基于高基元SSR构建黍稷种质资源的分子身份证

doi:10.3724/SP.J.1006.2022.14034

Abstract

Abstract:

In order to identify hog millet (Panicum miliaceum) germplasm rapidly, to establish a big data management platform, to provide a theoretical basis for germplasm identification and traceability management, 130 germplasms from four ecological cultivation areas were used as materials to construct a molecular ID based on 35 high-motif SSRs (21, 10, and 4 with four-, five-, and six-nucleotide repeats, respectively). The results showed that 30 out of the 35 SSRs could be used as core markers for the construction of molecular ID cards. Ninety allelic variants were detected; effective allelic variants (Ne) ranged from 2.3186 to 2.9982 with an average of 2.7607; Shannon diversity index (I) ranged from 0.9158 to 1.0873 with an average of 1.0472. The observed heterozygosity (Ho) was 0.5000-0.8678 with a mean of 0.7168; the expected observed heterozygosity (He) was 0.5710-0.6691 with a mean of 0.6386; the Nei’s gene diversity index (Nei) was 0.5687-0.6665 with a mean of 0.6360; the polymorphism information content (PIC) was 0.5151-0.7898, with a mean of 0.6966. All accessions were divided into three groups (Group I, II, and III) according to UPGMA analysis. In terms of Shanxi accessions, landraces and breeding varieties were classified into Group I and III, respectively, and farmers materials were distributed into three groups. Based on PCA analysis, all accessions were classified into four clusters, which were related to their geographical origin. As for the rule that the most germplasms were determined using the least markers, 3 markers were excluded due to their high similarity coefficient with others, namely RYW23, RYW49, and RYW51. Screening the remaining 27 markers, the combinations of 17 SSR (RYW35, RYW40, RYW37, RYW18, RYW30, RYW16, RYW20, RYW19, RYW8, RYW5, RYW3, RYW7, RYW1, RYW14, RYW9, RYW6, and RYW10) could identify all hog millet accessions. The DNA molecular identifications of character strings, bar code, and quick response (QR) codes were constructed via ID analysis 4.0, software online of bar code and QR codes technique (http://barcode.cnaidc.com/app/html/bcgcode128.php and https://cli.im/).

Key words: hog millet (Panicum miliaceum), SSR with high motif, molecular identity card, germplasm resources

CHEN Xiao-Hong, LIN Yuan-Xiang, WANG Qian, DING Min, WANG Hai-Gang, CHEN Ling, GAO Zhi-Jun, WANG Rui-Yun, QIAO Zhi-Jun. Development of DNA molecular ID card in hog millet germplasm based on high motif SSR[J].Acta Agronomica Sinica, 2022, 48(4): 908-919.

Figures/Tables 8

Table 1

Table 2

Table 3

SSR primers sequences and annealing temperatures"

