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作物学报

作物学报 ›› 2024, Vol. 50 ›› Issue (5): 1181-1192.doi: 10.3724/SP.J.1006.2024.34153

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

东北春播区糜子核心种质及其DNA分子身份证构建

丁艺冰1,2(), 辛旭霞2, 冯智尊2, 曹越2, 郭娟2, Dipak K SANTRA3, 王瑞云2,*(), 陈喜明1,4,*()   

  1. 1山西农业大学玉米研究所, 中国山西忻州 034000
    2山西农业大学农学院, 中国山西太谷 030801
    3内布拉斯加大学林肯分校农艺系小宗粮豆研究与推广中心, 美国内布拉斯加州斯克茨布拉夫 69361
    4山西农业大学杂粮种质创新与分子育种山西省重点实验室, 中国山西太原 030000
  • 收稿日期:2023-09-12 接受日期:2024-01-12 出版日期:2024-05-12 网络出版日期:2024-01-31
  • 通讯作者: 陈喜明, E-mail: 516834898@qq.com; 王瑞云, E-mail: wry925@126.com
  • 作者简介:E-mail: 2771284358@qq.com
  • 基金资助:
    山西农业大学杂粮种质创新与分子育种国家实验室(筹)项目(202204010910001);国家自然科学基金项目(31271791);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-06-14.5-A16);山西省现代农业产业技术体系建设专项资金项目(2023CYJSTX03-12);山西省重点研发项目(2022ZDYF110);山西农业大学杂粮种质创新与分子育种山西省重点实验室项目(K462202040);山西农业大学生物育种工程项目(YZGC069);山西农业大学农学院研究生教育改革项目(2023YJG05);山西省科技成果转化引导专项(201904D131056)

Core germplasm and DNA molecular identity card of proso millet in Northeast Spring sowing region in China

DING Yi-Bing1,2(), XIN Xu-Xia2, FENG Zhi-Zun2, CAO Yue2, GUO Juan2, Dipak K SANTRA3, WANG Rui-Yun2,*(), CHEN Xi-Ming1,4,*()   

  1. 1Corn Research Institute, Shanxi Agricultural University, Xinzhou 034000, Shanxi, China
    2College of Agronomy, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    3Panhandle Research and Extension Center, Department of Agronomy and Horticulture, University of Nebraska- Lincoln, Scottsbluff 69361, Nebraska, USA
    4Shanxi Key Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding, Shanxi Agricultural University, Taiyuan 030000, Shanxi, China
  • Received:2023-09-12 Accepted:2024-01-12 Published:2024-05-12 Published online:2024-01-31
  • Contact: E-mail: 516834898@qq.com; E-mail: wry925@126.com
  • Supported by:
    National Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding(202204010910001);National Natural Science Foundation of China(31271791);China Agriculture Research System of MOF and MARA(CARS-06-14.5-A16);Special Fund for Construction of Modern Agricultural Industrial Technology System of Shanxi Province(2023CYJSTX03-12);Key Research & Development Project of Shanxi Province(2022ZDYF110);Shanxi Key Laboratory of Innovation and Molecular Breeding, Shanxi Agricultural University(K462202040);Biological Breeding Project of Shanxi Agricultural University(YZGC069);Graduate Education Reform Project of Agronomy College in Shanxi Agricultural University(2023YJG05);Shanxi Province Scientific and Technological Achievements Transformation and Guidance Project(201904D131056)

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摘要：

本研究以500份东北春播区糜子资源为材料, 利用169个SSR标记, 采用UPGMA聚类分组, 进行分层抽样, 构建核心种质, 同时应用ID Analysis 4.0软件构建分子身份证。利用等位基因数(Na)等遗传多样性衡量指标评估核心种质的遗传差异, 并利用PCOA分析核心种质。结果表明, 对169对SSR引物进行筛选, 发现30对多态性好, 利用30对SSR引物构建的糜子核心种质包含190份材料, 占全部种质的38%, 全部种质与核心种质的均检测出91个等位变异, 保留了100%等位基因; 有效等位基因数为2.2977~2.9975和2.2872~3.0173, 平均值分别为2.8198和2.8297; Shannon多样性指数为0.9532~1.0990和0.9535~1.1162, 平均值为1.0645和1.0667; 观测杂合度为0.3434~0.8037和0.3162~0.7849, 平均值为0.5399和0.5359; 期望杂合度为0.5654~0.6672和0.5645~0.6707, 平均值为0.6448和0.6473; Nei’s基因多样性指数为0.5648~0.6664和0.5628~0.6686, 平均值为0.6441和0.6452; 多态性信息含量为0.6657~0.8356和0.6493~0.8340, 平均值为0.7974和0.7944。全部种质与核心种质的分子标记的相关指标进行t检验, 结果无显著性差异, 且PCOA分析表明核心种质与全部种质具有相似的遗传多样性和群体结构, 同时发现8个SSR标记(RYW5、RYW8、RYW16、RYW28、RYW40、RYW53、RYW62和RYW67)可区分190份核心种质, 构建了东北糜子核心种质的分子身份证。

