Nuclear factor Y (NF-Y) transcription factors regulate the expression of target genes by specifically binding to the CCAAT element in the promoter region of target genes, and play a role in plant responses to abiotic stresses such as drought and salinity. Broomcorn millet exhibits strong tolerance to drought and barrenness, serving as an indispensable crop for saline-alkali and marginal lands. Systematic elucidation of the functions of the NF-Y gene family in broomcorn millet will provide a theoretical basis for enriching the genetic theory of crop stress tolerance and promoting drought-resistant breeding practices.

Genome data of broomcorn millet and bioinformatics approaches were employed to identify NF-Y family members, physicochemical properties, gene structures, construct phylogenetic trees, and predict conserved domains as well as cis-acting elements in promoters. Reverse Transcription Quantitative PCR (RT-qPCR) was used to determine the expression of these genes in different plant tissues, and the expression characteristics of PmNF-YA8 under drought stress were verified through transgenic functional validation in Arabidopsis thaliana.

Based on the proso millet reference genome, 33 PmNF-Y genes (including 11 PmNF-YA, 12 PmNF-YB, and 10 PmNF-YC) were identified, encoding proteins with lengths of 122-571 amino acids, isoelectric points of 4.74-10.19, and molecular weights of 13.58-59.17 kDa. Chromosomal localization analysis showed that these 33 genes (PmNF-YA1-PmNF-YA11, PmNF-YB1-PmNF-YB12, and PmNF-YC1-PmNF-YC10) were distributed across 15 chromosomes. Two types of drought stress-responsive elements were predicted in the promoter regions of PmNF-Y family members. A total of 3, 47, and 59 NF-Y orthologous gene pairs were identified between broomcorn millet and three other plant species (Arabidopsis thaliana, rice, and maize), respectively. RT-qPCR analysis revealed that PmNF-Y genes were widely expressed in tissues such as roots, stems, and leaves. The expression level of PmNF-YA8 was up-regulated in both XHS and NM9 varieties after drought stress. Transgenic Arabidopsis thaliana lines overexpressing PmNF-YA8 were generated, and phenotypic and physiological analyses showed that under 300 mmol·L^-1 mannitol stress, the average root length of transgenic plants (14.03 mm) was longer than that of wild-type (WT) plants (9.07 mm). Additionally, transgenic plants had a lower MDA increase range (55.23%-57.12%) compared with WT plants (100.78%), with SOD and POD activities increased by 24.28%-27.43% and 163.57%-341.33%, respectively, and Pro content (151.31-175.14 μg·g^-1) higher than that of WT plants (143.78 μg·g^-1).

A total of 33 NF-Y family members were identified in broomcorn millet. The PmNF-YA8 gene was cloned from broomcorn millet and heterologously transformed into Arabidopsis thaliana. Its heterologous expression enhanced the drought stress tolerance of Arabidopsis thaliana plants by regulating the expression of stress-related genes, thus making PmNF-YA8 a key candidate gene for modulating drought tolerance in broomcorn millet.

As an ancient minor grain crop, broomcorn millet ( Panicum miliaceum L. ) is abundant in germplasm. The construction of their DNA molecular identity based on fluorescent SSR markers would provide theoretical basis and molecular detection tool for digital management of resources.

Two hundred and thirty five broomcorn millet core accessions from China were used as experimental material, polymerase chain reaction were conducted several times using the broomcorn millet specific SSR markers which developed previously by the Broomcorn Millet Crop Molecular Breeding Research Group of the Agronomy College in Shanxi Agricultural University, core markers were obtained. With the given reference genome information of broomcorn millet, the core markers were mapped on chromosomes through BLAST sequence alignment. Fluorescence (FAM/HEX) was labeled on the 5' end of the SSR primer, the genotype of the material was given by capillary electrophoresis. Using binary coding means of expression, “0, 1” was written representing the presence or absence of amplified bands, and the discrimination of the material was detected by the software ID Analysis 4.0. Decimal (0-9) coding methods were used to calculate the size of the amplified fragments so as to obtain the character string molecular identity card of the accession. Genetic diversity, genetic clustering and principal component analysis were performed using the softwares Popgene, Powermarker, MEGA and NTSYS. The two-dimensional code DNA molecular identity card of the accession was given using the two-dimensional code online software (https://cli.im/).

PCR amplification results showed that all the 235 accessions could be separated by 7 fluorescent SSR markers (RYW3, RYW6, RYW11, RYW18, RYW37, RYW43 and RYW125) combined together. BLAST results showed that RYW18 and RYW37 were distributed on Chromosome 2, located at 0.60 cM and 0.80 cM, respectively. RYW125 is located on Chromosome 4 at 10.40 cM. RYW43 and RYW6 were distributed on Chromosome 5, located at 52.80 cM and 53.00 cM, respectively. RYW11 and RYW3 were located on Chromosome 6 at 2.10 cM and 20.70 cM, respectively. Genetic diversity analysis showed that 87 alleles were detected at 7 loci among all accessions, 3 (RYW11)-25 (RYW6) alleles were detected at each locus, with an average of 12.4286. Shannon diversity index (I) was detected and ranged from 0.2055 (RYW18) to 2.0587 (RYW6), with an average of 1.1398. The observed heterozygosity (Ho) was 0.0086 (RYW11)-0.9455 (RYW18). The expected observed heterozygosity (He) was 0.0795 (RYW18)-0.7469 (RYW11). Nei’s gene diversity index (Nei) was 0.0793 (RYW18)-0.7452 (RYW6). The polymorphism information content (PIC) was 0.0334 (RYW11)-0.8071 (RYW6), with an average of 0.5185. The results of cluster analysis and principal component analysis showed that 235 accessions were classified into 8 groups. The electrophoretic bands were number coding, and 7 marker combinations were used to construct the character string and two-dimensional code DNA molecular ID of all the accessions.

Two hundred and thirty five broomcorn millet core germplasms from China were used as material, polymerase chain reaction and capillary electrophoresis were conducted, 7 core SSR markers were screened. With the given reference genome information of broomcorn millet, the above markers were mapped on 4 chromosomes. Used the above SSR markers, genetic diversity analysis of all accessions was conducted and genetic diversity parameters were obtained. Based on Cluster analysis, all accessions were classified into 8 groups. Principal component analysis result resolved the deviation occured in Cluster analysis. According to the principle of most accessions were tell apart using the least markers, decimal (0-9) coding methods were used to calculate the size of the amplified fragments so as to obtain the character string molecular identity card of the accession. Combined the phenotype data with the above character string, two-dimensional code DNA molecular ID of all the accessions were developed.