绿豆窄叶突变体<i>vrnl11</i>的精细定位

doi:10.7668/hbnxb.20194319

摘要/Abstract

摘要：

绿豆叶形突变体和叶形调控基因的鉴定,可为品种叶形的遗传改良提供种质资源,也有利于解析叶片发育的遗传调控机理。从皖科绿3号EMS诱变突变体库中筛选到窄叶突变体vrnl11,利用vrnl11/皖科绿3号和vrnl11/中绿1号的杂交后代进行遗传分析,用χ²检验确定F₂群体中不同表型植株的分离模式。以vrnl11和中绿1号及中绿5号构建的2个F₂群体为定位群体,利用BSA测序技术和图位克隆的方法完成vrnl11的精细定位。表型鉴定结果表明,vrnl11叶片宽和叶面积较野生型皖科绿3号分别减少25.7%和21.7%。遗传分析表明,vrnl11的窄叶表型受单隐性核基因调控。BSA测序分析将突变位点定位在第11染色体上15.0 Mb至末端4.7 Mb的区间内。利用新开发的多态性分子标记将vrnl11定位在标记nl-61和nl-46之间186.5 kb的区间内,包含9个预测基因。这些研究结果为克隆vrnl11和解析绿豆叶片生长发育的分子调控机理提供理论依据。

关键词: 绿豆, 窄叶, 突变体vrnl11, 基因定位

Abstract:

The identification of leaf shape mutant and genes controlling leaf shape can not only provide germplasm resources for genetic improvement of leaf shape,but also help to analyze the genetic regulation mechanism of leaf development.vrnl11 was identified from an EMS induced Wankelü 3(WK3)mutant library.Progeny populations derived from vrnl11/WK3 and vrnl11/Zhonglü 1 were used for genetic analysis,and the segregation pattern of different phenotypic plants in F₂ populations was determined by χ² test.Two F₂ populations constructed by crossing vrnl11 with Zhonglü 1 and Zhonglü 5 were used as mapping populations.Fine mapping for vrnl11 was completed by using BSA sequencing technology and map-based cloning strategy.Phenotype identification results showed that,compared with wild-type WK3,the leaf width and leaf area of vrnl11 decreased by 25.7% and 21.7%,respectively.Genetic analysis showed the narrow leaf phenotype of vrnl11 was controlled by a single recessive nuclear gene.BSA sequencing analysis located the mutation site within a 4.7 Mb interval from 15.0 Mb to the chromosomal end on chromosome 11.vrnl11 was finally located in the 186.5 kb interval between the markers nl-61 and nl-46 by using these newly developed polymorphic molecular markers.The mapping interval contained 9 predicted genes.These results provide a theoretical basis for cloning vrnl11 and understanding the molecular regulation mechanism of leaf development in mungbean.

Key words: Mungbean, Narrow leaf, Mutant vrnl11, Gene mapping

叶卫军, 吴泽江, 田东丰, 周斌. 绿豆窄叶突变体vrnl11的精细定位[J]. 华北农学报, 2024, 39(1): 1-7. doi: 10.7668/hbnxb.20194319.

YE Weijun, WU Zejiang, TIAN Dongfeng, ZHOU Bin. Fine Mapping of a Narrow Leaf Mutant vrnl11 in Mungbean[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(1): 1-7. doi: 10.7668/hbnxb.20194319.

