A total of 100 foxtail millet accessions were collected in 2016 from all the breeding programs in the North China summer foxtail millet region and Northwest and Northeast China spring foxtail millet regions. Those materials were initially evaluated for agronomically and economically-important traits and high yield potential at the breeding observation nursery of the Institute of Foxtail Millet Crops, Shijiazhuang, Hebei, China. After initial screening, 90 accessions were selected as basic breeding materials based on their yield and adaptation performance. Most of these materials were from eight foxtail millet growing provinces: Hebei, Henan, Shandong, Liaoning, Jilin, Shanxi, Inner Mongolia, Shaanxi (Supplemental Table 1).

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 A-PAGE gel formula was: 12% acrylamide, 0.4% methylene diacrylamide, 2% glacial acetic acid, 6% urea, 0.1% ascorbic acid, 0.075% glycine, and 0.004% ferrous sulfate; 2 μL hydrogen peroxide catalyst was used to prepare the gel with a thickness of 1 mm. A glycolic polymorphism was detected in a 15 μL reaction system with 0.4% glacial acetic acid and 0.04% glycine as the electrode solution at 500 V constant pressure and 15˚C for 80 min. After electrophoresis, acid-PAGE gel was stained with a 0.1% Coomassie Bright Blue R-250, 40% isopropyl alcohol, and 10% glacial acetic acid solution for 25 min. It was rinsed and decolorized with running water then photographs were taken with camera (Nikon, WJHH).

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) for the presence or absence of the bands and entered as a binary data matrix. Population structure was determined by STRUCTURE software v2.3.4 [29] [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].

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.

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). The group of foxtail millet accessions was divided into six subpopulations by the prolamin method (Figure 1B). Among the six subpopulations, the level of genetic diversity within pG6 (group 6 by the prolamin method) was the highest (26.67%), followed by pG1 (24.44%), pG3 (24.44), pG2 (11.11%), pG4 (7.78%), and pG5 (5.56%) (Table 1, Supplemental Table 2).

PG1 was collected from the summer foxtail millet region of central and southern Hebei Province. Riben60ri and 60rihuancang can be found in the pedigrees of most of these accessions. Nine accessions have definite ancestries, thirteen accessions did not. PG2 consisted of most accessions from central and southern Hebei Province and Liaoning Province. Riben60ri and Tulong can be found in the pedigrees of most of these accessions. Five accessions had definite ancestries and five accessions did not. PG3 was collected from Hebei Province. Most of these were the derivatives Riben60ri and Tulong, such as Yugu1 and its derivatives. Riben60ri is a Japanese landrace. Eighteen accessions had definite ancestries and four accessions did not. Riben60ri and Tulong can be found in the pedigrees of most of these accessions. PG4 was from Shanxi Province and was spring foxtail millet. Three can be traced back to founder-Moligu, two can be traced back to founder-Qitouhuang, one can be traced back to founder-Hainangu and Qinyuanmujizui. Six accessions had definite ancestries and one accession did not. The phylogeny of accessions was ambiguous. PG5 was collected from Shanxi Province, and two can be traced back to founder-Shanxidabaigu and two breeding materials that did not have clear pedigree information. One was breeding material from Hebei Province without clear pedigree information. Two accessions had common ancestors and three accessions did not. PG6 had diverse pedigrees and geographic origins. They include 8 accessions from Liaoning, 6 from Hebei, 3 from Shanxi, 3 from Neimenggu, 2 from Jilin, 1 from Henan, and 1 from America. Fifteen accessions had definite ancestries and nine accessions were unclear.

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 proportion of summer type (90.91%) and pG4 had the highest proportion of spring type (85.71%). All six groups consisted of accessions from different ecological regions, with pG1 and pG3 having the most accessions (81.82% and 77.24%, respectively) from the central and south Hebei Province, and accessions in pG4 and pG5 (84.6% and 80%) were mainly from central and south Shanxi Province. Accessions in pG2 consisted of most accessions from the central and south Hebei Province and Liaoning Province. Accessions in pG6 were from seven different regions with diverse ecological conditions. Only a small proportion of accessions (<35%) can be traced to each region.

Basic germplasm, parent-of-origin analysis (Supplemental Table 1), and pedigree analysis (Figure 3, Supplemental Table 3) indicated the following. Thirty-three accessions were derived from Riben60ri, and these accessions belonged to pG1 (proportion, 40.91%), pG2 (50%), pG3 (63.64%), pG4 (14.29%), pG6 (16.67%). Twenty-three accessions were derived from Tulong, and these accessions belonged to pG1 (13.64%), pG2 (40%), pG3 (63.64%), pG4 (14.29%), pG6 (4.17%). Thirteen accessions were derived from 60rihuancang, and these accessions belonged to pG1 (22.73%), pG2 (10%), pG3 (31.82%). Nine accessions were derived from Qinggouweicao, and these accessions belonged to pG1 (4.55%), pG2 (10%), pG3 (31.82%). Nine accessions were derived from Mihuanggu, and these accessions belonged to pG1 (22.73%), pG2 (10%), and pG3 (13.64%). Six accessions were derived from Xiaoliugen, and these accessions belonged to pG1 (4.55%), pG2 (10%), pG3 (18.18%). Five accessions were derived from Shuangguayin, and these accessions belonged to pG4 (14.29%) and pG6 (16.67%). Nineteen accessions were derived from Yugu1, and these accessions belonged to pG1 (22.73%), pG2 (30%), pG3 (40.91%), pG4 (14.29%), and pG6 (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 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% (Table 2) between the two methods. The accordant rates of groups 1-6 were 87.5%, 100.0%, 71.9%, 100%, 100%, and 100%.

