Shan et al BMC Genomics (2020) 21:96 https://doi.org/10.1186/s12864-020-6479-2 RESEARCH ARTICLE Open Access Genome-wide analysis of the NAC transcription factor family in broomcorn millet (Panicum miliaceum L.) and expression analysis under drought stress Zhongying Shan1,2, Yanmiao Jiang1,3, Haiquan Li1,3, Jinjie Guo1,3, Ming Dong1,3, Jianan Zhang1,3 and Guoqing Liu1,3* Abstract Background: Broomcorn millet is a drought-tolerant cereal that is widely cultivated in the semiarid regions of Asia, Europe, and other continents; however, the mechanisms underlying its drought-tolerance are poorly understood The NAM, ATAF1/2, and CUC2 (NAC) transcription factors form a large plant-specific gene family that is involved in the regulation of tissue development and abiotic stress To date, NAC transcription factors have not been systematically researched in broomcorn millet Results: In the present study, a total of 180 NAC (PmNAC) genes were identified from the broomcorn millet genome and named uniformly according to their chromosomal distribution Phylogenetic analysis demonstrated that the PmNACs clustered into 12 subgroups, including the broomcorn millet-specific subgroup Pm_NAC Gene structure and protein motif analyses indicated that closely clustered PmNAC genes were relatively conserved within each subgroup, while genome mapping analysis revealed that the PmNAC genes were unevenly distributed on broomcorn millet chromosomes Transcriptome analysis revealed that the PmNAC genes differed greatly in expression in various tissues and under different drought stress durations The expression of 10 selected genes under drought stress was analyzed using quantitative real-time PCR Conclusion: In this study, 180 NAC genes were identified in broomcorn millet, and their phylogenetic relationships, gene structures, protein motifs, chromosomal distribution, duplication, expression patterns in different tissues, and responses to drought stress were studied These results will be useful for the further study of the functional characteristics of PmNAC genes, particularly with regards to drought resistance Keywords: Broomcorn millet, NAC genes, Gene expression, Drought stress Background Transcription factors (TFs) play an important role in controlling a variety of vital growth and development processes, such as signal transduction, cellular morphogenesis, and response to environmental stressors, during the growth and development of plants [1, 2] The NAC family is one of the largest groups of plant-specific TFs [3] The term NAC derives from three proteins: no * Correspondence: guoqingliu@hotmail.com Institute of Millet Crops, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050035, Hebei, China Key Laboratory of Minor Crops in Hebei, Shijiazhuang 050035, Hebei, China Full list of author information is available at the end of the article apical meristem (NAM), Arabidopsis transcription activation factor (ATAF)1/2, and cup-shaped cotyledon (CUC2) Typical NAC proteins exhibit a highly conversed N-terminal DNA-binding domain containing ~ 150 amino acids, which is divided into five subdomains (A–E) [4] NAC TFs play important regulatory roles in various aspects of plant growth, development, and adaptation to the environment, including in shoot apical meristem formation [5], cell division and expansion [6, 7] nutrient remobilization [8], flower formation [9], lateral root development [10, 11], leaf senescence [12–16], secondary © The Author(s) 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Shan et al BMC Genomics (2020) 21:96 cell wall biosynthesis [3, 17–20], fiber development [19, 21, 22], fruit ripening [23, 24], seed development [25], response to pathogen infection [26–30], and abiotic stress tolerance [31–33] Many studies have confirmed that many NAC genes play crucial roles in the regulation of plant tolerance to drought Overexpression of CarNAC4, a chickpea TF, enhanced drought tolerance in Arabidopsis thaliana [34] TaNAC29, a NAC TF from wheat, enhanced salt and drought tolerance in transgenic A thaliana [35] Overexpression of the Miscanthus lutarioriparius NAC gene MlNAC5 conferred enhanced drought and cold tolerance in A thaliana [36] NAC genes have also been found to increase tolerance to drought stress in rice For instance, drought resistance in rice was enhanced by the overexpression of SNAC1 [37], ONAC022 [38], and ONAC045 [39] OsNAC10-overexpressing rice plants showed an increased grain yield under both normal and drought conditions [40] Therefore, NAC family genes are crucial regulators of plant tolerance to drought Broomcorn millet (Panicum miliaceum L.), also called proso millet, common millet, and hog millet, is a shortseason, drought-tolerant, and barren-tolerant cereal that is widely cultivated in the semiarid regions of Asia, Europe, and other continents The grains of broomcorn millet not only have high nutritional value, containing abundant proteins, starch, and a variety of vitamins and minerals, but also have medicinal value and are used in millet wine and other products The NAC gene family has been widely studied in many species, such as A thaliana [41], rice [41], wheat [42, 43], tartary buckwheat [44], maize [45, 46], foxtail millet [47], soybean [48], potato [49], Chinese cabbage [50], pepper [51], cassava [52], melon [53], physic nut [54], apple [55], and pigeon pea [56] However, no systematic study of the NAC family in broomcorn millet is available The genome of broomcorn millet was recently published, providing an important resource for further molecular research in this species [57] In addition, large RNA sequencing (RNA-seq) expression data from different tissues are also available [58] Based on these data, NAC gene family members in broomcorn millet were identified in the present study A phylogenetic tree was constructed, gene exon/intron and conversed motif structural analyses were performed, chromosomal location and synteny analysis were carried out, and the tissue-specific expression patterns of NAC genes in broomcorn millet were surveyed In addition, the differential expression of NAC genes was analyzed in broomcorn millet under drought conditions using transcriptomics and quantitative real-time PCR (qRT-PCR) This research is the first to detail the NAC gene family in broomcorn millet, which may help elucidate the molecular mechanisms underlying drought stress responses in this important food crop Page of 13 Results Identification and phylogenetic analysis of the PmNAC genes in broomcorn millet A total of 180 PmNAC genes were identified and were named PmNAC001 to PmNAC180 according to their chromosome location These were confirmed and used for further analyses (Additional file 1: Table S1) All the PmNAC proteins contained