关键词: 糜子, 东北春播区, SSR, 核心种质, DNA分子身份证

Abstract:

In this study, 500 proso millet resources in Northeast Spring Sowing Region were used as the experimental materials, 169 SSR markers, UPGMA clustering, and stratified sampling were used to construct core germplasm, and ID Analysis 4.0 software was used to construct molecular identity card. The genetic diversity of the core collection was evaluated by genetic diversity metrics such as allele number (Na), and the core collection was analyzed by PCOA. The results showed that 169 pairs of SSR primers were screened, and 30 pairs of SSR primers were found to have good polymorphism. The core collection of proso millet constructed by 30 pairs of SSR primers contained 190 materials, accounting for 38% of all germplasm. Ninety-one alleles were detected in all germplasm and core collection, and 100% alleles were retained. The number of effective alleles was 2.2977-2.9975 and 2.2872-3.0173, with an average of 2.8198 and 2.8297, respectively. The Shannon diversity index was 0.9532-1.0990 and 0.9535-1.1162, with an average of 1.0645 and 1.0667. The observed heterozygosity was 0.3434-0.8037 and 0.3162-0.7849, with an average of 0.5399 and 0.5359. The expected heterozygosity was 0.5654-0.6672 and 0.5645-0.6707, with an average of 0.6448 and 0.6473. Nei’s gene diversity index was 0.5648-0.6664 and 0.5628-0.6686, with an average of 0.6441 and 0.6452. The polymorphism information content was 0.6657-0.8356 and 0.6493-0.8340, with an average of 0.7974 and 0.7944. The results of t-test showed that there was no significant difference in the related indexes of molecular markers between all germplasm and core germplasm, and PCOA analysis showed that the core germplasm and all germplasm had similar genetic diversity and population structure. At the same time, 8 SSR markers (RYW5, RYW8, RYW16, RYW28, RYW40, RYW53, RYW62, and RYW67) were found to distinguish 190 core germplasms, and the molecular identity card of the core germplasms of Northeast proso millet was constructed, which providing a scientific basis for the efficient utilization and rapid traceability of proso millet germplasm.

Key words: Panicum miliaceum, the Northeast Spring sowing region, SSR, core collection, DNA molecular ID card

表1

用于筛选引物的12份材料"

材料编号
Serial
number		 统一编号
Unicode		 名称
Name		 来源
Source		 备注
Remark
1 00000018 黑糜子 Heimeizi 黑龙江克山 Keshan, Heilongjiang 地方品种
Landrace
2 00000173 黄糜子 Huangmeizi 黑龙江穆棱 Muling, Heilongjiang
3 00000081 黄糜子 Huangmeizi 黑龙江富锦 Fujin, Heilongjiang
4 00000001 64黍120 64 Shu 120 黑龙江嫩江 Nenjiang, Heilongjiang
5 00000501 通辽高粱黍Tongliaogaoliangshu 内蒙古通辽 Tongliao, Inner Mongolia
6 00000413 昌图红糜子Changtuhongmeizi 辽宁昌图 Changtu, Liaoning
7 00000452 海城紧穗 Haichengjinsui 辽宁海城 Haicheng, Liaoning
8 00000423 黑山红黍 Heishanhongshu 辽宁黑山 Heishan, Liaoning
9 00000401 红糜子 Hongmeizi 吉林通化 Tonghua, Jilin
10 00000331 红糜子 Hongmeizi 吉林白城 Baicheng, Jilin
11 00000386 糜子 Meizi 吉林安图 Antu, Jilin
12 00006562 燕头黑黍 Yantouheishu 吉林吉林 Jilin, Jilin

表2

构建核心种质的30对SSR引物"

