欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2021, Vol. 47 ›› Issue (2): 285-293.doi: 10.3724/SP.J.1006.2021.03015

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米籽粒突变体smk7的表型分析和基因定位

蒋成功1,2, 石慧敏2, 王红武2, 李坤2, 黄长玲2, 刘志芳2, 吴宇锦2, 李树强2, 胡小娇2,*, 马庆1,*   

  1. 1作物抗逆育种与减灾国家地方联合工程实验室, 安徽合肥 230036
    2中国农业科学院作物科学研究所 / 作物分子育种国家工程实验室, 北京 100081
  • 收稿日期:2020-03-11 接受日期:2020-08-19 出版日期:2021-02-12 网络出版日期:2020-09-10
  • 通讯作者: 胡小娇,马庆
  • 基金资助:
    国家自然科学基金项目(31500984);中国农业科学院科技创新工程和中国农业科学院重大科研联合攻关任务“玉米藏粮于技”项目(CAAS-ZDRW202004)资助

Phenotype analysis and gene mapping of small kernel 7 (smk7) mutant in maize

JIANG Cheng-Gong1,2, SHI Hui-Min2, WANG Hong-Wu2, LI Kun2, HUANG Chang-Ling2, LIU Zhi-Fang2, WU Yu-Jin2, LI Shu-Qiang2, HU Xiao-Jiao2,*, MA Qing1,*   

  1. 1National Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230036, Anhui, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, National Engineer Laboratory of Crop Molecular Breeding, Beijing 100081, China
  • Received:2020-03-11 Accepted:2020-08-19 Published:2021-02-12 Published online:2020-09-10
  • Contact: HU Xiao-Jiao,MA Qing
  • Supported by:
    National Natural Science Foundation of China(31500984);Agricultural Science and Technology Innovation Program of CAAS, and the Storing Grain in Technology for Maize (CAAS-ZDRW202004).

RichHTML

39

PDF

2191

摘要：

利用甲基磺酸乙酯(EMS)对玉米自交系B73进行诱变, 获得一个可以稳定遗传的小籽粒突变体smk7 (small kernel 7)。smk7成熟籽粒表现为体积变小, 胚和胚乳发育缺陷, 百粒重显著降低。突变籽粒发芽率仅为10%, 且幼苗黄化不能生长成正常植株。成熟smk7胚乳中淀粉、蛋白、油分含量与野生型籽粒相比无显著差异, 但突变体胚乳淀粉粒体积明显变小且形状不规则。smk7突变籽粒在授粉后12 d即可观察到明显的小籽粒和空瘪表型, 石蜡切片显微观察显示突变籽粒的胚和胚乳发育迟缓, 胚乳基部转移层细胞(BETL)相对于野生型细胞壁向内生长减少, 发育受阻。用杂合植株(+/smk7)与多个自交系分别杂交, 构建不同背景的F2分离群体, 遗传分析结果表明该性状受单隐性核基因控制。利用靶向测序基因型分型(genotyping by target sequencing, GBTS)技术将基因初定位于2号染色体短臂, 进一步精细定位发现该基因位于RM1433917和RM1535316两个标记之间约120 kb的物理范围内, 共有8个蛋白编码基因。本研究为进一步克隆和解析SMK7基因调控玉米籽粒发育的分子机制奠定了基础。

关键词: 玉米, 小粒突变体, 遗传分析, 基因定位

Abstract:

In this study, a stable small kernel mutant, named small kernel 7 (smk7), was isolated from ethylmethane sulfonate (EMS) mutagenesis of maize inbred line B73. Compared with wild type, the smk7 mutants showed smaller kernel size, defective embryo and endosperm development and a significant decrease in 100-kernel weight. The smk7 kernels showed a low level of germination rate at 10% and cannot grow into normal plants. No significant changes were detected in protein, starch and oil content between mature wild type and smk7 kernels, but the starch grains became significantly smaller and irregular in smk7 kernels compared with wild type. The smk7 kernels could be clearly distinguished from the wild type as early as 12 days after pollination (DAP), on the basis of their smaller and emptier phenotype. Microscopic inspection of the paraffin sections revealed that the development of embryo and endosperm were delayed, and the cell wall in growth in basal endosperm transfer layers (BETL) were arrested in smk7 compared with wild type. The F2 populations with multiple backgrounds were constructed by crossing heterozygous plants (+/smk7) with several other inbred lines. Genetic analysis showed that the mutant phenotype was controlled by a single recessive gene. Based on genotyping by target sequencing (GBTS) strategy, the SMK7 was initially mapped on the short arm of chromosome 2. The fine mapping results suggested that SMK7 was located between markers RM1433917 and RM1535316, with a physical distance of 120 kb. There were eight protein-coding genes in this region. This study laid a foundation for further genes cloning and research of the SMK7 function in regulating maize kernel development.