http://www.hbnxb.net/CN/Y2024/V39/I1/1

图/表 9

图1 野生型皖科绿3号与突变体vrnl11叶片表型

Fig.1 Leaf phenotype of wild-type WK3 and mutant vrnl11

表1 皖科绿3号和突变体vrnl11农艺性状比较

Tab.1 Agronomic traits comparison between WK3 and mutant vrnl11

农艺性状 Agronomic trait	野生型(皖科绿3号) Wild-type (WK3)		突变体(vrnl11) Mutant (vrnl11)
叶片长/cm Leaf length	13.6±	0.6A	13.8±	0.7A
叶片宽/cm Leaf width	11.3±	0.8A	8.4±	0.5B
叶片长宽比 Leaf length/width ratio	1.2±	0.1A	1.7±	0.1B
叶面积/cm² Leaf area	109.0±	6.1A	85.3±	5.3B
开花期/d Days from sowing to flowering	37.8±	0.6A	40.0±	0.5A
全生育天数/d Growth period days	62.8±	0.4A	63.7±	0.6A
株高/cm Plant height	66.4±	3.3A	64.0±	1.6A
分枝数 Branches	2.7±	0.8A	2.9±	0.7A
单株荚数 Number of pod per plant	25.8±	3.1A	25.7±	3.4A
单荚粒数 Number of seed per pod	11.0±	1.3A	10.7±	1.2A
百粒质量/g 100-seed weight	5.90±	0.07A	5.83±	0.06A
单株产量/g Yield per plant	15.6±	2.0A	15.5±	1.2A

表2 窄叶突变体vrnl11的遗传分析

Tab.2 Genetic analysis of the narrow leaf mutant vrnl11

组合 Combination	总株数 Total number	正常植株数 Number of normal leaf plants	窄叶植株数 Number of narrow leaf plants	期望比 Expected ratio	$χ 3 : 1 2$ 值 $χ 3 : 1 2$ value	P值 P value
vrnl11/WK3	156	114	42	3:1	0.31	0.58
vrnl11/ZL1	154	117	37	3:1	0.08	0.78

表2 窄叶突变体vrnl11的遗传分析

Tab.2 Genetic analysis of the narrow leaf mutant vrnl11

组合 Combination	总株数 Total number	正常植株数 Number of normal leaf plants	窄叶植株数 Number of narrow leaf plants	期望比 Expected ratio	$χ 3 : 1 2$ 值 $χ 3 : 1 2$ value	P值 P value
vrnl11/WK3	156	114	42	3:1	0.31	0.58
vrnl11/ZL1	154	117	37	3:1	0.08	0.78

图2 SNP-index和ΔSNP-index在染色体上的分布 SNP-index(A)和SNP-index(B)分别为突变混池和野生型混池的SNP-index分布图;图中的点即为SNP-index,红线为窗口下SNP-index 或ΔSNP-index的均值;绿线和橙线分别为95%和99%置信水平阈值线。

Fig.2 Distribution of SNP-index and ΔSNP-index value on chromosomes SNP-index(A)and SNP-index(B)were the distribution of SNP-index for mutant and wide-type bulk,respectively;dots in the graphs represent SNP-index,and the red lines represent the average values of SNP-index or ΔSNP-index in the sliding window;green and orange lines represent thresholds of 95% and 99% confidence levels,respectively.

表3 vrnl11精细定位所用分子标记

Tab.3 Molecular markers used for fine mapping of vrnl11

标记 Marker	正向引物(5'—3') Forward primer (5'—3')	反向引物(5'—3') Reverse primer (5'—3')
nl-12	TGTCAAAATGCAATCAACACAA	TCATCCCTTCAAAGTTCCATT
nl-18	CAGTTGTGCCCTTACCGCTA	TCCATGCTAATCAGGTTACACT
nl-19	TCAACATTTCTACCCCCACTACAAG	AACACCTGCTTCTACTTACATAC
nl-22	GTCGCATTCTCACATTTCACT	AATAGAACACCGCTATCTTGC
nl-38	CTACATCATGAAAAGGAAGGTGGTC	GTCCATAAACTTGGGATCTGAACAG
nl-39	ATAGACATCTCGTGCGTGATAGTAG	TGTCATCTCACAGGTTATCTCAATC
nl-61	CGTTTCATGCGGCGTTAGTT	TAGGCAAAAGCAGGAGGGTG
nl-40	TAAAAGGAAAGTTACAAGGGCAATG	ATTCTGTTAGCTTGTCGTCGAGTTT
nl-41	ATGAAAATGTATGTGGTGTTGTGGG	AACCAAAAACACACCTTGCTGATT
nl-46	GATCCTAAACATAACCACTAACTGC	TGATTAGTTGCATGTAGGTTTTG