引物
Primer name		 正向引物序列
Forward sequence (5′-3′)		 反向引物序列
Reverse sequence (5′-3′)		 退火温度 Tm (℃) 重复碱基
Repeat motif
RYW2 TTAGGGCTCTCCTGCATCC CAGCGAGTTCACCGTCAAG 56.8 (CGAAGC)5
RYW3 GGAGGCGTGACAATAAAAC GGCGTGAGGTGTTGTTTTT 52.5 (CTGCAA)5
RYW5 GACGATGCTCTTGACCTTGT CACCGTGAAATGTCTCTGCT 55.0 (CCTTT)5
RYW6 AGCCGATTTGCTGTGGAGT CTGCCTCCGATGAGTTGGT 57.4 (ACACC)5
RYW8 GGGTCAGAGAATACACAGCG GTAGGGAAGGAGAAGTGGGT 55.9 (AATAG)5
RYW12 ACCATCCCAGCACAAACCA TGCCTGAAGGAGAAGAGCG 57.5 (AGCT)5
RYW16 ATCTCCTCCGCCTTCTAACCC TGGCAATGGTCGTACAAACT 56.7 (GAGC)5
RYW17 TCAGCTACTTCGAACGGC GGATCATGCGATACATTTGG 53.0 (TTTC)5
RYW20 ACCTCTTGCCGCACACTAC TTCTACATCCCCGAACCAC 56.2 (TTGG)6
RYW28 CCAAGGCTGAGCAGAAAGAT ACAAGGTGAAACCCGAAGC 55.4 (AGGC)5
RYW29 CTTGATTTCTCACGCACCG TGTCCAGCAGTAGTCGTTCCT 56.2 (GCAG)5
RYW30 TAGCCTTCTTTGCCACCACT GCCCGTGATGATATTCGAC 54.9 (TTTC)5
RYW40 TGCTCTTCGGCTCTTCTCC ATCAGCTCATCGTGACCCC 57.4 (CAGC)6
RYW42 AGACACCCTGGGCAACATC CTGGACTGGGCTTCGTTCT 57.8 (GGCT)5
RYW43 GGAGATGCTTGCTTGGTTG CAGGAATCGCAAGGAACAG 54.0 (GGAG)5
RYW47 TTGTTTTTGCTGCTGCCTC TGCTGGACTTCTTTTTGCC 53.7 (GCCT)5
RYW50 CAAGGCAGATAGGGCAAGT TCGTCTGCTGCTGGTTTGT 56.1 (GGAG)5
RYW52 AGTAGTCCTCCACCGCCAT CTCTTCCTCGTTCTCGGCT 57.8 (TACC)5
RYW53 ATGCCTCCGATGTAGATGC GCCGCCTTCTCTTCATTCT 54.9 (GAGG)5
RYW55 CTGGTGGTGGTAGTTAGCG TTATGCCACCCACCGTAGC 56.9 (TAGC)5
RYW62 GTTTAGAGAGCAGGAGGCG AGCCCTGTCCACCCTAATC 56.4 (GCTC)5
RYW67 GAAGGAAACGCACCAGAGT TTGGGTTTGTGCTTGGAGT 54.9 (TGCG)5
RYW99 CGGAGTTCTTGGTGGCTT GCGTTCGCCAAAGAGCAT 56.1 (CCA)5
RYW125 TTGACGACGACTGTGTGC TGTTGGTGGAGTTGAGGAC 55.1 (GGC)5
RYW145 CTTTTTCTGCTGCTCCCT TGATGCCATACCCAACTG 52.2 (GAC)6
RYW146 TGATGCTTCTTGGGTTCG CGCCGTCCACTTCTGTAT 53.6 (GCG)6
RYW149 CAGGACTTGGGTGATTGC GAGCGGAGGAGGAAACTA 53.7 (AGC)7
RYW156 TTTACAACCCTTCCCGCC AGGACTTTCCGCCTCTACCC 57.5 (CCG)5
RYW158 GGTAGGGTTCAAGGTGGTT CAGGCAATCTCTTCAGGC 54.0 (CCG)5
RYW164 AGACAGCCATTCAACCACG CCATCTCCTCATCCACCA 54.6 (GA)6

图1

RYW40引物对部分材料扩增的聚丙烯凝胶电泳图 1~20为黑龙江材料, 21~35为吉林材料, 36~45为辽宁材料, 46~50为内蒙古材料。"

图2

基于30对SSR数据对4个糜子种质资源群体的聚类分析图 HLJ: 黑龙江; JL: 吉林; LN: 辽宁; IM: 内蒙古。"

表3

种质资源与核心种质遗传参数的对比"

