中国农业科学 ›› 2018, Vol. 51 ›› Issue (8): 1421-1430.doi: 10.3864/j.issn.0578-1752.2018.08.001

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

玉米SLAF标记的开发及其在玉米果皮纤维素含量BSA分析中的应用

杜龙岗,王美兴   

  1. 浙江省农业科学院作物与核技术利用研究所,杭州 310021
  • 收稿日期:2017-12-06 出版日期:2018-04-16 发布日期:2018-04-16
  • 通讯作者: 王美兴,E-mail:wangmeixing1975@126.com
  • 作者简介:杜龙岗,E-mail:51968582@qq.com
  • 基金资助:
    浙江省农业(粮食)新品种选育重大科技专项(2016C02050-9-2)

SLAF-marker Development and Its Application in BSA Analysis of Cellulose Content in Pericarp of Maize Kernel

DU LongGang, WANG MeiXing   

  1. Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
  • Received:2017-12-06 Online:2018-04-16 Published:2018-04-16

摘要: 【目的】降低果皮纤维素是甜玉米品质改良的重要目标,然而玉米果皮纤维素含量调控的研究甚少,相关调控基因尚未定位。利用纤维素含量差异的重组自交系(RILs)群体,通过特异位点扩增片段(specific-locus amplified fragment-sequencing,SLAF)测序和分离混池分析(bulked segregant analysis,BSA)定位控制玉米果皮纤维素含量的染色体区段,鉴定调控玉米果皮纤维素含量的候选基因。【方法】以果皮纤维素含量显著差异的E327和G5-1为亲本,构建重组自交系(RILs)。对RILs群体进行果皮纤维素含量的测定,并根据纤维素含量的结果选择纤维素含量高、低的样本进行混池,用于SLAF标签的鉴定和BSA分析。在BSA分析中,首先对两混池和2个亲本DNA用HaeⅢ和Hpy166Ⅱ进行酶切,回收414—464 bp的酶切片段进行Illumina建库,并进行SLAF测序,然后根据多态性SLAF标签开发SNP标记,利用SNP标记对玉米果皮纤维素含量进行关联分析,鉴定调控甜玉米果皮纤维素含量的染色体区段。分析这些区段所包含的玉米基因,并找到它们对应的拟南芥同源基因,通过查阅拟南芥相关基因功能研究的文献,进一步鉴定控制玉米果皮纤维素含量的候选基因。【结果】两亲本和2个混池SLAF建库测序得到的SLAF符合预期,基于SLAF测序数据,鉴定了73 786个多态性SLAF标签,这些SLAF标签均匀分布在玉米的10条染色体上。在这些多态性标签中得到了523 395 SNP位点信息。通过关联分析,调控果皮纤维素变异的基因被定位到玉米基因组的6个染色区段,都位于玉米的第5染色体上。在这些区段上,一共有47个玉米基因。通过进一步的研究分析,在这些关联的染色体区段最终确定了9个候选基因。【结论】定位到调控玉米果皮纤维素的含量的基因,表明此方法可以用于基因定位。