Key words: maize, small kernel mutant, genetic analysis, gene mapping

图1

smk7突变体表型 A: M4代果穗; B: smk7与野生型籽粒表型比较; C: smk7和野生型幼苗表型比较。"

图2

突变体籽粒和野生型籽粒的百粒重 **表示野生型与突变体在0.01水平差异显著。"

图3

smk7及野生型成熟籽粒扫描电镜照片 A: 野生型胚乳; B: smk7胚乳; SG为淀粉粒; PB为蛋白体; 标尺为20 μm。"

表1

基因定位引物序列"

引物
Primer		 正向序列
Forward sequence (5°-3′)		 反向序列
Reverse sequence (5′-3′)
In4.3 AACGCATCATCCTATGTCCAAC GGGTGAAGCCAGCCATTATTT
In1.9 ATGGTACGATCAACATAAAGGGAA GGCGTCACCGAAGAAATACAC
In0.83 GCAAGAAGCACCAGCCCT CGAGCGAAAGAAAGGAATGT
In1.16 ACATGACCCACGATCCAGACA TGCAGCCACTCTCCTTATGGT
SNP1 TTAGGGTGGAGTTGCTTCGC TTATGAATGAAGCACGGAAATGA
SNP2 TTAGGGTGGAGTTGCTTCGC TTATGAATGAAGCACGGAAATGA
SNP3 AAGGAATGGAGGCTTGGGTT ACAGCCGCCTTCGGATTC
SNP4 AGCGGTCCTTGACTTTATTTGA CATTACACGACCAATACAGCCA
SNP5 TGGCTTTTCATACCCTCCTCC CCTTCGCTGTGACTTGGATGT

图4

野生型籽粒和smk7籽粒的成分测定 **表示野生型与突变体在0.01水平差异显著。"

图5

授粉后不同时期胚和胚乳观察分析 A: 野生型籽粒在12、15、18、21、25和30 DAP胚和胚乳及完整种子的观察分析; 标尺为3 mm。B: 突变型籽粒(smk7)在12、15、18、21、25和30 DAP胚和胚乳及完整种子的观察分析; DAP: 授粉后天数; 标尺为3 mm。"

图6

授粉后不同天数野生型和突变体籽粒石蜡切片观察 A: 野生型授粉后12 d籽粒; 标尺为 1000 μm。B: smk7授粉后12 d籽粒; 标尺为1000 μm。C: WT授粉后12 d籽粒胚乳基部转移; 标尺为150 μm; D: smk7授粉后12 d籽粒胚乳基部转移层; 标尺为150 μm; BETL: 胚乳基部转移层细胞。"

表2

不同群体的遗传分离比检验"

世代
Populations		 总粒数
Total kernels		 正常籽粒数
Normal kernels		 突变籽粒数
Mutant kernels		 实际比例
Actual ratio		 理论比例
Theoretical ratio		 卡方值
χ2
M2 6372 4742 1630 2.91:1 3:1 1.1151
M3 9821 7412 2409 3.08:1 3:1 1.1367
F2 (Mo17×smk7) 5123 3870 1253 3.09:1 3:1 0.7730
F2 (Z58×smk7) 2016 1512 504 3:1 3:1 0.1120

图7

多态性SNP标记的数目"

图8

SNP index全基因组频率分布图"

图9

SMK7基因的精细定位 群体大小; Recombinants: 重组单株数。"

表3

候选区间内基因信息"

