techniques have drawbacks. The sensitivity of RFLP to DNA polymorphism detection is not high, it requires large quantities of DNA and a DNA fragment as a probe, and employs radioisotope and nucleic acid hybridization technology which are neither safe nor easy to automate. The stability and repeatability of RAPD experiments are poor and very sensitive to reaction conditions, such as template and Mg2+ concentrations. AFLP requires high genomic purity and reaction conditions, and SSR detection relies on a series of standard primers with a high polymorphism that covers all chromosomes in the genome, and the detection and analysis depends on a large number of samples.
Knowledge of genetic diversity is used for efficient germplasm management and utilization, genetic fingerprinting, and genotype selection [14]. prolamin, a heterogeneous group of alcohol-soluble storage proteins, is encoded by highly conserved multigenic families. An original methodology for their electrophoretic separation is acid polyacrylamide gel electrophoresis (acid-PAGE) [15] that can be used to detect the complex polymorphisms of prolamins. As prolamin is genotype-specific, the entire process includes protein extraction, electrophoresis, and band analysis-simple, repeatable, relatively cheap, and independent of environmental variation [16] or stage of plant ontogenesis. Genetic polymorphisms G. X. Ma  have been used to evaluate genetic diversity in many plants, such as wheat [17], barley [18] [19], Leymus [20], tall fescue cultivars [21], triticale [22], vetches [16] [23] and rice [24].
Clustering results based on prolamin banding patterns and SSR analysis are not always in agreement [17], but both methods produced similar total genetic diversity results for Chinse wheat landrace [25]. The application of prolamin in foxtail millet varieties is not as advanced as in other crops, because of a limited heterogeneity in the genetic background [26] [27] and protein content is lower than other crops. As the foxtail millet germline source is not clear,   Table 1).

Protein Extraction and Electrophoresis
Electrophoresis of prolamin was performed based on Wrigley's method [28] with some modifications. For the analysis, 20 healthy seeds were randomly selected in each accession. prolamins were extracted from the individually milled seed by adding 200 μL sample extract solution (70% isopropanol, 15% sucrose) into 1.5 mL tubes (Eppendorf, Germany) that were then incubated at 220 rpm (60˚C) for 60 min. The extract was then centrifuged at 12000 r/min (4˚C) for 10 min. The supernatant was transferred into a new tube and 100 μL of methylene green solution (80% glycerin, 0.02% methyl green) was added for pre-staining.
The solution was heated in an oven at 60˚C for 30 min, during which it was taken out and shaken every 10 min, and then put in a thermostat at 4˚C. The

Statistical Analysis
To detect population genetic structure and assign individuals to subpopulations, the data obtained from acid-PAGE was scored based on the results of electrophoretic band spectra (Supplemental Table 2 [30], which uses a Bayesian approach to identify clusters based on a fit to the Hardy-Weinberg equilibrium model and linkage equilibrium. Ten independent runs for each number of subpopulations value (k), which ranged from 3 to 13, were performed after the admixture model with 100,000 replicates for burn-in and 100,000 replicates during analysis. The optimal subgroup (k) value was determined based on 1) likelihood plots of these models, 2) stability of grouping patterns across the ten runs, and 3) information about the materials used in the study. The output was exported into Structure Harvester [31] to determine the most likely number of K clusters (K = 6 was optimum for this analysis, Figure 1A using Evanno's ΔK method [31].
Results from 10 independent STRUCTURE runs for the most likely K were assessed with the software CLUMPP [32] and plotted using DISTRUCT [32].

Genetic Diversity in Foxtail Millet
The prolamin contents were examined by acid-PAGE. Analysis of variance showed that 5 to 22 prolamin bands (Supplemental Table 2, Figure 2) appeared in tested varieties, of which were polymorphic. The results indicated that the genetic diversity of the breeding materials used in this study was high and should be valuable for breeding application.

Population Structure
STRUCTURE analysis of the population structure of the 90 foxtail millet accessions showed that the most appropriate grouping was six subpopulations with a ΔK peak of 6 ( Figure 1A, Supplemental Table 1 Table 2).