图3 利用标记nl-18对F₂群体中21个突变体单株进行基因型分析 1.中绿1号;2.vrnl11;3.F₁;4—24.F₂群体中突变体表型的单株;8,14.单交换单株。

Fig.3 Genotype analysis for the 21 F₂ plants with mutant phenotype using the marker nl-18 1.ZL1;2.vrnl11;3.F₁;4—24.Individuals with mutant phenotype in the F₂ population;8,14.Single crossing over plants.

图4 分子标记nl-46筛选出的3个交换单株测序峰图 447,464,478为3个交换单株。

Fig.4 Sequencing chromatograms of three single crossing over plants screened by molecular marker nl-46 447,464 and 478 were three single crossing over plants.

图5 vrnl11的精细定位

Fig.5 Fine mapping for vrnl11

表4 定位区间内基因功能注释

Tab.4 Function description of genes in the mapping interval

编号 No.	基因 Gene	基因功能描述 Gene function description
1	Vradi11g13330	重金属转运/解毒超家族蛋白
2	Vradi11g13340	核心-2/I-β分支-1,6-N-乙酰氨基葡萄糖转移酶家族蛋白
3	Vradi11g13350	重金属异戊二烯化相关的类植物蛋白26
4	Vradi11g13360	DNA旋转酶A
5	Vradi11g13370	SCP1-类小磷酸酶5
6	Vradi11g13380	MYB转录因子MYB64
7	Vradi11g13390	MYB转录因子MYB64
8	Vradi11g13400	MYB转录因子MYB64
9	Vradi11g13410	MYB转录因子MYB64