基因名称
Locus name		 基因注释
Gene annotation
ZM00001D001818 Probable polyol transporter 4
ZM00001D001819 N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase family protein
ZM00001D001820 Protochlorophyllide reductase1
ZM00001D001821 Unknown
ZM00001D001822 Unknown
ZM00001D001823 Anthranilate synthase beta subunit 1 chloroplastic
ZM00001D001824 Dof zinc finger protein DOF1.6
ZM00001D001825 Transducin/WD40 repeat-like superfamily protein
[1] Nuss E T, Tanumihardjo S A. Quality protein maize for Africa: closing the protein inadequacy gap in vulnerable populations. Adv Nutr, 2011,2:217-224.
doi: 10.3945/an.110.000182 pmid: 22332054
[2] Berger F. Endosperm development. Curr Opin Plant Biol, 1999,2:28-32.
doi: 10.1016/s1369-5266(99)80006-5 pmid: 10047564
[3] Guo Z F, Wang , Tao J J, Ren Y H, Xu C, Wu K S, Zou C, Zhang J A, Xu Y B. Development of multiple SNP marker panels affordable to breeders through genotyping by target sequencing (GBTS) in maize. Mol Breed, 2019,39:37-49.
[4] Sun X, Shantharaj D, Kang X, Ni M. Transcriptional and hormonal signaling control of Arabidopsis seed development. Curr Opin Plant Biol, 2010,13:611-620.
doi: 10.1016/j.pbi.2010.08.009 pmid: 20875768
[5] 任雪梅. 玉米籽粒发育基因Emp11及ZmSmk3的克隆及功能分析. 华中农业大学博士学位论文, 湖北武汉, 2018.
Ren X M. Cloning and Functional Analysis of Emp11 and ZmSmk3 Controlling Kernel Development in Maize PhD Dissertation of Huazhong Agricultural University, Wuhan, Hubei, China, 2018 (in Chinese with English abstract).
[6] 李见坤. 玉米籽粒发育基因UBL1的克隆与功能分析. 中国农业大学博士学位论文, 北京, 2016.
Li J K. Cloning and Characterization of UBL1 Controlling Kernel Development in Maize PhD Dissertation of China Agricultural University, Beijing, China, 2016 (in Chinese with English abstract).
[7] Neuffer M G, Sheridan W F. Defective kernel mutants of maize: I. Genetic and lethality studies. Genetics, 1980,95:929-944.
pmid: 17249053
[8] Wang G, Sun X, Wang G, Wang F, Gao Q, Sun X, Tang Y, Chang C, Lai J, Zhu L, Xu Z, Song R. Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm. Genetics, 2011,189:1281-1295.
doi: 10.1534/genetics.111.133967
[9] 姚东升. 玉米opaque10突变体基因的图位克隆和功能分析. 上海大学博士学位论文, 上海, 2016.
Yao D S. The Map-based Cloning and Functional Analysis of Maize opaque10 Gene PhD Dissertation of Shanghai University, Shanghai, China, 2016 (in Chinese with English abstract).
[10] Yao D, Qi W, Li X, Yang Q, Yan S, Ling H, Wang G, Wang G, Song R. Maize opaque10 encodes a cereal-specific protein that is essential for the proper distribution of zeins in endosperm protein bodies. PLoS Genet, 2016,12:e1006270.
doi: 10.1371/journal.pgen.1006270 pmid: 27541862
[11] Holding D R, Otegui M S, Li B, Meeley R B, Dam T, Hunter B G, Jung R, Larkins B A, Larkins B A. The maize floury1 gene encodes a novel endoplasmic reticulum protein involved in zein protein body formation. Plant Cell, 2007,19:2569-2582.
doi: 10.1105/tpc.107.053538 pmid: 17693529
[12] Mertz E T, Bates L S, Nelson O E. Mutant gene that changes protein composition and increases lysine content of maize endosperm. Science, 1964,145:279-280.
pmid: 14171571
[13] Schmidt R J, Burr F A, Aukerman M J, Burr B. Maize regulatory gene opaque-2 encodes a protein with a“leucine-zipper”motif that binds to zein DNA. Proc Natl Acad Sci USA, 1990,87:46-50.