a conserved NAC domain (PF01849) or NAM domain (PF02365) Phylogenetic analysis of the NAC proteins from broomcorn millet and Arabidopsis was conducted to explore the evolutionary relationships among broomcorn millet NAC proteins The results demonstrated that the PmNAC proteins could be divided into 12 subgroups according to clade and the classification from Arabidopsis, including a broomcorn millet-specific subgroup named Pm_NAC (Fig 1, 2a) In our analysis, no NAC members from the subgroups ANAC001, SEUN5, ANAC3, and ANAC011 were identified Subgroup ANAC063 contained the most PmNAC proteins, namely 52, while subgroup TIP contained only two PmNAC proteins Protein properties and sequence analyses The protein properties were analysed, and the results are summarized in Additional file 1: Table S2 The length and molecular mass of the PmNAC proteins varied greatly, with lengths ranging from 67 to 1435 amino acids (aa) and molecular weights (MWs) ranging from 7.53 to 162.48 kDa The theoretical isoelectric point (pI) varied greatly from 4.13 to 11.38 Most PmNAC proteins (149 out of 180) were considered unstable due to the instability index being higher than 40 The PmNAC proteins contained a predicted aliphatic index ranging from 45.59 to 101.24 All PmNAC proteins were predicted to be hydrophilic due to the relatively low average hydropathy (GRAVY) value (< 0), with the exception of PmNAC005 and PmNAC165 Gene structural diversity is an important component of gene family evolution and further supports phylogenetic groupings [59] Gene structure analysis indicated that the number of introns of PmNAC genes varied from to 14 (Fig 2b), with 44 PmNAC genes lacking introns Most PmNAC genes (134 out of 180) contained one to six introns PmNAC41 had the highest number of introns (14), followed by PmNAC148 with 13 introns Generally, closely clustered PmNAC genes in the phylogenetic tree exhibited similar exon-intron structures Most PmNAC genes had no introns in subgroup ANAC063, and there was an average of four introns in subgroup ONAC003 To further investigate the structural features of broomcorn millet NACs, the conserved motif distributions were analysed A total of 20 conserved motifs (referred to as motifs 1–20) were identified by MEME, with Shan et al BMC Genomics (2020) 21:96 Page of 13 ANAC01 100 100 51 89 52 100 d ON AC 003 99 ifie 20 00 AC AN ss 02 22 AC C0 74 AN NA C0 C06 A A N A 12 A mN AC 15 P mN AC 10 P mN AC 175 P mN AC 46 P mN AC0 91 10 P mN C0 57 P NA 043 98 Pm AC 066 AN AC C07 94 AN NA C156 Pm NA 12 0 10 Pm AC0 33 AN AC0 079 0 91 AN NAC 70 76 Pm NAC0 22 Pm NAC1 99 Pm NAC14 0 Pm C015 00 66 100 ANA C070 ANA AC102 100 PmN AC177 99 PmN 030 100 ANAC 101 ANAC 007 99 ANAC 88 100 ANAC02 66 PmNAC0 43 97 100 96 PmNAC1 PmNAC008 83 100 PmNAC042 99 ANAC105 99 99 ANAC037 ANAC076 100 PmNAC069 61 PmNAC126 100 100 PmNAC076 97 PmNAC157 100 PmNAC044 PmNAC056 100 PmNAC0 78 58 PmNAC0 86 100 PmNAC 62 100 85 PmNA 083 PmNA C074 100 PmN C125 100 100 PmN AC072 PmN AC123 100 PmN AC073 PmN AC071 89 100 Pm AC12 Pm NAC13 100 10 Pm NAC1 87 Pm NAC0 69 Pm NAC 47 Pm NAC 097 99 AN NAC 137 58 81 AN AC0 168 10 10 AN AC 63 Pm AC 093 00 N Pm A 064 10 P NA C0 P mN C1 57 PmmNA AC0 42 P N C 15 P mN AC 117 P mN AC 05 P mN AC 08 Pm mN AC 12 Pm N AC 05 N AC 08 AC 05 36 02 AC N C06 00 Pm NA AC C03 A mN A 03 P mN AC 02 P mN AC 14 P N C 04 PmmNA AC1 78 P mN AC1 38 