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1 作物学报 ACTA AGRONOMICA SINICA 2017, 43(7): ISSN ; CODEN TSHPA9 DOI: /SP.J 绿豆分子遗传图谱构建及若干农艺性状的 QTL 定位分析王建花 1,2,3 张耀文 3 程须珍 2,* 王丽侠 2,* 1, ; 2, ; 3, : F 2, 95 SSR, 11, cm, cm, QTL, QTL 1, 8.49%~66.64%; QTL3, 60.32%~80.36% ; QTL 4, 69.06%~87.35%; QTL, 26, 58.32%~99.51% QTL LG1 LG2 LG4 LG8 LG10, LG1, 1 QTL, LG4, 6 15 QTL; QTL, : ; ; SSR ; ; QTL; Construction of Genetic Map and Identification of QTLs Related to Agronomic Traits in Mung Bean WANG Jian-Hua 1,2,3, ZHANG Yao-Wen 3, CHENG Xu-Zhen 2,*, and WANG Li-Xia 2,* 1 Shanxi University, Taiyuan , China; 2 Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing , China; 3 Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan , China Abstract: Two hundreds and eight individuals of F 2 population, derived from a cross between two mung bean genotypes (Huaye 1 and Zijing 1) were used to construct genetic map, and to identify QTLs related to important agronomic traits. This genetic map contained 11 linkage groups with a total length of cm and an average interval of cm. QTLs mapping was conducted for plant height, young stem color, main stem color, growth habit, podding habit, trilobate leaf shape and mature leaf color using composite interval mapping method. Only one QTL for each trait was detected including plant height, young stem color, main stem color and trilobate leaf shape, and with a contribution ranging from 8.49% to 66.64%. Three QTLs with high contribution rates from 60.32% to 80.36% were identified for the trait of pod habit in mung bean. Four QTLs related to mature leaf color showed at contribution rate from 69.06% to 87.35%. There were 26 QTLs related to growth habit, the most of the tested QTLs, with a contribution rate each from 58.32% to 99.51%. The present QTLs for seven agronomic traits distributed on LG1, LG2, LG4, LG8, and LG10, respectively, could be used in molecular breeding based on marker-assisted selection in mung bean, and also lay a foundation for further study of the inheritance of these traits. Keywords: Mung bean; Genetic linkage map; SSR markers; Composite interval mapping (CIM); QTL; Contribution rate [Vigna radiata (L.) Wilczek](2n=2x=22) (Leguminosae) (Papilionaceae) (Phaseoleae) (Vigna),,,, Kang [1] MAKER pipeline [2] 421 Mbp ( 80%), ( 470 Mbp) [3], (CARS-09) This study was supported by the China Agriculture Research System (CARS-09) and the Agricultural Science and Technology Innovation Program of CAAS * (Corresponding authors):, chengxuzhen@caas.cn,, wanglixia03@caas.cn, Tel: : @qq.com Received(): ; Accepted(): ; Published online(): URL:

2 7 : QTL 1097,,,, Humphry [4] Reddy [5] Parinya [6] Chaitieng [7] Young [8], Reddy [5] 3 (V4718 V4758 V4785),, Pm1 Pm2 [9] [10] [11] [12] Berken/ACC41, cm, C220 Vr cm 1.7 cm, [9] Berken/ACC41, [13] QTLNetwork 2.0 [14] QTL, 9 33 ( [10] [11] ),, 1 1, QTL,,, ; 1 材料与方法 ,, F 2, 1 m, 10 cm, 208 F 2 (PH)(YSC)(MSC) (GH) (PHA) (MLC)( : ) (TLS), [15] ( 3 ) SSR SSR SSR, [15], (Vr1) 2007, (Vr2) 1696, (Vr3) 1485, DNA PCR F 2, Retsch MM400, CTAB [16] DNA, DNA PCR 10 μl, 30 ng DNA, 1 Taq (20 mmol L 1 Tris-HCl, ph 8.8; 10 mmol L 1 KCl; 10 mmol L 1 (NH 4 ) 2 SO 4 ; 1.5 mmol L 1 MgCl 2 ; 0.1% Triton X-100), 1 mmol L 1 dntps, 0.25 μmol L 1 1 U Taq DNA PCR 95 5 min, s, s, s, 35, 72 5 min, 4 EDC-810 PCR 8%, 200 V, SNP, 1.0~1.5 h 2, 0.2% AgNO 3 10 min, 2 1.5% NaOH 0.5%, 2 1.4, A B H - Marker (600 bp DNA marker) SSR, , JoinMap 4.0 F QTL QTL IciMapping V4.0 QTL, Permutation 1000, LOD 3.68 QTL QTL, JoinMap 4.0 MapChart 2 结果与分析 2.1 SSR 5188 SSR, 4281, 99 Vr1 1651, 82.26%, 64 ; Vr2 1309, 77.18%, 55 ; Vr3 1321, 88.96%, 32, (4.20%), (3.88%), (2.42%) SSR F 2, JoinMap 4.0, 95 SSR ( Vr3-985 Vr2-657 Vr1-574 Vr ), 11(LG), cm, 5.52~

3 Type of primers 表 1 不同引物在 F 2 群体亲本中的扩增 Table 1 Amplification of different markers in parents of F 2 No. of primer pairs No. of amplification primers Ratio (%) No. of polymorphic primers Ratio (%) Mono-nucleiotide repeat SR (Vr1) Di-nucleiotide repeat SSR (Vr2) Tri-nucleiotide repeat SSR (Vr3) SSR from cowpea SSR from mung bean Linkage group 表 2 遗传图谱分析 Table 2 Analysis of genetic map Length (cm) Number of markers Average distance (cm) Total or mean cm, LG6, LG4 2~28, 1.38~21.92 cm,, (LG1) 11, Vr2-401 Chr.6; LG2 19, 70% Chr.9; LG3 7, 43% Chr.1; LG4 28, 7 Chr.4, 8 Chr.5, 7 Chr.8; LG5 3, Chr.1; LG6 4, 2 Chr.1, 2 Chr.7; LG7 2, Chr.8; LG8 4, Chr.7; LG9 5, 3 Chr.11; LG10 7, ; LG11 5, Chr F 2, F 2, 5.92,, F 2 ( 1) 6, SAS F 2 208, (PH)(YSC)(MSC) (GH) (PHA) (TLS)(MLC) (P=0.05)( 1) QTL, LOD QTL ( 3) 1 QTL, 8 Vr3-932 Vr (11.79 cm), Vr cm, 8.49% 1 QTL, 4 Vr Vr (63.74 cm), Vr cm, 66.64% 1 QTL, 4 Vr Vr (45.49 cm), Vr cm, 38.68% 3 QTL, 2 Vr3-837 Vr2-440 (25.54 cm), Vr cm, 60.32%; 2 4 Vr1-545 Vr (63.36 cm), 346 cm, Vr cm; 361 cm, Vr cm, 67.82% 80.36%

4 7 : QTL 1099 Fig. 1 图 1 F 2 群体中数量性状与质量性状的分布 Distribution of quantitative and quality traits in F 2 population 26 QTL, 1 Vr3-791 Vr3-303 (3.55 cm), Vr cm, 58.32%; 16 2 Vr3-837 Vr2-440 (25.54 cm) Vr2-440 Vr (53.86 cm), 99.51%; 8 4 Vr Vr (98.33 cm), 99.51%; 1 10 Vr Vr3-40 (17.87 cm), Vr cm, 99.51% QTL, 4 Vr1-598~ Vr (67.79 cm), Vr cm, 54.96% 4 QTL, 2 2, Vr Vr3-107 (52.14 cm), Vr cm; Vr2-440 Vr (53.86 cm), Vr cm, 87.35% 69.06%; 2 4, Vr1-598 Vr (67.79 cm), Vr cm; Vr Vr (45.49 cm), Vr cm, 87.2% 84.3% 3 讨论 3.1, RFLP 14, 9 cm [18], 2 12 [19], RFLP 9 [20] Wang [21] , 45.2~117.0 cm, cm, 1.3 cm, [11] cm, [10] cm, Humphry [22] Wang [21] 3.0 cm 1.3 cm,,,,,,, F 2 208, RIL,