doi: 10.1073/pnas.87.1.46 pmid: 2296602
[14] Schmidt R J, Ketudat M, Aukerman M J, Hoschek G. Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes. Plant Cell, 1992,4:689-700.
doi: 10.1105/tpc.4.6.689 pmid: 1392590
[15] Cord-Neto G, Yunes J A, da Silva M J, Vettore A L, Arruda P, Leite A. The involvement of opaque 2 on beta-prolamin gene regulation in maize and coix suggests a more general role for this transcriptional activator. Plant Mol Biol, 1995,27:1015-1029.
doi: 10.1007/BF00037028 pmid: 7766871
[16] Li Q, Wang J, Ye J, Zheng X, Xiang X, Li C, Fu M, Wang Q, Zhang Z, Wu Y. The maize imprinted gene FLOURY3 encodes a PLATZ protein required for tRNA and 5S rRNA transcription through interaction with RNA polymerase III. Plant Cell, 2017,29:2661-2675.
doi: 10.1105/tpc.17.00576 pmid: 28874509
[17] Silva-Sanchez C, Chen S, Li J, Chourey P S. A comparative glycoproteome study of developing endosperm in the hexose-deficient miniature1 (mn1) seed mutant and its wild type Mn1 in maize. Front Plant Sci, 2014,5:63-77.
doi: 10.3389/fpls.2014.00063 pmid: 24616729
[18] Zhu C, Jin G, Fang P, Zhang Y, Feng X, Tang Y, Qi W, Song R. Maize pentatricopeptide repeat protein DEK41 affects cis-splicing of mitochondrial nad4 intron 3 and is required for normal seed development. J Exp Bot, 2019,70:3795-3808.
doi: 10.1093/jxb/erz193 pmid: 31020318
[19] Tian Q, Olsen L, Sun B, Lid S E, Brown R C, Lemmon B E, Fosnes K, Gruis D F, Opsahl-Sorteberg H G, Otegui M S, Olsen O A. Subcellular localization and functional domain studies of DEFECTIVE KERNEL1 in maize and Arabidopsis suggest a model for aleurone cell fate specification involving CRINKLY4 and SUPERNUMERARY ALEURONE LAYER1. Plant Cell, 2007,19:3127-3145.
pmid: 17933905
[20] Yuan N, Wang J, Zhou Y, An D, Xiao Q, Wang W, Wu Y. EMB-7L is required for embryogenesis and plant development in maize involved in RNA splicing of multiple chloroplast genes. Plant Sci, 2019,287:110203.
doi: 10.1016/j.plantsci.2019.110203 pmid: 31481208
[21] Shen Y, Li C, McCarty D R, Meeley R, Tan B C. Embryo defective12 encodes the plastid initiation factor 3 and is essential for embryogenesis in maize. Plant J, 2013,74:792-804.
doi: 10.1111/tpj.12161 pmid: 23451851
[22] Ding S, Liu X Y, Wang H C, Wang Y, Tang J J, Yang Y Z, Tan B C. SMK6 mediates the C-to-U editing at multiple sites in maize mitochondria. J Plant Physiol, 2019,240:152992.
pmid: 31234031
[23] Wang H C, Sayyed A, Liu X Y, Yang Y Z, Sun F, Wang Y, Wang M, Tan B C. SMALL KERNEL4 is required for mitochondrial cox1 transcript editing and seed development in maize. J Integr Plant Biol, 2019.
pmid: 33215867
[24] Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, Bruyere C, Caboche M, Debast C, Gualberto J, Hoffmann B, Lecharny A, Le Ret M, Martin-Magniette M L, Mireau H, Peeters N, Renou J P, Szurek B, Taconnat L, Small I. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell, 2004,16:2089-2103.
doi: 10.1105/tpc.104.022236 pmid: 15269332
[25] Wang Y, Liu W, Wang H, Du Q, Fu Z, Li W X, Tang J. ZmEHD1 is required for kernel development and vegetative growth through regulating auxin homeostasis. Plant Physiol, 2019,182:1467-1480.
doi: 10.1104/pp.19.01336 pmid: 31857426