P mN C1 66 10 P NA C1 10 Pm NA C14 Pm NA C15 10 Pm NA C020 Pm NA 140 99 Pm NAC 164 Pm NAC 021 0 Pm NAC 39 10 10 Pm NAC1 03 Pm NAC1 100 Pm NAC16 100 Pm AC01 100 99 100 PmN AC038 PmN AC096 90 PmN AC130 100 PmN AC029 PmN C030 100 PmNA C179 PmNA 031 100 PmNAC 11 PmNAC1 80 100 100 99 PmNAC1 PmNAC110 PmNAC154 PmNAC174 PmNAC005 PmNAC028 PmNAC039 RN TE cla 59 C22 ONA Un 84 A A NA AN NA C0 C Pm AN AC 02 Pm NA AC 024 NA C0 023 A Pm NA C0 95 61 Pm NA C0 48 10 N C 75 AN AC 159 10 AN AC 080 A AC 09 Pm NAC 010 99 10 Pm NAC 073 Pm NAC 089 75 Pm NAC 052 Pm NAC0 07 10 59 Pm NAC1 24 N 100 10 Pm AC0 05 99 N 61 54 Pm AC15 NAC PmN 145 A 100 100 ANA C067 98 ANA C085 99 C ANA 044 100 78 PmN C008 A PmNA C158 100 C 100 PmNA 077 93 100 C 99 PmNAC 131 100 065 PmNAC0 41 PmNAC0 45 96 PmNAC009 100 PmNAC037 88 100 PmNAC004 PmNAC001 PmNAC043 100 NAC2 SEU N5 TIP AN AC AT AF P AN Pm mN AC A AN NAC C1 25 A Pm N AC 01 Pm NA AC0 056 Pm NA C1 18 10 Pm NA C0 20 Pm NA C03 18 10 Pm NA C00 73 10 Pm NA C08 NA C17 98 10 AN C1 0 10 AN AC1 35 0 AN AC0 Pm AC 03 N 10 AN AC08 10 AN AC00 A AN C055 A 79 60 87 64 ANA C019 100 ANA C072 ANA C083 C ANA C 100 ANA 091 C 100 ANA 062 89 PmNA C014 98 C118 PmNA C01 100 ANAC01 ANAC01 100 100 ANAC01 PmNAC116 90 100 PmNAC014 PmNAC061 89 PmNAC133 100 100 PmNAC171 PmNAC134 97 ANAC042 97 ANAC094 100 PmNAC119 100 100 ANAC009 PmNAC161 82 95 100 PmNAC0 03 AC AN 100 ANAC03 00 03 C ANA C176 100 PmNA C060 A PmN C109 100 A PmN AC027 100 PmN AC097 AN C104 95 ANA C149 NA Pm AC12 N 99 Pm AC1 84 97 N Pm NAC0 06 A AC0 89 10 73 AN AC0967 10 N 82 Pm NAC 058 93 0 Pm NAC 090 Pm NACC061 A A 00 10 AN AC 004 00 AN AC 048 AN AC 00 AN AC 00 AN AC 04 AN NAC C06 68 A NA C0 67 A NA C0 95 A NA C0 A NA A C1 NA 100 ANAC100 100 ANAC080 53 ANAC079 96 ANAC020 76 ANAC028 100 ANAC057 ANAC08 99 100 ANAC04 ANAC09 07 56 69 87 100 ANAC 011 ANAC 052 ANAC 0 C051 ANA 050 56 C 99 ANA C077 100 ANA C078 98 ANA C053 100 10 ANA C172 99 NA 98 Pm AC0 32 N 87 Pm NAC1 63 Pm NAC0103 10 Pm NAC 082 A 10 C A 10 AN C1 NA 05 10 Pm NACC108 88 Pm NA C02 Pm NA C11 10 Pm NA C01 40 Pm NA C0 06 Pm NA AC0 093 Pm N C 162 Pm mNA AC 019 P mN AC 04 P mN AC 02 P AN AC AN P NA ANAC059 ANAC092 PmNAC007 ANAC046 100 100 ANAC087 PmNAC0 100 PmNAC1 68 84 PmNAC 28 PmNA 147 51 PmNA C150 PmN C163 PmN AC051 95 100 PmN AC050 10 PmN AC092 100 PmN AC017 Pm AC04 100 AN NAC09 AN AC038 10 Pm AC03 Pm NAC 67 N A N AC 13 10 AN AC0 115 72 AN AC0 40 AN AC0 60 83 93 Pm AC 89 95 Pm NA 058 10 A NA C0 10 PmNAC C1 99 A N 03 73 A NA AC P NA C0 078 Pm mN C09 54 P N AC P mN A Pm mN AC C16 81 N AC 15 AC 11 32 NAM C NA Pm_NAC Fig Phylogenetic analysis of NAC proteins of broomcorn millet and Arabidopsis The phylogenetic tree was constructed using the neighbourjoining method in MEGA-X more motifs located within the N-terminal region than within the C-terminal region (Fig 2c) The features of these protein motifs are listed in Additional file 1: Table S3 In this analysis, most of the closely related members in the phylogenetic tree showed common motifs in terms of alignment and position, which suggested that the NAC members that clustered along the same branch may possess similar biological functions Chromosomal location and synteny analysis of PmNAC genes All the PmNAC genes were unevenly distributed on broomcorn millet chromosomes, except PmNAC178– 180 (Fig 3) Chromosome (Chr5) contained the highest number of PmNAC genes (n = 