5 Trait Table 3 表 3 绿豆 7 个产量相关性状的 QTL 及其遗传分析 QTLs for seven yield-related traits and their genetic analysis in F 2 population QTL Linkage group Position (cm) Flanking markers PVE (%) Plant height qph Vr3-932 Vr Young stem color qysc Vr Vr Main stem color qmsc Vr Vr Podding habit qpha Vr3-837 Vr qpha Vr1-545 Vr Growth habit qpha Vr1-545 Vr qgh Vr3-791 Vr qgh Vr3-837 Vr qgh Vr3-837 Vr qgh Vr3-837 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr2-440 Vr qgh Vr Vr qgh Vr1-545 Vr qgh Vr1-545 Vr qgh Vr1-545 Vr qgh Vr1-545 Vr qgh Vr1-545 Vr qgh Vr1-545 Vr qgh Vr1-545 Vr qgh Vr Vr Trilobate leaf shape qtls Vr1-598 Vr Mature leaf color qmlc Vr Vr qmlc Vr2-440 Vr qmlc Vr1-598 Vr qmlc Vr Vr

6 7 : QTL 1101 图 2 检测到的主效 QTL 在连锁群上的分布 (LG) Fig. 2 Location of additive effects QTLs on linkage groups GH: growth habit; MLC: mature leaf color; PHA: podding habit; TLS: trilobate leaf shape; MSC: main stem color; YSC: young stem color; PH: plant height. 3.2 QTL [10] [11] QTL, QTL LG8, [23] ( ), 6 QTL, QTL 2 4, 2 Vr3-837~Vr cm, 16 QTL, 2~15 cm, 4 Vr3-1117~Vr cm, 8 QTL, 2~31 cm [11], QTL 9 P3-334~P (TLS) QTL LG4 Vr1-598~Vr1-2059, cm, Vr , Vr , Jiao [24] (lma) Chr.3,, LG2= Chr.9; LG3 43% LG5 1, LG3 LG5, LG3+ LG5=Chr.1; LG8 LG11 7, LG8 LG11, LG8+LG11=Chr.7; LG9 60% 11, LG9=Chr.11; LG4 Chr.4 Chr.5 Chr.8, 3, Chr.4 Chr.5 Chr.8,, 7 LG1 LG2 LG4 LG8 LG10, LG1, LG4 ; QTL NIL,,,,