参考文献 25

[1]	Somta P, Laosatit K, Yuan X X, Chen X. Thirty years of mungbean genome research:where do we stand and what have we learned?[J]. Frontiers in Plant Science, 2022, 13:944721.doi:10.3389/fpls.2022.944721. URL
[2]	王丽侠, 程须珍, 王素华. 绿豆种质资源、育种及遗传研究进展[J]. 中国农业科学, 2009, 42(5):1519-1527.doi:10.3864/j.issn.0578-1752.2009.05.003.
	Wang L X, Cheng X Z, Wang S H. Advances in research on genetic resources,breeding and genetics of mungbean(Vigna radiata L.)[J]. Scientia Agricultura Sinica, 2009, 42(5):1519-1527.
[3]	田静, 程须珍, 范保杰, 王丽侠, 刘建军, 刘长友, 王素华, 曹志敏, 陈红霖, 王彦, 王珅. 我国绿豆品种现状及发展趋势[J]. 作物杂志, 2021(6):15-21.doi:10.16035/j.issn.1001-7283.2021.06.003.
	Tian J, Cheng X Z, Fan B J, Wang L X, Liu J J, Liu C Y, Wang S H, Cao Z M, Chen H L, Wang Y, Wang S. Current situation and development trend of mungbean varieties in China[J]. Crops, 2021(6):15-21.
[4]	Yaqub M, Mahmood T, Akhtar M, Iqbal M M, Ali S. Induction of mungbean(Vigna radiata (L.)Wilczek)as a grain legume in the annual rice-wheat double cropping system[J]. Pakistan Journal of Botany, 2010, 42(5):3125-3135.
[5]	Alam M K, Islam M M, Salahin N, Hasanuzzaman M. Effect of tillage practices on soil properties and crop productivity in wheat-mungbean-rice cropping system under subtropical climatic conditions[J]. The Scientific World Journal, 2014, 2014:437283.doi:10.1155/2014/437283.
[6]	马秀杰. 间作对绿豆生物性状、产量和品质的影响[J]. 核农学报, 2014, 28(3):546-551.doi:10.11869/j.issn.100-8551.2014.03.0546.
	Ma X J. Effects of intercropping on biological features,yield and qualities of mungbean[J]. Journal of Nuclear Agricultural Sciences, 2014, 28(3):546-551.
[7]	段玉, 邢弘擎, 刘国栋, 王婷, 刘乐峰, 朱旭君, 钟增涛, 房婉萍. 茶树-绿豆/大豆间作对茶园土壤和茶叶品质的影响[J]. 南京农业大学学报, 2022, 45(3):511-520.doi:10.7685/jnau.202106039.
	Duan Y, Xing H Q, Liu G D, Wang T, Liu L F, Zhu X J, Zhong Z T, Fang W P. The effects of tea plants-mung bean/soybean intercropped on soil physicochemical properties and tea quality in tea plantation[J]. Journal of Nanjing Agricultural University, 2022, 45(3):511-520.
[8]	赵雪英, 张泽燕, 朱慧珺, 闫虎斌, 张春明. 绿豆全生育期抗旱性鉴定及形态指标研究[J]. 华北农学报, 2017, 32(S1):180-184.doi:10.7668/hbnxb.2017.S1.031.
	Zhao X Y, Zhang Z Y, Zhu H J, Yan H B, Zhang C M. Analysis of drought resistance and morphological index of the whole growth of mung bean[J]. Acta Agriculturae Boreali-Sinica, 2017, 32(S1):180-184. doi: 10.7668/hbnxb.2017.S1.031
[9]	Nair R M, Schafleitner R, Kenyon L, Srinivasan R, Easdown W, Ebert A W, Hanson P. Genetic improvement of mung bean[J]. SABRAO Journal of Breeding and Genetics, 2012, 44(44):177-190.
[10]	程须珍, 王素华, 王丽侠. 绿豆种质资源描述规范和数据标准[M]. 北京: 中国农业出版社, 2006:13-20.
	Cheng X Z, Wang S H, Wang L X. Descriptors and data standard for mungbean(Vigna radiata (L.))Wilczek[M]. Beijing: China Agriculture Press, 2006:13-20.
[11]	Hou D Z, Yousaf L, Xue Y, Hu J R, Wu J H, Hu X S, Feng N H, Shen Q. Mung bean(Vigna radiata L.):bioactive polyphenols,polysaccharides,peptides,and health benefits[J]. Nutrients, 2019, 11(6):1238.doi:10.3390/nu11061238. URL
[12]	周素梅, 李若凝, 唐健, 侯殿志, 芦晶. 绿豆营养功能特性及其在植物基食品开发中的应用[J]. 粮油食品科技, 2022, 30(2):16-23,12.doi:10.16210/j.cnki.1007-7561.2022.02.003.
	Zhou S M, Li R N, Tang J, Hou D Z, Lu J. Nutritional components and health functions of mung bean and its application in the development of plant-based food[J]. Oils and Foods, 2022, 30(2):16-23,12.
[13]	Kang Y J, Kim S K, Kim M Y, Lestari P, Kim K H, Ha B K, Jun T H, Hwang W J, Lee T, Lee J, Shim S, Yoon M Y, Jang Y E, Han K S, Taeprayoon P, Yoon N, Somta P, Tanya P, Kim K S, Gwag J G, Moon J K, Lee Y H, Park B S, Bombarely A, Doyle J J, Jackson S A, Schafleitner R, Srinives P, Varshney R K, Lee S H. Genome sequence of mungbean and insights into evolution within Vigna species[J]. Nature Communications, 2014, 5:5443.doi:10.1038/ncomms6443.