17), followed by Chr8 (n = 15) and then Chr3, which had 14 members In addition, 13, 12, and 11 PmNAC genes were detected on Chr4, Chr6, and Chr12, respectively Ten PmNAC genes Shan et al BMC Genomics (2020) 21:96 Page of 13 Phylogenetic Tree 96 100 84 92 100 54 100 98 100 100 100 100 100 100 ANAC063 100 98 100 100 100 84 100 100 100 89 100 100 100 100 82 100 85 100 74 100 57 100 59 100 99 100 99 99 100 100 100 100 ONAC003 82 100 100 100 93 100 100 100 80 95 100 100 100 100 100 Pm_NAC 100 85 100 100 100 100 52 99 100 61 100 96 NAC2 ONAC22 TERN 100 97 86 100 100 50 100 78 100 100 100 100 93 100 100 ATAF 98 100 67 63 72 100 NAP 100 100 50 100 99 NAC2 100 86 100 99 93 100 63 100 100 OsNAC7 55 100 99 100 55 100 92 98 100 100 TIP 76 100 100 NAC1 96 91 100 100 51 100 57 68 100 100 NAM 100 100 100 55 66 100 56 78 99 Gene Structure Motif Pattern PmNAC054 PmNAC088 PmNAC121 PmNAC036 PmNAC055 PmNAC087 PmNAC053 PmNAC025 PmNAC144 PmNAC151 PmNAC002 PmNAC034 PmNAC035 PmNAC104 PmNAC178 PmNAC138 PmNAC166 PmNAC020 PmNAC140 PmNAC164 PmNAC021 PmNAC139 PmNAC103 PmNAC165 PmNAC022 PmNAC146 PmNAC057 PmNAC142 PmNAC015 PmNAC117 PmNAC047 PmNAC097 PmNAC137 PmNAC168 PmNAC136 PmNAC169 PmNAC071 PmNAC124 PmNAC073 PmNAC072 PmNAC123 PmNAC074 PmNAC125 PmNAC062 PmNAC083 PmNAC056 PmNAC086 PmNAC069 PmNAC126 PmNAC076 PmNAC157 PmNAC044 PmNAC024 PmNAC107 PmNAC052 PmNAC089 PmNAC080 PmNAC159 PmNAC023 PmNAC105 PmNAC048 PmNAC095 PmNAC145 PmNAC152 PmNAC067 PmNAC065 PmNAC131 PmNAC077 PmNAC158 PmNAC041 PmNAC010 PmNAC038 PmNAC096 PmNAC130 PmNAC110 PmNAC029 PmNAC030 PmNAC179 PmNAC031 PmNAC111 PmNAC180 PmNAC005 PmNAC154 PmNAC028 PmNAC039 PmNAC043 PmNAC001 PmNAC004 PmNAC037 PmNAC009 PmNAC045 PmNAC090 PmNAC058 PmNAC167 PmNAC127 PmNAC149 PmNAC106 PmNAC119 PmNAC082 PmNAC161 PmNAC176 PmNAC060 PmNAC027 PmNAC109 PmNAC134 PmNAC171 PmNAC133 PmNAC061 PmNAC014 PmNAC116 PmNAC003 PmNAC033 PmNAC018 PmNAC120 PmNAC085 PmNAC084 PmNAC135 PmNAC170 PmNAC012 PmNAC114 PmNAC019 PmNAC162 PmNAC093 PmNAC006 PmNAC040 PmNAC011 PmNAC113 PmNAC026 PmNAC108 PmNAC059 PmNAC141 PmNAC063 PmNAC132 PmNAC098 PmNAC172 PmNAC008 PmNAC042 PmNAC066 PmNAC143 PmNAC102 PmNAC177 PmNAC075 PmNAC156 PmNAC079 PmNAC070 PmNAC122 PmNAC148 PmNAC046 PmNAC091 PmNAC016 PmNAC118 PmNAC163 PmNAC174 PmNAC081 PmNAC160 PmNAC100 PmNAC155 PmNAC032 PmNAC112 PmNAC064 PmNAC129 PmNAC153 PmNAC101 PmNAC175 PmNAC013 PmNAC115 PmNAC078 PmNAC099 PmNAC173 PmNAC068 PmNAC128 PmNAC147 PmNAC150 PmNAC007 PmNAC051 PmNAC050 PmNAC092 PmNAC017 PmNAC049 PmNAC094 Intron Fig (See legend on next page.) Motif Motif Motif Motif Motif Motif Motif Motif 14 Motif 11 Motif 12 Motif 10 Motif 17 Motif 15 Motif 19 Motif Motif Motif 18 Motif 20 Motif 16 Motif 13 CDS 100 200 300 400 500 600 700 800 900 1000 1100 200 400 600 800 1000 1200 1400 1600 Shan et al BMC Genomics (2020) 21:96 Page of 13 (See figure on previous page.) Fig Phylogenetic relationships, gene structure, and architecture of conserved protein motifs in NAC genes from broomcorn millet a A phylogenetic tree was constructed by MEGA-X using the NJ method b Structures of the 180 putative broomcorn millet NAC genes Light green boxes indicate exons and black lines indicate introns c Motif distribution of NAC proteins Different motifs are indicated by different colours for motifs 1–20 The sequence information for each motif is provided in Additional file 1: Table S3 were found on each of Chr1, Chr10, and Chr17 Nine