关键词: 玉米, 果皮, 纤维素, SLAF测序, BSA分析

Abstract: 【Objective】 Reducing cellulose content in pericarp is one of the important goals for quality improvement of sweet maize. However, the research focusing on cellulose content in pericarp of maize is limited and the related gene has not been identified. In order to map the chromosome regions and candidate genes controlling cellulose content in pericarp of sweet maize, specific-locus amplified fragment-sequencing (SLAF-seq) and bulked segregant analysis (BSA) were conducted in this study. 【Method】 A recombinant inbred line (RIL) population was constructed using E327 and G5-1 as parents. In F6 generation of RILs, the cellulose contents in pericarp of each line were detected. According to cellulose content results, the lines with relatively high and low cellulose contents in pericarp were selected for SLAF tags identification and BSA. For BSA, DNA was extracted from two bulked populations as well as two parent lines and digested with HaeⅢ and Hpy166Ⅱ restriction enzymes. After digestion, the 414-464 bp DNA fragments were recovered and used for Illumina library construction which were subsequently sequenced by 2nd generation sequencing technology. Based on the polymorphic SLAF tags developed in this analysis, the SNPs-cellulose content association analysis was performed to map the chromosome regions associated with cellulose content in pericarp of sweet maize. Then, the genes located on the associated regions were found. To further reduce the number of candidate genes, the genes in Arabidopsis homological to these genes were identified and annotated. Based on the published works related on the functional analysis of these Arabidopsis genes, we further identified the candidate genes regulating cellulose content in pericarp of sweet maize. 【Result】The sequencing results of Illumina libraries were consistent with expected. As a result, 73 786 polymorphic SLAF tags were identified, which were uniformly distributed on 10 chromosomes. A total of 523 395 SNPs in those polymorphic tags were identified from the SLAF-sequencing data. Genes responsible for cellulose content in pericarp were mapped onto 6 chromosome regions of maize genome via association analysis and all these 6 chromosome regions were located on the 5th chromosome. In total, 47 gene loci in those regions and 9 genes in these associated regions were identified as candidate genes in further analysis. 【Conclusion】Through SLAF-sequencing based bulking segregated analysis, the cellulose content related genes in pericarp of sweet maize were mapped, suggesting that this method can be applied in gene mapping for other traits.

Key words: maize, pericarp, cellulose, specific-locus amplified fragment-sequencing, bulked segregant analysis