In sG1 (group 1 by the SSR method), Jigu25 and Gu10A were the accessions with inconsistent groupings. The parent-of-origin of Jigu25 is WR1 × Shi181-5, and the main germplasm base of Jigu25 are Riben60ri, 60rihuancang, Tulong, and Qinggouweicao. The main germplasm base rates (Table 1, Table 2, Figure 3) of sG1 are Riben60ri (68.75%), Tulong (68.75%), and Qinggouweicao (43.75%); Riben60ri (50%), Tulong (40%), and Qinggouweicao (10%) in pG2, therefore Jigu25 belonged to sG1. Data indicated that the SSR method is more robust. In sG3, the parent-of-origin of Heng968, Datong14, Datong28, and Datong27 are Lugu5 (7112 × (male-sterile lines × Riben60ri) × Lugu2) × 91101, Xiannong3 × Jingu9 (Shanxidabaigu), (Huangruangu × Zhangchunyi) F2 × Jingu9

(Shanxidabaigu) and (73-50 × Zao1) × Yi17, respectively. The main germplasm bases of Heng968, Datong14, and Datong28 are (Riben60ri, 60rihuancang), (Xiannong3, Shanxidabaigu) and (Huangruangu, Zhangchunyi, Shanxidabaigu), respectively. The main germplasm bases of pG5 are Shanxidabaigu (40%), Xainnong3 (20%), Huangruangu (20%) and Zhangchunyi (20%), whereas they were Riben60ri (15.63%); Shanxidabaigu (6.25%); 60rihuancang (3.13%), Huangruangu (3.13%), Xainnong3 (3.13%), and Zhangchunyi (3.13%) in sG3. They showed that Heng968 belonged to pG1, and Datong14, Datong28, and Datong27 belonged to pG5 by the main germplasm base identified by the two methods. These data indicated the A-PAGE prolamins method is closer to the pedigree analysis. Because the main germplasm bases of Gu10A, Tiedalihuang, Jinzhougu14, shi207191, Datong27, Datong30, and Jigu28 are not distinct, it is difficult to judge which of the two methods is preferred.

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.

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. For example, structure analysis can estimate the number of subpopulations and the genetic relatedness among accessions. Cluster analysis can also group the assayed accessions into different groups. In this study, structure analysis identified six groups, 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, and the origins of accessions would differentiate among accessions if they are all landraces.

The extensive exchange of genetic resources breaks barriers among the three major ecological areas in China. Following germplasm being introduced into a new ecological environment, it goes through continuous domestication and hybridization with local breeding lines and eventually results in new progenies. All the groups contain both summer and spring types. Riben60ri was introduced to the North China summer foxtail millet region and became major parent cultivars in this region. After that, Riben60ri and its derivatives were usually hybridized with a locally adapted parent. Breeders from different regions selected different genotypes according to their preferences. Thus, it is expected that many new cultivars are interrelated and some geographically distant accessions may also be genetically related.

Structure analysis mainly was based on the genetic relatedness among accessions. Thus, the grouping based on these two approaches may not necessarily match with their geographic origin and ecotype. In pG4, most of the accessions derived from Qitouhuang and Moligu, which originated from central and southern Shanxi Province. Most of the accessions in pG1, pG2, and pG3 were selected from crosses between Riben60ri and locally adapted materials. For example, Qingdaolao and Yugu1 were derived by crossing Riben60ri to the Chinese landrace Mihuanggu derivative Xinnong724 and Japanese landrace Tulong. Further, Yugu1 was crossed to other locally adapted materials, and cultivars were selected for their adaptation to different growth areas, which generated pG1, pG2, and pG3. Qingdaolao derivatives mainly contributed to accessions in pG1, while accessions in pG3 were mainly transition types between Yugu1 derivatives and Qingdaolao derivatives. These included Yugu1 and Qingdaolao derivatives and progenies from the cross between the two cultivars. PG5 was mainly selected from locally adapted materials, including Shanxidabaigu, which originated from central and southern Shanxi Province. PG6 was scattered in several regions, but most accessions were from Northeast China and the nearby Northwest spring foxtail millet region.

Insufficient water resources seriously affected agricultural production. Foxtail millet is a highly drought-tolerant crop. The research of genetic diversity and the population structure of foxtail millet germplasm resources will accelerate the effective utilization of the limited resources for breeding. The foxtail millet materials used in this study were derived from the main foxtail millet-producing area in China. Many accessions (38.89%) had no pedigree records; this study elucidated the genetic relationship between these unknown accessions and those with a known pedigree to determine origins.

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.