Fine Mapping of a Narrow Leaf Mutant vrnl11 in Mungbean

doi:10.7668/hbnxb.20194319

Abstract

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

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.

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Figures/Tables 9

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

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

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

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

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.

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

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.

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.

Fig.5 Fine mapping for vrnl11

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

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