[1]    葛欣然. 我国玉米产业发展现状及政策调整. 新农业, 2017, 10: 7-8.
GE X R. Current situation and policy adjustment of corn industry in China. New Agriculture, 2017, 10: 7-8. (in Chinese)
[2]    丁鑫. 我国玉米产业现状及发展趋势. 现代农村科技, 2017, 10: 10-11.
DING X. Current situation and development trend of corn industry in China. Modern Rural Science and Technology,2017, 10: 10-11. (in Chinese)
[3]    赵华, 王子明. 高产优质甜玉米新品种筛选. 玉米科学, 2017, 25(3): 38-42.
ZHAO H, WANG Z M. Screening of new sweet corn varieties with high yield and good quality. Journal of Maize Sciences,2017, 25(3): 38-42. (in Chinese)
[4]    石明亮, 薛林, 胡加如, 黄小兰, 陈国清, 陆虎华. 玉米和特用玉米的营养保健作用及加工利用途径. 中国食物与营养, 2011, 17(2): 66-71.
SHI M L, XIE L, HU J R, HUANG X L, CHEN G Q, LU H H. Nutritional and health effects of maize and special corn and its processing and utilization. Food and Nutrition in China, 2011, 17(2): 66-71. (in Chinese)
[5]    Dewanto V, Wu X Z, Liu R H. Processed sweet corn has higher antioxidant activity. Journal Agricultural and Food Chemistry,2002, 50(17): 4959-4964.
[6]    岳武. 甜玉米育种中几个问题的探讨. 辽宁农业科学, 2017(1): 62 -63.
YUE W. Discussion on several problems in sweet corn breeding. Liaoning Agricultural Sciences, 2017(1): 62 -63. (in Chinese)
[7]    戴惠学, 施泽平. 优质高产超甜玉米晶甜3号的选育报告. 玉米科学, 2006, 14(2): 64-65.
DAI H X, SHI Z P. Reports on the selecting & breeding of super sweet corn Jingtian No.3 with high yield and good quality. Journal of Maize Sciences,2006, 14(2): 64-65. (in Chinese)
[8]    姚文华, 韩学莉, 汪燕芬, 谭静, 徐春霞, 陈洪梅, 番兴明. 我国甜玉米育种研究现状与发展对策. 中国农业科技导报, 2011, 13(2): 1-8.
YAO W H, HAN X L, WANG Y F, TAN J,XU C X, CHEN H M, FAN X M. Research status and development strategy for sweet corn breeding in China. Journal of Agricultural Science and Technology, 2011, 13(2): 1-8. (in Chinese)
[9]    林建新, 卢和顶, 廖长见. 甜玉米新品种闽甜4号的选育. 福建农业学报, 2012, 27(6): 611-616.
LIN J X, LU H D, LIAO C J. Breeding a new sweet corn variety, Mintian4. FujianJournal of Agricultural Sciences,2012, 27(6): 611-616. (in Chinese)
[10]   王蕴波, 杨泉女, 陈文胜, 甄畅迪. 华南地区甜玉米的育种目标初探. 佛山科学技术学院学报(自然科学版), 2010, 28(2): 68-72.
WANG Y B, YANG Q N, CHEN W S, ZHEN C D. Breeding objectives of sweet corn in South China. Journal of Foshan University (Natural Science Edition),2010, 28(2): 68-72. (in Chinese)
[11]   林金元, 王慧, 潘玲玲, 孙大鹏, 于典司, 施标, 卢有林, 郑洪建. 超甜玉米新品种“金银898”的选育. 上海农业学报, 2015, 31(3): 94-97.
Lin J Y, WANG H, PAN L L, SUN D P, YU D S, SHI B, LU Y L, ZHENG H J. Breeding of a new super-sweet corn cultivar ‘Jinyin 898’. Acta Agriculture Shanghai, 2015, 31(3): 94-97. (in Chinese)
[12]   孙萍东, 潘玲玲, 徐声宇, 周美华, 王慧, 孙大鹏, 于典司, 郑洪建, 林金元. 超甜玉米新品种“沪甜1”的选育. 上海农业学报, 2016, 32 (3): 164-167.
SUN P D, PAN L L, XU S Y, ZHOU M H, WANG H, SUN D P, YU D S, ZHENG H J, LIN J Y. Breeding of a new super sweet corn cultivar ‘Hutian 1’. Acta Agriculture Shanghai, 2016, 32(3): 164-167. (in Chinese)
[13]   林建新, 陈山虎, 廖长见, 卢和顶. 甜玉米新品种“闽甜6855”的选育. 福建农业学报, 2016, 31(11): 1171-1174.
LIN J X, CHEN S H, LIAO C J, LU H D. Breeding a new sweet corn variety, Mintian6855. Fujian Journal of Agricultural Sciences, 2016, 31(11): 1171-1174. (in Chinese)
[14]   张士龙, 贺正华, 黄益勤. 超甜玉米子粒果皮柔嫩度的变化规律. 湖北农业科学, 2016, 55(5): 1105-1108.
ZHANG S L, HE Z H, HUANG Y Q. Variation law of pericarp tenderness of super sweet corn kernel. HubeiAgricultural Sciences,2016, 55(5): 1105-1108. (in Chinese)