genes were located on Chr11 and seven NAC genes were located on Chr15 Eight PmNAC genes were distributed on each of Chr2, Chr7, Chr13, and Chr14, and six PmNAC genes were found on both Chr16 and Chr18 There were only five PmNAC genes on Chr9 Chr1 Mb PmNAC001 PmNAC003 PmNAC002 PmNAC012 PmNAC004 16 Mb Chr2 PmNAC011 PmNAC013 PmNAC015 PmNAC017 PmNAC014 PmNAC016 To identify the duplication events in PmNAC genes, a collinearity analysis was performed using MCScanX software There were 84 pairs of segmentally duplicated PmNAC genes (Fig 4) and five groups of tandem duplicated PmNAC genes (Figs 3, 4; PmNAC029/030/031, PmNAC053/054/055, PmNAC087/088, PmNAC136/137, Chr3 PmNAC019 PmNAC021 PmNAC023 PmNAC025 PmNAC027 PmNAC020 PmNAC022 PmNAC024 Chr4 PmNAC033 PmNAC035 PmNAC034 PmNAC036 Chr5 PmNAC046 PmNAC048 PmNAC050 PmNAC037 PmNAC047 PmNAC049 PmNAC051 PmNAC038 PmNAC026 PmNAC028 Chr6 PmNAC063 PmNAC065 PmNAC067 PmNAC069 PmNAC064 PmNAC066 PmNAC068 32 Mb 48 Mb PmNAC018 PmNAC005 PmNAC007 PmNAC006 64 Mb PmNAC009 PmNAC008 PmNAC010 Chr7 Mb Chr8 PmNAC083 PmNAC085 PmNAC075 PmNAC076 PmNAC087 PmNAC089 PmNAC029 PmNAC031 PmNAC030 PmNAC032 Chr9 PmNAC098 PmNAC084 PmNAC086 PmNAC088 PmNAC099 16 Mb PmNAC039 PmNAC040 PmNAC041 PmNAC043 PmNAC045 PmNAC042 PmNAC044 Chr10 PmNAC103 PmNAC104 PmNAC105 PmNAC107 PmNAC106 PmNAC109 PmNAC108 PmNAC052 PmNAC054 PmNAC056 PmNAC058 PmNAC060 PmNAC062 PmNAC053 PmNAC055 PmNAC057 PmNAC059 PmNAC061 PmNAC071 PmNAC073 PmNAC070 PmNAC072 PmNAC074 Chr11 Chr12 PmNAC113 PmNAC114 PmNAC115 PmNAC117 PmNAC116 PmNAC119 PmNAC118 PmNAC122 PmNAC124 PmNAC123 PmNAC125 PmNAC077 PmNAC078 32 Mb PmNAC079 PmNAC090 PmNAC080 PmNAC081 PmNAC082 48 Mb PmNAC091 PmNAC093 PmNAC095 PmNAC097 PmNAC092 PmNAC094 PmNAC096 PmNAC100 PmNAC120 PmNAC101 PmNAC111 PmNAC102 PmNAC110 PmNAC112 PmNAC121 PmNAC126 PmNAC128 PmNAC130 PmNAC127 PmNAC129 PmNAC131 PmNAC132 64 Mb Chr13 Mb Chr14 Chr15 PmNAC141 PmNAC133 PmNAC134 16 Mb PmNAC143 PmNAC145 PmNAC147 PmNAC156 PmNAC157 PmNAC142 PmNAC144 PmNAC146 PmNAC148 PmNAC149 PmNAC151 PmNAC153 PmNAC155 32 Mb PmNAC135 PmNAC137 PmNAC139 Chr16 Chr18 Chr17 PmNAC162 PmNAC164 PmNAC166 PmNAC168 PmNAC170 PmNAC163 PmNAC165 PmNAC167 PmNAC169 PmNAC172 PmNAC173 PmNAC158 PmNAC150 PmNAC152 PmNAC154 PmNAC159 PmNAC160 PmNAC161 PmNAC171 PmNAC174 PmNAC176 PmNAC136 PmNAC138 PmNAC140 48 Mb 64 Mb Fig Positions of NAC gene family members on broomcorn millet chromosomes Tandem duplicated genes are indicated in red PmNAC175 PmNAC177 Shan et al BMC Genomics (2020) 21:96 Page of 13 25 50 NA Pm C008009 Pm NA Pm 25 C PmNAC004 PmNAC003 Pm NA C00 Pm NAC 011 N 01 P AC PmmNA 013 NA C0 ,01 C0 15 16 57 C1 156 NA AC Pm mN P Ch r1 171 AC17 PmN C172 A PmN AC PmN -17 68 66 C1 4-1 NA 16 Pm NACAC1 61 PmPmN C1 60 NA C1 Pm NA C15 Pm NA Pm 25 25 PmNAC177 PmNAC175 25 r1 Ch Chr1 17 Chr 25 Chr18 50 Pm Pm NA NA C1 C1 52 49 ,15 -1 51 Ch r2 01 C NA Pm 02 9- C0 23 C0 24 NA C0 PmmNA 6,027 P 28 C0 NA C0 Pm NA Pm NA Chr 14 PmN PmN AC1 47 AC1 50 43-1 45 9-031 AC02 PmN PmNA PmNACC138-140 135-13 32 NAC0 Pm C033 PmNA 25 25 PmNAC134 PmNAC132 PmNAC131 PmNAC0 39,040 29,13 PmNAC1 -128 C126 PmNA 04 23 AC1 PmNNAC12 Pm Chr1 2,045 PmN PmN AC047 PmN AC048 AC0 49 25 20 C1 NA Pm N Pm AC05 NA 2-05 Pm NAC C056 Pm NAC 59,06 06 Pm NA P C P m NA 50 PmmN NA C0 63 NA AC C06 65 C0 69 Pm Pm 18 C1 11 NA Pm - 15 C1 14 C1 13 NA C1 Pm NA C11 Pm NA Pm N Pm PmN PmNAAC099 C09 PmNAC PmNAC0097 95 PmNAC094 0 r6 25 25 