Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (12): 2249-2261.doi: 10.3864/j.issn.0578-1752.2023.12.002


Construction of DNA Molecular Identity Card of Core Germplasm of Broomcorn Millet in China Based on Fluorescence SSR

XUE YaPeng1(), DING YiBing1, WANG YuZhuo1, WANG XiaoDan1, CAO XiaoNing2, SANTRA Dipak K3, CHEN Ling2, QIAO ZhiJun2(), WANG RuiYun1,2()   

  1. 1 College of Agronomy, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    2 Center for Agricultural Genetic Resources Research, Shanxi Agricultural University/Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture and Rural Affairs/Shanxi Key Laboratory of Genetic Resources and Genetic Improvement of Minor Crops, Taiyuan 030031, Shanxi, China
    3 Panhandle Research & Extension Center, Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Scottsbluff, 69361, Nebraska, USA
  • Received:2023-02-01 Accepted:2023-04-01 Online:2023-06-16 Published:2023-06-27


【Objective】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. 【Method】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 ( 【Result】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.【Conclusion】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.

Key words: broomcorn millet, capillary electrophoresis, fluorescent SSR, DNA molecular identification

Table 1

Information of 14 accessions of broomcorn millet in this experiment"

Serial number
1 00000082 黄糜子Huangmeizi 中国黑龙江Heilongjiang, China 地方品种Landrace
2 00000840 六十天小红黍60-day Xiaohongshu 中国山西Shanxi, China 地方品种Landrace
3 00000710 皋兰鸭蛋青Gaolanyadanqing 中国甘肃Gansu, China 地方品种Landrace
4 00003117 黄糜Huangmei 中国新疆Xinjiang, China 地方品种Landrace
5 00006438 乌克兰黍Ukraine shu 中国内蒙古Inner Mongolia, China 地方品种Landrace
6 00004198 金守黍Jinshoushu 中国广东Guangdong, China 地方品种Landrace
7 00007131 白黍子Baishuzi 中国河北Hebei, China 地方品种Landrace
8 00006681 札达糜Zhadamei 中国西藏Tibet, China 地方品种Landrace
9 00007258 黄糜子Huangmeizi 中国辽宁Liaoning, China 地方品种Landrace
10 00007375 790035 印度India 地方品种Landrace
11 00005191 塞盖德斯Saigaidesi 中国山东Shandong, China 地方品种Landrace
12 00005671 扶余白糜子Fuyubaimeizi 中国吉林Jilin, China 地方品种Landrace
13 00005452 夯糜子Hangmeizi 中国陕西Shaanxi, China 地方品种Landrace
14 00002641 西吉小黄糜Xijixiaohuangmei 中国宁夏Ningxia, China 地方品种Landrace

Table 2

Information of 15 fluorescently labeled SSR primers"

Forward primer (5′-3′)
Reverse primer (5′-3′)
5′ end modification
Repeated motif
Tm (℃)

Fig. 1

The site of 7 markers located on the chromosome of broomcorn millet"

Fig. 2

Electrophoregrams of capillary polymorphism in some accessions amplyfied by RYW43"

Table 3

Amplification results and code typing of 7 pairs of core primers detected with capillary electrophoresis"