[15]   Schmidt D H, Tracy W F. Effects of starchy sugary and sugary sugary endosperm on pericarp thickness in sweet corn. Hortscience,1988, 23(5): 885-886.
[16]   乐素菊, 刘厚诚, 张璧, 王晓明. 超甜玉米籽粒乳熟期碳水化合物变化及食用品质. 华南农业大学学报(自然科学版), 2003, 24(2): 9-11.
YUE S J, LIU H C, ZHANG B, WANG X M. Changes of carbohydrate and taste quality in the kernels of super-sweet corn in the milky maturity stage. Journal of South China Agricultural University (Natural Science Edition),2003, 24(2): 9-11. (in Chinese)
[17]   王世恒, 冯凤琴, 徐仁政. 超甜玉米营养品质分析. 玉米科学, 2004, 12(1): 61-62.
WANG S H, FENG F Q, XU R Z. Analysis on nutritional quality of super-sweet corn. Journal of Maize Sciences, 2004, 12(1): 61-62. (in Chinese)
[18]   王林风, 程远超. 硝酸乙醇法测定纤维素含量. 化学研究, 2011, 22(4): 52-55.
WANG L F, CHENG Y C. Determination the content of cellulose by nitric acid-ethanol method. Chemical Research, 2011, 22(4): 52-55. (in Chinese)
[19]   Kozich J J, Westcott S L, Baxter N T, HIGHLANDER S K, SCHLOSS P D.Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and environmental microbiology, 2013, 79(17): 5112-5120.
[20]   Sun X, Liu D, Zhang X, LI W, LIU H, HONG W, JIANG C, GUAN N, MA C, ZENG H, XU C, SONG J, HUANG L, WANG C, SHI J, WANG R, ZHENG X, LU C, WANG X, ZHEGN H. SLAF-seq: an efficient method of large-scale De novo SNP discovery and genotyping using high-throughput sequencing. PloS one,2013, 8(3): e58700.
[21]   Hill J T, Demarest B L, Bisgrove B W, Gorsi B, Su Y C, Yost H J. MMAPPR: Mutation mapping analysis pipeline for pooled RNA-seq. Genomes Research, 2013. 23: 687-697.
[22]   Li R, Li Y, Kristiansen K, WANG J. SOAP: short oligonucleotide alignment program. Bioinformatics, 2008, 24(5): 713-714.
[23]   Xia C, CHEN L, RONG T, LI R, XIANG Y, WANG P, LIU C, DONG X, LIU B, ZHAO D, WEI R, LAN H. Identification of a new maize inflorescence meristem mutant and association analysis using SLAF-seq method. Euphytica,2015, 202: 35-44.
[24]   Won S K, Lee Y J, Lee H Y, HEO Y K, CHO M, CHO H T. Cis-element-and transcriptome-based screening of root hair-specific genes and their functional characterization in Arabidopsis. Plant physiology, 2009, 150(3): 1459-1473.
[25]   Baumberger N, Steiner M, Ryser U, KELLER B, RINGLI C. Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in cell wall formation during root hair development. The Plant Journal, 2003, 35(1): 71-81.
[26]   Yadav V K, Yadav V K, Pant P, singh s p, maurya r, sable a, sawant s v. GhMYB1 regulates SCW stage-specific expression of the GhGDSL promoter in the fibres of Gossypium hirsutum L.. Plant Biotechnology Journal,2017, 15(9): 1163-1174.
[27]   Passardi F, Cosio C, Penel C, DUNAND C. Peroxidases have more functions than a Swiss army knife. Plant cell reports, 2005, 24(5): 255-265.
[28]   Liszkay A, Kenk B, Schopfer P. Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth. Planta,2003, 217(4): 658-667.
[29]   Van H L, Kuraishi S, Sakurai N. Aluminum-induced rapid root inhibition and changes in cell-wall components of squash seedlings. Plant Physiology, 1994, 106(3): 971-976.
[30]   Goodwin S B, Sutter T R. Microarray analysis of Arabidopsis genome response to aluminum stress. Biologia Plantarum,2009, 53(1): 85-99.