25 50 25 25 Ch 75 C0 76 C0 NA 50 25 Chr NA Pm Pm PmNAC087-89 r1 80 AC0 PmN C081 PmNA C082 PmNA 083 PmNAC PmNAC086 Ch 70 C0 71 NA AC0 PmmN P AC P PmmN 108, NAAC1 109 Pm C 10 07 NA Pm C103 , Pm NAC 104 NA 102 C1 01 NA Pm r11 Ch 25 50 PmNAC r5 Chr4 PmNAC037 Ch Chr13 25 r3 25 Ch Pm Chr Chr8 Fig Gene duplication of PmNAC genes Gray lines indicate all synteny blocks in the broomcorn millet genome, the green lines indicate duplicated NAC gene pairs, and the red lines indicate tandem duplicated genes PmNAC168/169) Duplicated genes were the most common on Chr3, followed by Chr5 and Chr10 (Fig 4) Expression pattern analysis of PmNAC genes in different tissues To better understand the function of PmNAC genes in broomcorn millet, the transcription levels of PmNAC genes in different tissues, i.e., seedlings at one week of age, shoots at three weeks of age, leaf blades, leaf sheaths, stems, inflorescences, and roots at the eight weeks of age, and mature seeds at 12 weeks of age, were investigated using publicly available transcriptome datasets [58] The Fragments Per Kilobase per Million mapped reads (FPKM) values of the PmNAC genes are listed in Additional file 1: Table S4, and a hierarchical clustering analysis and heatmap were generated to display the expression patterns of the PmNAC genes (Fig 5) The expression of three PmNAC genes (PmNAC055, 178, and 179) was not detected in any analysed tissue, possibly due to variations in spatial and temporal expression patterns The expression of 15 PmNAC genes Shan et al BMC Genomics (2020) 21:96 Page of 13 PmNAC043 PmNAC084 PmNAC003 PmNAC033 PmNAC039 PmNAC009 PmNAC045 PmNAC026 PmNAC108 PmNAC004 PmNAC037 PmNAC063 PmNAC001 PmNAC028 PmNAC018 PmNAC120 PmNAC007 PmNAC091 PmNAC014 PmNAC116 PmNAC098 PmNAC172 PmNAC101 PmNAC132 PmNAC041 PmNAC175 PmNAC148 PmNAC176 PmNAC006 PmNAC040 PmNAC128 PmNAC135 PmNAC170 PmNAC161 PmNAC061 PmNAC083 PmNAC081 PmNAC160 PmNAC147 PmNAC150 PmNAC059 PmNAC048 PmNAC095 PmNAC032 PmNAC112 PmNAC111 PmNAC031 PmNAC117 PmNAC072 PmNAC119 PmNAC065 PmNAC106 PmNAC025 PmNAC123 PmNAC015 PmNAC151 PmNAC078 PmNAC099 PmNAC070 PmNAC145 PmNAC103 PmNAC165 PmNAC022 PmNAC126 PmNAC139 PmNAC021 PmNAC076 PmNAC137 PmNAC157 PmNAC169 PmNAC057 PmNAC086 PmNAC136 PmNAC164 PmNAC074 PmNAC049 PmNAC125 PmNAC020 PmNAC046 PmNAC138 PmNAC047 PmNAC050 PmNAC096 PmNAC140 PmNAC142 PmNAC152 PmNAC008 PmNAC066 PmNAC042 PmNAC130 PmNAC153 PmNAC036 PmNAC094 PmNAC030 PmNAC064 PmNAC062 PmNAC155 PmNAC105 PmNAC080 PmNAC159 PmNAC129 PmNAC075 PmNAC156 PmNAC093 PmNAC163 PmNAC013 PmNAC115 PmNAC058 PmNAC082 PmNAC174 PmNAC110 PmNAC027 PmNAC109 PmNAC044 PmNAC100 PmNAC102 PmNAC143 PmNAC167 PmNAC060 PmNAC067 PmNAC097 PmNAC177 PmNAC127 PmNAC149 PmNAC154 PmNAC024 PmNAC107 PmNAC068 PmNAC052 PmNAC089 PmNAC012 PmNAC114 PmNAC011 PmNAC023 PmNAC131 PmNAC113 PmNAC054 PmNAC088 PmNAC019 PmNAC158 PmNAC141 PmNAC085 PmNAC162 FPKM 127 24 2.6 0.3 Seed_mature Root_8w Inflorescences_8w Stem_8w Leaf_sheath_8w Leaf_blade_8w Shoot_3w Seedling_1w Fig Expression patterns of NAC genes in different tissues Seedling_1w, Shoot_3w, Leaf_8w, Sheath_8w, Stem_8w, Inflorescences_8w, Root_8w, and Seed_mature indicate tissues from seedlings at one week of age, shoots at three weeks of age, leaf blades, leaf sheaths, stems, inflorescences, and roots at eight weeks of age, and seeds at 12 weeks of age, respectively FPKM values were used to generate the heatmap with