位点
Locus		 Na Ne I Ho He Nei PIC
CC EC CC EC CC EC CC EC CC EC CC EC CC EC
RYW2 3 3 2.8424 2.7698 1.0695 1.0561 0.4785 0.5023 0.6502 0.6397 0.6482 0.6390 0.8241 0.8202
RYW3 3 3 2.8456 2.8320 1.0705 1.0692 0.6785 0.7271 0.6506 0.6476 0.6486 0.6469 0.7663 0.7438
RYW5 3 3 2.9708 2.9804 1.0936 1.0953 0.3162 0.3434 0.6658 0.6655 0.6634 0.6645 0.7867 0.7830
RYW6 3 3 2.9285 2.9128 1.0864 1.0841 0.3791 0.3652 0.6607 0.6575 0.6585 0.6567 0.8213 0.8154
RYW8 3 3 2.7788 2.7895 1.0552 1.0576 0.4938 0.4635 0.6421 0.6423 0.6401 0.6415 0.7989 0.8133
RYW12 3 3 2.8112 2.7954 1.0654 1.0631 0.5875 0.5704 0.6463 0.6430 0.6443 0.6423 0.8100 0.8093
RYW16 3 3 2.7053 2.7785 1.0443 1.0562 0.3664 0.3610 0.6328 0.6410 0.6304 0.6401 0.7701 0.7703
RYW17 3 3 2.9691 2.9921 1.0933 1.0973 0.4908 0.5274 0.6652 0.6666 0.6632 0.6658 0.8340 0.8356
RYW20 3 3 2.9224 2.8151 1.0848 1.0634 0.4931 0.4558 0.6601 0.6456 0.6578 0.6448 0.8075 0.8130
RYW28 3 3 2.9102 2.8245 1.0828 1.0663 0.4522 0.4638 0.6587 0.6467 0.6566 0.6460 0.8227 0.8168
RYW29 3 3 2.8740 2.8909 1.0762 1.0803 0.5739 0.6388 0.6539 0.6548 0.6521 0.6541 0.7935 0.8021
RYW30 4 4 3.0173 2.9609 1.1162 1.0990 0.5380 0.5217 0.6707 0.6631 0.6686 0.6623 0.8307 0.8226
RYW40 3 3 2.7040 2.7078 1.0376 1.0385 0.6095 0.6147 0.6320 0.6314 0.6302 0.6307 0.7453 0.7762
RYW42 3 3 2.9503 2.9008 1.0904 1.0821 0.5944 0.6116 0.6629 0.6560 0.6610 0.6553 0.8119 0.8035
RYW43 3 3 2.5607 2.5420 1.0080 1.0029 0.6080 0.6122 0.6112 0.6073 0.6095 0.6066 0.7538 0.7616
RYW47 3 3 2.7607 2.7934 1.0534 1.0591 0.7849 0.8037 0.6395 0.6427 0.6378 0.6420 0.6493 0.6657
RYW50 3 3 2.9990 2.9975 1.0984 1.0982 0.5395 0.5205 0.6688 0.6672 0.6666 0.6664 0.8223 0.8245
RYW52 3 3 2.2872 2.2977 0.9535 0.9532 0.5868 0.5494 0.5645 0.5654 0.5628 0.5648 0.7468 0.7599
RYW53 3 3 2.9822 2.9776 1.0956 1.0949 0.4326 0.4820 0.6670 0.6651 0.6647 0.6642 0.7931 0.8051
RYW55 3 3 2.9655 2.9109 1.0927 1.0834 0.5385 0.5323 0.6649 0.6573 0.6628 0.6565 0.8253 0.8191
RYW62 3 3 2.7967 2.8045 1.0638 1.0651 0.6628 0.6645 0.6443 0.6441 0.6424 0.6434 0.7774 0.7773
RYW67 3 3 2.7732 2.8980 1.0596 1.0816 0.5148 0.5583 0.6413 0.6557 0.6394 0.6549 0.8114 0.8169
RYW99 3 3 2.4995 2.5108 1.0050 1.0073 0.4765 0.4834 0.6017 0.6024 0.5999 0.6017 0.7887 0.7958
RYW125 3 3 2.8868 2.8902 1.0796 1.0803 0.5723 0.5429 0.6556 0.6548 0.6536 0.6540 0.8132 0.8219
RYW145 3 3 2.9704 2.9767 1.0936 1.0946 0.6312 0.6320 0.6654 0.6649 0.6633 0.6641 0.8154 0.8223
RYW146 3 3 2.9161 2.9046 1.0841 1.0824 0.4324 0.3854 0.6593 0.6566 0.6571 0.6557 0.8156 0.8093
RYW149 3 3 2.5402 2.4947 1.0006 0.9880 0.5756 0.6000 0.6081 0.5998 0.6063 0.5991 0.7617 0.7645
RYW156 3 3 2.9045 2.8552 1.0824 1.0743 0.5893 0.5597 0.6577 0.6505 0.6557 0.6498 0.8085 0.8134
RYW158 3 3 2.8640 2.8123 1.0751 1.0663 0.5098 0.4975 0.6530 0.6452 0.6508 0.6444 0.8139 0.8195
RYW164 3 3 2.9527 2.9776 1.0905 1.0948 0.5765 0.6050 0.6633 0.6649 0.6613 0.6642 0.8128 0.8207
平均Mean 3.0333 3.0333 2.8297 2.8198 1.0667 1.0645 0.5359 0.5399 0.6473 0.6448 0.6452 0.6441 0.7944 0.7974
合计Total 91 91 — — — — — — — — — — — —
St.Dev 0.1826 0.1826 0.1704 0.1661 0.0354 0.0350 0.0979 0.1047 0.0238 0.0232 0.0237 0.0232 — —