位点 Locus	正向引物 Forward primer (5'-3')	反向引物 Reverse primer (5'-3')	退火温度 T_m (℃)	重复基序 Repeated motif
RYW1	TAACGCTTCACCTTCAGACC	TGAGATGGAGTTGGCTGATG	55.7	(TCATCT)6
RYW2	TTAGGGCTCTCCTGCATCC	CAGCGAGTTCACCGTCAAG	57.2	(CGAAGC)5
RYW3	GGAGGCGTGACAATAAAAC	GGCGTGAGGTGTTGTTTTT	54.3	(CCTTCC)5
RYW4	AATCCACAACGCACACGAC	ATTTGCTCCTCTCGTCGGT	56.9	(GTGCCG)5
RYW5	GACGATGCTCTTGACCTTGT	CACCGTGAAATGTCTCTGCT	55.2	(CCTTT)5
RYW6	AGCCGATTTGCTGTGGAGT	CTGCCTCCGATGAGTTGGT	57.1	(ACACC)5
RYW7	TCCACTCATCCATTGCTCGT	GATGGATTCAAAGGGACGCT	58.7	(CGCGC)5
RYW8	GGGTCAGAGAATACACAGCG	GTAGGGAAGGAGAAGTGGGT	55.7	(AATAG)5
RYW9	GGACCCTTCCCTCACAGATT	TCCAGTTGCTCTTGCCGTT	58.3	(CTAG)6
RYW10	TGGATTGGGTGGTGGTAT	AAGGACGGCAGCACAAAT	53.0	(CGAG)5
RYW14	CGCACAACGACCACAAGAG	ATACACCAGAGGAGCACGC	56.7	(GGCC)5
RYW16	ATCTCCTCCGCCTTCTAACCC	TGGCAATGGTCGTACAAACT	58.4	(GAGC)5
RYW18	CTCCCTCTTTGTCCTCGTT	GCTGCCTCTTCGCTATCTT	54.3	(AGTT)6
RYW19	GAATGATAGGTCCGCAAGG	CAGCCTTTGTTCAGTTGTCTC	55.1	(TTAT)5
RYW20	ACCTCTTGCCGCACACTAC	TTCTACATCCCCGAACCAC	55.4	(TTGG)6
RYW21	CCCTCCTACTGCTCCCTTT	ATTACTCGTTCTCGCCTCG	55.6	(CGGA)6
RYW23	AGGAACAGCAGAGAGAGGG	CAGAACACCACGAAACACC	53.7	(GGAA)5
RYW26	TAAGGGTGGCGTTGGATAG	AACCCAACAGGTCCTCCAT	56.1	(AGGA)6
RYW28	CCAAGGCTGAGCAGAAAGAT	ACAAGGTGAAACCCGAAGC	57.2	(AGGC)5
RYW29	CTTGATTTCTCACGCACCG	TGTCCAGCAGTAGTCGTTCCT	57.2	(GCAG)5
RYW30	TAGCCTTCTTTGCCACCACT	GCCCGTGATGATATTCGAC	56.6	(TTTC)5
RYW31	ACCCAGAGTCCAGAGAAGC	GATGTCCTCCTCCTTCTCC	53.0	(AGCG)5
RYW32	CAGGTTATGGGAGGACGAG	GGTGCTACGGTTACAGGGT	54.9	(ATCTT)5
RYW33	CGATTCTACACCGACGAGG	TGTAGGGTTCCATTCATCTCC	54.6	(CCATC)5
RYW34	TCCCCCGATTAGGAAAGAT	CTGGTGAGGTGATGAAGCC	55.8	(CGATT)5
RYW35	ATTAGCATCCCCCTCCAC	ATCCGCTTTCCCAACCAC	54.7	(CGTGC)5
RYW36	TATTGTCCTTCCGCTCCC	ATGACTACTCTCCCCCCCT	55.0	(GGCTT)5
RYW37	CATTCCGTTCCTTGTCTTCC	CAGTCTCACTCCTGCGATGT	55.3	(GCGAT)5
RYW39	GTTGGGCGAGGTCAATCTG	TAGGGAGCCGAAGCAGAAG	58.2	(TCCT)5
RYW40	TGCTCTTCGGCTCTTCTCC	ATCAGCTCATCGTGACCCC	57.6	(CAGC)6
RYW43	GGAGATGCTTGCTTGGTTG	CAGGAATCGCAAGGAACAG	56.2	(GGAG)5
RYW47	TTGTTTTTGCTGCTGCCTC	TGCTGGACTTCTTTTTGCC	56.7	(GCCT)5
RYW49	GCTAAATCCGCTGATGAGGT	TGTATGTTGCTCCAGCCTTG	57.0	(TATC)6
RYW50	CAAGGCAGATAGGGCAAGT	TCGTCTGCTGCTGGTTTGT	56.1	(GGAG)5
RYW51	TATCGCCGCACCTTACAAC	TGAGCCTGCTTCCATCTTG	56.8	(CTGC)5

Table 3

Fig. 1

Table 4

Genetic parameters of the 30 polymorphic SSR markers used in the study"