hierarchical clustering analysis The scale represents the relative signal intensity of the FPKM values (PmNAC005, 010, 038, 051, 071, 073, 087, 092, 104, 118, 124, 144, 166, 171, and 180) was detected only in one tissue Thirteen PmNAC genes (PmNAC001, 004, 009, 026, 028, 037, 039, 045, 063, 101, 108, 132, and 175) exhibited high expression (FPKM > 20) in all the tested tissues, suggesting key roles of these genes in tissue development In addition, of all the analyzed tissues, there were 10, 16, 10, 12, 18, 29, 55, and 27 PmNAC genes exhibiting the highest expression in seedlings at one week of age, shoots at three weeks of age, and the leaf blades, leaf sheaths, stems, inflorescences, and roots at eight weeks of age, and mature seed at 12 weeks of age, respectively, demonstrating that different PmNAC genes may play different roles in the growth and tissue development of broomcorn millet The high expression of many PmNAC genes (31%) in the roots may be one explanation for the observed rapid response of broomcorn millet to drought stress Understanding the expression patterns of PmNAC genes in different tissues can provide a foundation for identifying functional genes in broomcorn millet Responses of PmNAC genes to drought treatments To detect the dynamic changes in the transcription levels of broomcorn millet NAC genes under drought stress, transcriptomic analysis was conducted at h (CK), h (T1), h (T2), and h (T3) under drought treatment (Additional file 1: Table S5) There were 117 PmNAC genes detected in the experiment A heatmap representing the expressions of the detected PmNAC genes was constructed (Fig 6a), and trend analysis was carried out to explore the time-related dynamic changes under drought stress (Fig 6b) The expression of 27 PmNAC genes (PmNAC003, 014, 019, 026, 028, 030, 033, 037, 048, 068, 081, 083, 084, 091, 095, 098, 101, 108, 113, 116, 128, 132, 160, 162, 163, 172, and 175) increased as the time under drought stress progressed in profile1 The expression of 22 PmNAC genes (PmNAC001, 008, 025, 039, 041, 054, 059, 063, 067, 075, 082, 085, 088, 089, 102, 110, 120, 142, 150, 154, 156, and 161) showed an ‘N’ shape and peaked at h in profile2, while the expression of 10 PmNAC genes (PmNAC006, 013, 040, 050, 106, 109, 112, 147, 174, and 177) demonstrated an opposite trend in profile5 The expressions of 11 (PmNAC004, 007, 009, 023, 045, 060, 093, 107, 141, 152, and 170) and seven (PmNAC018, 043, 044, 061, 072, 135, and 148) PmNAC genes showed similar ‘V’ shape trends in profiles and 6, respectively The expressions of 10 (PmNAC011, 027, 032, 042, 057, 062, 064, 097, 105, and 131) and seven (PmNAC012, 052, 058, 158, 165, 167, and 176) PmNAC genes first increased but then decreased in profiles and 7, respectively ... conditions [40] Therefore, NAC family genes are crucial regulators of plant tolerance to drought Broomcorn millet (Panicum miliaceum L. ), also called proso millet, common millet, and hog millet, is a... nutritional value, containing abundant proteins, starch, and a variety of vitamins and minerals, but also have medicinal value and are used in millet wine and other products The NAC gene family has... (Additional file 1: Table S 1) All the PmNAC proteins contained a conserved NAC domain (PF0184 9) or NAM domain (PF0236 5) Phylogenetic analysis of the NAC proteins from broomcorn millet and Arabidopsis