表4

种质资源与核心种质之间SSR和群体遗传参数的t检验"

t检验
t-test		 Na Ne I Ho He Nei PIC
SSR 1.000 0.822 0.806 0.887 0.691 0.847 0.746
群体Group 1.000 0.680 0.614 0.715 0.862 0.658 0.575

表5

不同来源糜子的遗传多样性参数"

指标
Index		 群体
Group		 黑龙江
Heilongjiang		 吉林
Jilin		 辽宁
Liaoning		 内蒙古
Inner Mongolia
Na EC 3.0333±0.1826 3.0000±0.0000 3.0000±0.0000 2.9667±0.1826
CC 3.0333±0.1826 3.0000±0.0000 3.0000±0.0000 2.6333±0.4901
Ne EC 2.7912±0.2025 2.8123±0.1458 2.7715±0.1611 2.4734±0.3518
CC 2.7958±0.1980 2.7720±0.1761 2.7665±0.2072 2.2392±0.3878
I EC 1.0572±0.0465 1.0637±0.0287 1.0551±0.0326 0.9669±0.1148
CC 1.0582±0.0474 1.0560±0.0347 1.0526±0.0457 0.8568±0.1764
Ho EC 0.5442±0.1137 0.5260±0.1182 0.5472±0.1475 0.5165±0.2476
CC 0.5387±0.1178 0.5106±0.1203 0.5576±0.1465 0.5461±0.3237
He EC 0.6410±0.0298 0.6462±0.0196 0.6452±0.0220 0.6157±0.0728
CC 0.6446±0.0292 0.6441±0.0247 0.6484±0.0313 0.6013±0.0923
Nei EC 0.6397±0.0297 0.6434±0.0194 0.6380±0.0215 0.5863±0.0700
CC 0.6403±0.0289 0.6378±0.0242 0.6363±0.0303 0.5400±0.0814
PIC EC 0.7932 0.7896 0.7778 0.6715
CC 0.7805 0.7840 0.7610 0.5572

图3

核心种质与原种质的主坐标图 EC: 种质资源; CC: 核心种质。"

表6

8对SSR引物的遗传多样性参数"

引物Primer Na Ne I Ho He Nei PIC
RYW5 3.0000 2.9335 1.0873 0.3841 0.6613 0.6591 0.8213
RYW8 3.0000 2.7817 1.0559 0.4969 0.6425 0.6405 0.7989
RYW16 3.0000 2.7052 1.0442 0.3643 0.6328 0.6303 0.7701
RYW28 3.0000 2.9053 1.0815 0.4452 0.6579 0.6558 0.8227
RYW40 3.0000 2.7007 1.0368 0.6048 0.6316 0.6297 0.7453
RYW53 3.0000 2.9793 1.0951 0.4317 0.6668 0.6644 0.7931
RYW62 3.0000 2.7981 1.0641 0.6588 0.6445 0.6426 0.7774
RYW67 3.0000 2.7664 1.0585 0.5150 0.6404 0.6385 0.8114
平均Mean 3.0000 2.8213 1.0654 0.4876 0.6472 0.6451 0.7925
合计Total 24 — — — — — —
标准差SD 0.0000 0.1054 0.0208 0.1033 0.0132 0.0131 —

图4

190份糜子核心种质的主成分分析 HLJ: 黑龙江; JL: 吉林; LN: 辽宁; IM: 内蒙古。"

图5

糜子材料1品种条形码(A)和二维码(B)DNA分子身份证"

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