[31]   Fernandezperez F, Pomar F, Pedreno M A, NOVO-UZAL E. The suppression of AtPrx52 affects fibers but not xylem lignification in Arabidopsis by altering the proportion of syringyl units. Physiologia Plantarum,2015, 154(3): 395-406.
[32]   Zhang Q, Cheetamun R, Dhugga K S, Rafalski J A, Tingey S V, SHIRLEY N J, TAYLOR J, HAYES K, BEATTY M, BACIC A, BURTON R A, FINCHER G B. Spatial gradients in cell wall composition and transcriptional profiles along elongating maize internodes. BMC plant biology,2014, 14(1): 27.
[33]   Eulgem T, Rushton P J, Robatzek S, SOMSSICH I E. The WRKY superfamily of plant transcription factors. Trends in Plant Science,2000, 5(5): 199-206.
[34]   Yu D, Chen C, Chen Z. Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. The Plant Cell, 2001, 13(7): 1527-1540.
[35]   Dong J, Chen C, Chen Z. Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Molecular Biology,2003, 51(1): 21-37.
[36]   Miao Y, Laun T M, Zimmermann P, ZENTGRAF U. Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Molecular Biology,2004, 55(6): 853-867.
[37]   Xu X, Chen C, Fan B, CHEN Z. Physical and Functional Interactions between Pathogen-Induced Arabidopsis WRKY18, WRKY40, and WRKY60 Transcription Factors. The Plant Cell, 2006, 18(5): 1310-1326.
[38]   Lu S, Li L, Yi X, JOSHI C P, CHIANG V L. Differential expression of three eucalyptus secondary cell wall-related cellulose synthase genes in response to tension stress. Journal of experimental botany, 2008, 59(3): 681-695.
[39]   Wang H, Avci U, Nakashima J, Hahn M G, Chen F, Dixon R A. Mutation of WRKY transcription factors initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107: 22338-22343.
[40]   Yu F, Huaxia Y, Lu W, WU C, CAO X, GUO X. GhWRKY15, a member of the WRKY transcription factor family identified from cotton (Gossypium hirsutum L.), is involved in disease resistance and plant development. BMC Plant Biology, 2012, 12(1): 144.
[1] 陈鹏飞,李刚,石雅娇,徐志涛,杨粉团,曹庆军. 一款无人机高光谱传感器的验证及其在玉米叶面积指数反演中的应用[J]. 中国农业科学, 2018, 51(8): 1464-1474.
[2] 李婷,李世清,占爱,刘建亮. 地膜覆盖、氮肥与密度及其互作对黄土旱塬春玉米氮素吸收、转运及生产效率的影响[J]. 中国农业科学, 2018, 51(8): 1504-1517.
[3] 杨云马,孙彦铭,贾良良,贾树龙,孟春香. 磷肥施用深度对夏玉米产量及根系分布的影响[J]. 中国农业科学, 2018, 51(8): 1518-1526.
[4] 周丽平,杨俐苹,白由路,卢艳丽,王磊. 夏玉米施用不同缓释化处理氮肥的效果及氮肥去向[J]. 中国农业科学, 2018, 51(8): 1527-1536.
[5] 任胜茂,邓榆川,文凤君,Sajad Hussain,蒲全明,于晓波,刘卫国,杨文钰. 套作对大豆苗期茎秆纤维素合成相关糖类物质转化的影响及其与叶片光合的关系[J]. 中国农业科学, 2018, 51(7): 1272-1282.
[6] 孙粲然,张雪海,马指挥,郭战勇,汤继华,付志远. 玉米籽粒淀粉粒密度基因tw1的精细定位[J]. 中国农业科学, 2018, 51(7): 1233-1243.
[7] 温维亮,郭新宇,赵春江,肖伯祥,王勇健. 基于三维数字化的玉米株型参数提取方法研究[J]. 中国农业科学, 2018, 51(6): 1034-1044.
[8] 刘坤,张雪海,孙高阳,闫鹏帅,郭海平,陈思远,薛亚东,郭战勇,谢惠玲,汤继华,李卫华. 玉米株型相关性状的全基因组关联分析[J]. 中国农业科学, 2018, 51(5): 821-834.
[9] 银敏华,李援农,陈朋朋,徐路全,申胜龙,王星垚. 基于Meta-analysis的中国北方地区免耕玉米产量效应研究[J]. 中国农业科学, 2018, 51(5): 843-854.
[10] 赵久然,李春辉,宋伟,王元东,张如养,王继东,王凤格,田红丽,王蕊. 基于SNP芯片揭示中国玉米育种种质的遗传多样性与群体遗传结构[J]. 中国农业科学, 2018, 51(4): 626-634.
[11] 王寅,徐卓,李博凝,高强,冯国忠,李翠兰,焉莉,王少杰. 尿素硝铵溶液对黑土区春玉米产量和氮素吸收利用的影响[J]. 中国农业科学, 2018, 51(4): 718-727.
[12] 冯小杰,郑子成,李廷轩,范丽. 暴雨条件下紫色土区玉米季坡耕地氮素流失特征[J]. 中国农业科学, 2018, 51(4): 738-749.
[13] 周敏,周雪梅,杨立杰,黄丽波,冯蕾,邵明慧,杨晨,杨维仁,杨在宾,姜淑贞 . 玉米赤霉烯酮对断奶小母猪子宫形态学及热应激蛋白70分布和表达的影响[J]. 中国农业科学, 2018, 51(4): 778-788.
[14] 陈先敏,梁效贵,赵雪,高震,吴巩,申思,林珊,周丽丽,周顺利. 历年国审玉米品种产量和品质性状变化趋势分析[J]. 中国农业科学, 2018, 51(21): 4020-4029.
[15] 刘兆辉,吴小宾,谭德水,李彦,江丽华. 一次性施肥在我国主要粮食作物中的应用与环境效应[J]. 中国农业科学, 2018, 51(20): 3827-3839.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!