[26] Li X J, Zhang Y F, Hou M, Sun F, Shen Y, Xiu Z H, Wang X, Chen Z L, Sun S S, Small I, Tan B C. Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa). Plant J, 2014,79:797-809.
doi: 10.1111/tpj.12584 pmid: 24923534
[27] Yang Y Z, Ding S, Wang Y, Li C L, Shen Y, Meeley R, McCarty D R, Tan B C. Small kernel2 encodes a glutaminase in Vitamin B6 biosynthesis essential for maize seed development. Plant Physiol, 2017,174:1127-1138.
pmid: 28408540
[28] Pan Z, Ren X, Zhao H, Liu L, Tan Z, Qiu F. A mitochondrial transcription termination factor, ZmSmk3, is required for nad1 intron4 and nad4 intron1 splicing and kernel development in maize. G3: Genes Genom Genet, 2019,9:2677-2686.
[29] Pan Z, Liu M, Xiao Z, Ren X, Zhao H, Gong D, Liang K, Tan Z, Shao Y, Qiu F. ZmSMK9, a pentatricopeptide repeat protein, is involved in the cis-splicing of nad5, kernel development and plant architecture in maize. Plant Sci, 2019,288:110205.
doi: 10.1016/j.plantsci.2019.110205 pmid: 31521217
[30] Handa N, Terada T, Kamewari Y, Hamana H, Tame J R, Park S Y, Kinoshita K, Ota M, Nakamura H, Kuramitsu S, Shirouzu M, Yokoyama S. Crystal structure of the conserved protein TT1542 from thermus thermophilus HB8. Protein Sci, 2003,12:1621-1632.
doi: 10.1110/gad.03104003 pmid: 12876312
[31] 王家利, 刘冬成, 郭小丽, 张爱民. 生长素合成途径的研究进展. 植物学报, 2012,47:292-301.
Wang J L, Liu D C, Guo X L, Zhang A M. Research progress of auxin synthesis pathway. Chin Bull Bot, 2012,47:292-301 (in Chinese with English abstract).
[32] Bashline L, Li S, Zhu X, Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis. Proc Natl Acad Sci USA, 2015,112:12870-12875.
doi: 10.1073/pnas.1509292112 pmid: 26417106
[1] 肖颖妮, 于永涛, 谢利华, 祁喜涛, 李春艳, 文天祥, 李高科, 胡建广. 基于SNP标记揭示中国鲜食玉米品种的遗传多样性[J]. 作物学报, 2022, 48(6): 1301-1311.
[2] 崔连花, 詹为民, 杨陆浩, 王少瓷, 马文奇, 姜良良, 张艳培, 杨建平, 杨青华. 2个玉米ZmCOP1基因的克隆及其转录丰度对不同光质处理的响应[J]. 作物学报, 2022, 48(6): 1312-1324.
[3] 郑崇珂, 周冠华, 牛淑琳, 和亚男, 孙伟, 谢先芝. 水稻早衰突变体esl-H5的表型鉴定与基因定位[J]. 作物学报, 2022, 48(6): 1389-1400.
[4] 王丹, 周宝元, 马玮, 葛均筑, 丁在松, 李从锋, 赵明. 长江中游双季玉米种植模式周年气候资源分配与利用特征[J]. 作物学报, 2022, 48(6): 1437-1450.
[5] 杨欢, 周颖, 陈平, 杜青, 郑本川, 蒲甜, 温晶, 杨文钰, 雍太文. 玉米-豆科作物带状间套作对养分吸收利用及产量优势的影响[J]. 作物学报, 2022, 48(6): 1476-1487.
[6] 陈静, 任佰朝, 赵斌, 刘鹏, 张吉旺. 叶面喷施甜菜碱对不同播期夏玉米产量形成及抗氧化能力的调控[J]. 作物学报, 2022, 48(6): 1502-1515.
[7] 徐田军, 张勇, 赵久然, 王荣焕, 吕天放, 刘月娥, 蔡万涛, 刘宏伟, 陈传永, 王元东. 宜机收籽粒玉米品种冠层结构、光合及灌浆脱水特性[J]. 作物学报, 2022, 48(6): 1526-1536.
[8] 单露英, 李俊, 李亮, 张丽, 王颢潜, 高佳琪, 吴刚, 武玉花, 张秀杰. 转基因玉米NK603基体标准物质研制[J]. 作物学报, 2022, 48(5): 1059-1070.
[9] 王好让, 张勇, 于春淼, 董全中, 李微微, 胡凯凤, 张明明, 薛红, 杨梦平, 宋继玲, 王磊, 杨兴勇, 邱丽娟. 大豆突变体ygl2黄绿叶基因的精细定位[J]. 作物学报, 2022, 48(4): 791-800.
[10] 许静, 高景阳, 李程成, 宋云霞, 董朝沛, 王昭, 李云梦, 栾一凡, 陈甲法, 周子键, 吴建宇. 过表达ZmCIPKHT基因增强植物耐热性[J]. 作物学报, 2022, 48(4): 851-859.
[11] 刘磊, 詹为民, 丁武思, 刘通, 崔连花, 姜良良, 张艳培, 杨建平. 玉米矮化突变体gad39的遗传分析与分子鉴定[J]. 作物学报, 2022, 48(4): 886-895.
[12] 闫宇婷, 宋秋来, 闫超, 刘爽, 张宇辉, 田静芬, 邓钰璇, 马春梅. 连作秸秆还田下玉米氮素积累与氮肥替代效应研究[J]. 作物学报, 2022, 48(4): 962-974.
[13] 徐宁坤, 李冰, 陈晓艳, 魏亚康, 刘子龙, 薛永康, 陈洪宇, 王桂凤. 一个新的玉米Bt2基因突变体的遗传分析和分子鉴定[J]. 作物学报, 2022, 48(3): 572-579.
[14] 宋仕勤, 杨清龙, 王丹, 吕艳杰, 徐文华, 魏雯雯, 刘小丹, 姚凡云, 曹玉军, 王永军, 王立春. 东北主推玉米品种种子形态及贮藏物质与萌发期耐冷性的关系[J]. 作物学报, 2022, 48(3): 726-738.
[15] 渠建洲, 冯文豪, 张兴华, 徐淑兔, 薛吉全. 基于全基因组关联分析解析玉米籽粒大小的遗传结构[J]. 作物学报, 2022, 48(2): 304-319.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2