[14]	Yan Q, Wang Q, Cheng X Z, Wang L X, Somta P, Xue C C, Chen J B, Wu R R, Lin Y, Yuan X X, Chen X. High-quality genome assembly,annotation and evolutionary analysis of the mungbean(Vigna radiata)genome[J]. Authorea, 2020.doi:10.22541/au.160587196.63922177/v1.
[15]	Ha J, Satyawan D, Jeong H, Lee E, Cho K H, Kim M Y, Lee S H. A near-complete genome sequence of mungbean(Vigna radiata L.)provides key insights into the modern breeding program[J]. The Plant Genome, 2021, 14(3):e20121.doi:10.1002/tpg2.20121. URL
[16]	Jiao K Y, Li X, Guo W X, Yuan X X, Cui X Y, Chen X. Genome re-sequencing of two accessions and fine mapping the locus of lobed leaflet margins in mungbean[J]. Molecular Breeding, 2016, 36(9):128.doi:10.1007/s11032-016-0552-1. URL
[17]	Wang J, Li J L, Liu Z X, Yuan X X, Wang S H, Chen H L, Chen X, Cheng X Z, Wang L X. Construction of a high-density genetic map and its application for QTL mapping of leaflet shapes in mung bean(Vigna radiata L.)[J]. Frontiers in Genetics, 2020, 11:1032.doi:10.3389/fgene.2020.01032. pmid: 33133136
[18]	Jiao K Y, Li X, Su S H, Guo W X, Guo Y F, Guan Y N, Hu Z B, Shen Z G, Luo D. Genetic control of compound leaf development in the mungbean(Vigna radiata L.)[J]. Horticulture Research, 2019, 6:23.doi:10.1038/s41438-018-0088-0.
[19]	Jiao K Y, Li X, Guo Y F, Guan Y N, Guo W X, Luo D, Hu Z B, Shen Z G. Regulation of compound leaf development in mungbean(Vigna radiata L.)by cup-shaped cotyledon/no apical meristem(CUC/NAM)gene[J]. Planta, 2019, 249(3):765-774.doi:10.1007/s00425-018-3038-z.
[20]	Fekih R, Takagi H, Tamiru M, Abe A, Natsume S, Yaegashi H, Sharma S, Sharma S, Kanzaki H, Matsumura H, Saitoh H, Mitsuoka C, Utsushi H, Uemura A, Kanzaki E, Kosugi S, Yoshida K, Cano L, Kamoun S, Terauchi R. MutMap⁺:genetic mapping and mutant identification without crossing in rice[J]. PLoS One, 2013, 8(7):e68529.doi:10.1371/journal.pone.0068529. URL
[21]	Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, Uemura A, Utsushi H, Tamiru M, Takuno S, Innan H, Cano L M, Kamoun S, Terauchi R. QTL-seq:rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations[J]. The Plant Journal, 2013, 74(1):174-183.doi:10.1111/tpj.12105. URL
[22]	Jeong N, Suh S J, Kim M H, Lee S, Moon J K, Kim H S, Jeong S C. Ln is a key regulator of leaflet shape and number of seeds per pod in soybean[J]. The Plant Cell, 2012, 24(12):4807-4818.doi:10.1105/tpc.112.104968. URL
[23]	杨霞, 高金珊, 杨素欣. 豆科复叶发育分子遗传机制的研究进展[J]. 植物生理学报, 2017, 53(6):905-915.doi:10.13592/j.cnki.ppj.2017.1026.
	Yang X, Gao J S, Yang S X. Progress of molecular mechanism of compound leaf development in legume plants[J]. Plant Physiology Journal, 2017, 53(6):905-915.
[24]	Timmermans M C, Hudson A, Becraft P W, Nelson T. ROUGH SHEATH2:a Myb protein that represses Knox homeobox genes in maize lateral organ primordia[J]. Science, 1999, 284(5411):151-153.doi:10.1126/science.284.5411.151. pmid: 10102816
[25]	Zhang G H, Xu Q, Zhu X D, Qian Q, Xue H W. SHALLOT-LIKE1 is a KANADI transcription factor that modulates rice leaf rolling by regulating leaf abaxial cell development[J]. The Plant Cell, 2009, 21(3):719-735.doi:10.1105/tpc.108.061457. URL

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绿豆窄叶突变体vrnl11的精细定位

Fine Mapping of a Narrow Leaf Mutant vrnl11 in Mungbean

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