引物 Locus	观测等位变异 Na	有效等位变异 Ne	Shannon多样性指数 I	观测杂合度 Ho	期望杂合度 He	Nei’s基因多样性指数 Nei	多态性信息含量 PIC
RYW1	3.0000	2.9844	1.0960	0.6614	0.6675	0.6649	0.6635
RYW3	3.0000	2.9982	1.0983	0.6160	0.6691	0.6665	0.7839
RYW5	3.0000	2.9587	1.0917	0.7520	0.6647	0.6620	0.7306
RYW6	3.0000	2.8314	1.0677	0.7059	0.6495	0.6468	0.7451
RYW7	3.0000	2.5901	1.0140	0.7049	0.6164	0.6139	0.7009
RYW8	3.0000	2.9753	1.0944	0.7155	0.6668	0.6639	0.7870
RYW9	3.0000	2.8813	1.0780	0.7661	0.6556	0.6529	0.7372
RYW10	3.0000	2.5276	0.9892	0.7227	0.6069	0.6044	0.6913
RYW14	3.0000	2.8069	1.0613	0.7258	0.6463	0.6437	0.7370
RYW16	3.0000	2.9801	1.0952	0.6239	0.6673	0.6644	0.7775
RYW18	3.0000	2.9499	1.0899	0.5391	0.6636	0.6610	0.8043
RYW19	3.0000	2.9132	1.0830	0.6218	0.6595	0.6567	0.7999
RYW20	3.0000	2.8143	1.0655	0.6822	0.6472	0.6447	0.7056
RYW21	3.0000	2.5147	0.9878	0.7680	0.6048	0.6023	0.5907
RYW23	3.0000	2.5244	0.9886	0.8537	0.6063	0.6039	0.5886
RYW26	3.0000	2.3186	0.9158	0.6829	0.5710	0.5687	0.6302
RYW28	3.0000	2.5846	1.0193	0.5000	0.6155	0.6131	0.7735
RYW29	3.0000	2.6400	1.0228	0.7578	0.6237	0.6212	0.6209
引物 Locus	观测等位变异 Na	有效等位变异 Ne	Shannon多样性指数 I	观测杂合度 Ho	期望杂合度 He	Nei’s基因多样性指数 Nei	多态性信息含量 PIC
RYW30	3.0000	2.9599	1.0918	0.6198	0.6649	0.6621	0.7898
RYW31	3.0000	2.6094	1.0166	0.6532	0.6193	0.6168	0.7237
RYW33	3.0000	2.7255	1.0428	0.7949	0.6358	0.6331	0.6856
RYW35	3.0000	2.9496	1.0902	0.7179	0.6638	0.6610	0.7457
RYW37	3.0000	2.9802	1.0952	0.8374	0.6672	0.6645	0.7301
RYW39	3.0000	2.7768	1.0548	0.6880	0.6424	0.6399	0.6999
RYW40	3.0000	2.8700	1.0757	0.7458	0.6543	0.6516	0.7362
RYW43	3.0000	2.6909	1.0343	0.8678	0.6310	0.6284	0.6592
RYW32	3.0000	2.6620	1.0270	0.8661	0.6268	0.6243	0.5473
RYW47	3.0000	2.6426	1.0256	0.7812	0.6240	0.6216	0.6584
RYW50	3.0000	2.4418	0.9614	0.7563	0.5930	0.5905	0.5151
RYW51	3.0000	2.7418	1.0413	0.7752	0.6347	0.6322	0.5401
Mean	3.0000	2.7607	1.0472	0.7168	0.6386	0.6360	0.6966

Table 4

Fig. 2

Fig. 3

Fig. 4

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生态区 Ecotope area	来源 Origin	数量 Accession number
黄土高原春夏糜子区LPSS Loess Plateau Spring & Summer Sowing Hog Millet Ecotope	山西 Shanxi, China 宁夏 Ningxia, China 河北 Hebei, China 甘肃 Gansu, China 青海 Qinghai, China 陕西 Shaanxi, China	83 3 1 2 1 2
北方春糜子区 NCSP Northern China Spring Sowing Hog Millet Ecotope	山西 Shanxi, China 陕西 Shaanxi, China 内蒙古 Inner Mongolia, China 甘肃 Gansu, China 宁夏 Ningxia, China	22 2 4 4 1
华北夏糜子区 NCSU North China Summer Sowing Hog Millet Ecotope	河南 Henan, China 河北 Hebei, China	1 2
东北春糜子区 NECS Northeast China Spring Sowing Hog Millet Ecotope	内蒙古 Inner Mongolia, China 辽宁 Liaoning, China	1 1
合计 Total		130

编号 Serial number	生态区 Ecotope	统一编号 Unicode	名称 Name	原产地 Origin	备注 Remark
1	NCSP	00000832	鸭爪白 Yazhuabai	山西大同市 Datong, Shanxi, China	地方品种 Landrace
2	NCSP	00001637	糜子 Meizi	陕西榆林市 Yulin, Shaanxi, China	地方品种 Landrace
3	NCSP	00002250	临河黄糜子 Linhehuangmeizi	内蒙古临河市 Linhe, Inner Mongolia, China	地方品种 Landrace
4	LPSS	00006780	黑城小黑糜 Heichengxiaoheimei	宁夏固原市 Guyuan, Ningxia, China	地方品种 Landrace
5	NCSP	00006573	宁朔紫秆小红糜 Ningshuoziganxiaohongmei	甘肃 Gansu, China	地方品种 Landrace
6	NCSU	00007118	糜子 Meizi	河北省承德市 Chengde, Hebei, China	地方品种 Landrace

Development of DNA molecular ID card in hog millet germplasm based on high motif SSR

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