Population Structure, Pedigree, and Geographic and Ecological Distributions
There was no tight association between structure and ecological group (summer or spring foxtail millet) (Supplemental Table 1) based on grouping results from structure analysis. No relationships among genetic diversity, geographic origin, ecological group (summer or spring foxtail millet) (Supplemental Table 1), and the genotypes were observed based on grouping results from structure analysis.
In each structure group, both summer and spring foxtail millet types were identified. However, the majority of accessions in pG1, pG2, pG3 and pG6 were the summer type and pG4, pG5 and pG6 were spring type. PG1 had the highest  (4.17%). Nine accessions were derived from WR1, and these accessions belonged to pG1 (4.55%), pG2 (10%), and pG3 (13.64%). Eight accessions were derived from shi181-5, and these accessions belonged to pG1 (4.55%), pG2 (10%), and pG3 (27.27%). These data indicated that Riben60ri and Tulong were the major germplasm of three ecological areas, pG1, pG2, and pG3, mainly derived from Yugu1 which is a derivative of Riben60ri (Supplemental Table 1). They also showed that pG1, pG2, and pG3 were close affinities, pG4 and G6 were close affinities, and Riben60ri was the source of these five groups; pG5 is relatively independent of the other groups.

The Consistency between the SSR and A-PAGE Prolamins Methods
The group of 90 foxtail millet accessions was both divided into six subpopulations by the SSR method and the A-PAGE prolamins method. Consistency analysis indicated that the accordant rate reached 87.8% (

Prolamin and Genetic Diversity
Prolamin is the main storage protein of plant seeds, a gene expression product at a specific stage of seed development. The number and combination of its electrophoresis bands are controlled by genes, minimally affected by environmental factors, and thus can reflect the differences in gene coding sites of different crop varieties [33] [34]. Therefore, the analysis of plant varieties by prolamin can reveal specific genetic differences among varieties ( Figure 2). The application of prolamin to the study of plant genetic resources has the advantages of simplicity, convenience, and accuracy. Lang et al. [35] found the glycolic homology degree in wheat generally reflects the distance of the genetic relationship among the main popularized wheat varieties in China and can be further used to guide the selection of parents.
In this research, a high level of polymorphism was identified for prolamin across the 90 accessions. It showed that 5 to 22 prolamin bands appeared in tested varieties. Structure analysis identified six groups, which matches with their pedigree information, but not with their geographic origins. The grouping consistency was 87.78% between the SSR method and the acid-PAGE prolamin method [13]. This might be due to highly diverse accessions which were collected from three major foxtail millet ecological regions.

Genetic Diversity and Population Structure of Chinese Foxtail Millet
Because it is genotype-specific, simple, repeatable, cheap, and independent of environmental variation nature, prolamin has frequently been used as a tool to examine the dynamics of genetic differentiation in a population. which matches with their germplasm information and SSR method grouping [13]. Basic germplasm and parent-of-origin analysis (Supplemental Table 1) indicated that there were not associated with a particular ecological environment,

The Implications of Genetic Improvement of Foxtail Millet
Insufficient water resources seriously affected agricultural production. The study indicated that when breeding foxtail millet, prolamin analysis was simple, accurate, and efficient, which will improve breeding project design and selection accuracy. In past, male-sterile parents and geographical unrelated materials were used for hybrid breeding. The results from this study indicated that accessions separated by great geographical distance may not necessarily be genetically distant. Classification of 90 accessions into six groups matched with their genetic relatedness and thus provides a good reference for designing crosses to improve hybrid-breeding efficiency. Accessions in pG4 and pG5 have unique geographic origins and pedigrees that are different from other groups; therefore, cross accessions among pG4, pG5 and other groups are more likely to obtain expected recombination for developing both conventional and hybrid cultivars. Further research may be needed to evaluate the combining results among groups to determine the combinations of accessions from different groups with the best heterosis. Although accessions in pG6 had the Riben60ri consanguinity, they have the most diverse origins and the greatest variation within the group. Accessions in pG5 were an independent group with five other groups, and crosses between accessions within pG5 and the other five groups may generate useful heterosis. Thus, further research on accessions may facilitate effective the use of germplasm in this group.
The acid-PAGE prolamin method is reliable, and the grouping consistency was 87.78% between the SSR method and the acid-PAGE method. The classification is closer to the germline source of foxtail millet. Furthermore, the acid-PAGE prolamin method is not needed for designing a large number of specific PCR primers or high-quality genomic DNA, and it is a simpler operation with a lower cost. In sum, it is an effective method for the breeding, identification, and evaluation of new varieties of foxtail millet.

Conclusion
In general, the acid-PAGE prolamin method is reliable and advantageous in breeding, identification, and evaluation of new varieties of foxtail millet, which has highly consistent with SSR method in group classification and is closer to the germline source of foxtail millet. In addition, the acid-PAGE prolamin method is more convenient than SSR method, don't have to design or select a large number of specific PCR primers or high-quality genomic DNA.