Fluorescent group
Polymorphism bands
Length and code of electrophoresis fragments (bp)
RYW3 6-羧基荧光素
FAM (blue)
19 149(01),440(02),132/149(03),132/149/156(04),138/149/178(05),143/149/156(06),143/149(07),143/149/178(08),143/149/216(09),149/156(10),149/156/216(11),149/169/198(12),149/178(13),149/211/216(14),149/216(15),156/169(16),272/344/440/474(17),440/474(18),143/149/184(19)
RYW6 6-羧基荧光素
FAM (blue)
38 242(01),255(02),262(03),300(04),308(05),323(06),333(07),387(08),444(09),452(10),174/195/242/372(11),187/195/242/372(12),195/202/242(13),195/202/242/372(14),195/242(15),195/242/267/372(16),195/242/347/372(17),195/242/372(18),195/242/387(19),195/242/401(20),202/333(21),211/242/372(22),211/333(23),242/323(24),242/333(25),242/387(26),242/425(27),252/257(28),255/267(29),296/308(30),300/308(31),323/328(32),323/333(33),323/338(34),328/333(35),333/338(36),387/425(37),444/452(38)
RYW11 6-羧基荧光素FAM (blue) 3 180(01),222(02),213/222(03)
RYW18 6-羧基荧光素FAM (blue) 5 376(01),369/381(02),376/381(03),381/386(04),381(05)
RYW37 6-羧基荧光素 FAM (blue) 6 203(01),209(02),196/203(03),196/203/209(04),196/203/209/216(05),203/209(06)
RYW125 6-羧基荧光素
FAM (blue)
24 196(01),202(02),207(03),292(04),333(05),105/202/323(06),122/211/292(07),130/151/202/333(08),130/202/219/339(09),130/202/323/333(10),130/323(11),141/259/292/372(12),141/299/323(13),196/202(14),196/219(15),202/207(16),202/211(17),202/227(18),219/223(19),259/323/333/393(20),292/333(21),299/339(22),323/339(23),323/402(24)
RYW43 六氯-6-甲基荧光素
HEX (green)
30 109(01),120(02),128(03),181(04),204(05),109/115(06),109/115/181(07),109/120/128/147(08),109/128/181/204(09),109/128(10),109/128/181(11),109/138(12),109/138/181(13),109/155/181(14),109/181(15),109/181/204(16),109/181/226/293(17),109/181/293(18),109/181/293/405 (19),109/226/293(20),109/226/293/491(21),109/226/483(22),109/226/491(23),109/293(24),128/181(25),138/181(26),170/181(27),181/204(28),103/109/181/204(29),109/204(30)

Table 4

Genetic parameters of the 7 fluorescently labeled SSR markers used in the study"

Observed number of alleles (Na)
Shannon 多样性指数
Shannon’s diversity index(I)
Observed heterozygosity (Ho)
Expected heterozygosity (He)
Nei’s 基因多样性指数
Nei's expected heterozygosity (Nei)
多态性信息含量Polymorphism information content (PIC)
RYW3 15.0000 0.9874 0.8136 0.4114 0.4105 0.5073
RYW6 25.0000 2.0587 0.5238 0.7469 0.7452 0.8071
RYW11 3.0000 0.7178 0.0086 0.5053 0.5043 0.0334
RYW18 4.0000 0.2055 0.9455 0.0795 0.0793 0.2103
RYW37 4.0000 1.1777 0.3376 0.6700 0.6686 0.6495
RYW125 16.0000 1.6084 0.4414 0.6940 0.6916 0.7681
RYW43 20.0000 1.2230 0.5399 0.5161 0.5149 0.6540
合计Total 87.0000 - - - - -
平均Mean 12.4286 1.1398 0.5158 0.5176 0.5163 0.5185

Table 5

Discrimination of broomcorn millet accessions by 7 pairs of primer and their combinations"

No. of germplasm distinguished
Rate of discrimination (%)
RYW3 10 4.26
RYW6 17 7.23
RYW11 2 0.85
RYW18 0 0.00
RYW37 2 0.85
RYW43 16 6.81
RYW125 16 6.81
RYW3+RYW6 46 19.57
RYW3+RYW6+RYW11 48 20.42
RYW3+RYW6+RYW11+RYW18 62 26.38
RYW3+RYW6+RYW11+RYW18+RYW37 88 37.45
RYW3+RYW6+RYW11+RYW18+RYW37+RYW43 161 68.51
RYW3+RYW6+RYW11+RYW18+RYW37+RYW43+RYW125 235 100.00

Fig. 3

Cluster map of 235 broomcorn millet accessions based on UPGMA The accession number is the same as the Supplemental Table 1"

Fig. 4

Principal component analysis of 235 broomcorn millet accessions based on SSR"

Fig. 5

Two-dimensional code DNA molecular identification of broomcorn millet accessions of No. 1-No. 12"

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