7 References [1] Kang Y J, Kim S K, Kim M Y. Genome sequence of mungbean and insights into evolution within Vigna species. Nat Commun, 2014, 11: 1 7 [2] Cantarel B L, Korf I, Robb S M, Parra G, Ross E, Moore B, Holt C, Sánchez Alvarado A, Yandell M. MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Res, 2008, 18: [3],,.. :,, pp Zhao J R, Wang X L, Bai Y N. Advances in Comparative Genomics of Leguminous Plants. In: Proceedings of Hainan Biotechnology Research and Development Symposium, Sanya, China, pp (in Chinese with English abstract) [4] Humphry M E, Magner T, McIntyre C L, Aitken E A B, Liu C J. Identification of a major locus conferring resistance to powdery mildew (Erysiphe polygoni DC) in mungbean (Vigna radiata L. Wilczek) by QTL analysis. Genome, 2003, 46: [5] Reddy S K. Identification and inheritance of a new gene for powdery mildew resistance in mungbean (Vigna radiata L. Wilczek). Plant Breed, 2009, 128: [6] Khajudparn P, Wongkaew S. Identification of genes for powdery mildew resistance in mungbean. J Life Sci, 2010, 4(8): 25 [7] Chaitieng B, Kaga A, Han O K, Wang X W, Wongkaew S, Laosuwan P, Tomooka N, Vaughan D A. Mapping a new source of resistance to powdery mildew in mungbean. Plant Breed, 2002, 121: [8] Young N D, Danesh D, Menancio-Hautea D, Kumar L. Mapping oligogenic resistance to powdery mildew in mungbean with RFLPs. Theor Appl Genet, 1993, 87: [9],,,,,.., 2007, 33: Mei L, Wang S H, Wang L X, Liu C Y, Sun L, Xu N. Mapping of genes resistant to Bruchid in mungbean using recombinant inbred lines population. Acta Agron Sin, 2007, 33: (in Chinese with English abstract) [10],,,,.., 2010, 36: Zhao D, Cheng X Z, Wang L X, Wang S H, Ma Y L. Integration of mungbean (Vigna radiata) genetic linkage map. Acta Agron Sin, 2010, 36: (in Chinese with English abstract) [11]..,, 2012 Zhong M. Construction of Genetic Linkage Map and Fine Mapping of Bruchid-resistant Gene (Br1) of Mungbean. MS Thesis of Chinese Academy of Agricultural Sciences, Beijing, China, 2012 (in Chinese with English abstract) [12],,,,,,.., 2014, 47: Wu C S, Wang L X, Wang S H, Chen H L, Wu J X, Cheng X Z, Yang X M. Construction of a genetic linkage map in mungbean. Chin Agric Sci, 2014, 47: (in Chinese with English abstract) [13] Wang D L, Zhu J, Li Z K, Paterson A H. Mapping QTLs with epistatic effects and QTL environment interactions by mixed linear model approaches. Theor Appl Genet, 1999, 99: [14] Yang J, Zhu J. Predicting superior genotypes in multiple environments based on QTL effects. Theor Appl Genet, 2005, 110: [15] Wang L X, Baidouri M E, Abernathy B, Chen H L, Wang S H, Cheng X Z. Distribution and analysis of SSR in mung bean (Vigna radiata L.) genome based on an SSR-enriched library. Mol Breed, 2015, 35: 25, DOI: /s [16] Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull, 1987, 19: [17],,.. :, pp 3 38 Cheng X Z, Wang S H, Wang L X. Descriptors and Data Standard for Mungbean. Beijing: China Agriculture Press, (in Chinese) [18] Menancio-Hautea D, Fatokun C A, Kumar L, Danesh D, Young N D. Comparative genome analysis of mungbean (Vigna radiata L. Wilczek) and cowpea (V. unguiculata L. Walpers) using RFLP mapping data. Theor Appl Genet, 1993, 86: [19] Lambrides D J, Lawn R J, Godwin I D, Manners J, Imrie B C. Two genetic linkage maps of mungbean using RFLP and RAPD markers. Aust J Agric Res, 2000, 51: [20] Hautea D M, Legume I. Molecular mapping of drought resistance in mungbean [Vigna radiata (L.) Wilczek]: 1. Linkage map in mungbean using AFLP markers, J Bioteknologi Pertanian, 2002: 7: [21] Wang L X, Wu C S. Construction of an integrated map and location of a bruchid resistance gene in mung bean. Crop J, 2016, 4: [22] Humphry M E, Konduri V, Lambrides C J, Magner T, McIntyre C L, Aitken E A B, Liu C. Development of a mungbean (Vigna radiata) RFLP linkage map and its comparison with lablab (Lablab purpureus) reveals a high level of colinearity between the two genomes. Theor Appl Genet, 2002, 105: [23]. QTL.,, 2007 Mei L. QTL Analysis of Bruchid Resistance, Seed Dormancy and Other Important Agronomic Traits in Mungbean. MS Thesis of Chinese Academy of Agricultural Sciences, Beijing, China, 2007 (in Chinese with English abstract) [24] 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. Mol Breed, 2016, 36: 128

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L 2012 13 2 201-206 Journal of Plant Genetic Resources / 071001 41 SSR 21 SSR 41 52 2 ~ 4 2. 5 Shannon 0. 21 ~ 1. 40 0. 73 5. 54 3 7 9 13. 16% ~ 186. 49% 5. 48 6 11 SSR Genetic Diversity of Peanut Arachis