Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (9): 1730-1742.doi: 10.3864/j.issn.0578-1752.2020.09.004

• SPECIAL FOCUS: APPLICATIONS OF RESTRICTED TWO-STAGE MULTI-LOCUS GENOME-WIDE ASSOCIATION ANALYSIS • Previous Articles     Next Articles

Detection Power of RTM-GWAS Applied to 100-Seed Weight QTL Identification in a Recombinant Inbred Lines Population of Soybean

LiYuan PAN1,JianBo HE1(),JinMing ZHAO1,WuBin WANG1,GuangNan XING1,DeYue YU1,XiaoYan ZHANG3,ChunYan LI3,ShouYi CHEN2,JunYi GAI1()   

  1. 1 Soybean Research Institute, Nanjing Agricultural University /National Center for Soybean Improvement /Key Laboratory of Biology and Genetic Improvement of Soybean (General), Ministry of Agriculture/State Key Laboratory for Crop Genetics and Germplasm Enhancement/Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing 210095;
    2 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences/State Key Laboratory of Plant Genomics, Beijing 100101;
    3 Shandong Shofine Seed Technology Co. Ltd., Jiaxiang 272400, Shandong
  • Received:2019-08-24 Accepted:2020-01-02 Online:2020-05-01 Published:2020-05-13
  • Contact: JianBo HE,JunYi GAI E-mail:hjbxyz@gmail.com;sri@njau.edu.cn

Abstract:

【Objective】To thoroughly dissect the QTL system conferring 100-seed weight in a recombinant inbred lines population, the restricted two-stage multi-locus genome-wide association analysis (RTM-GWAS) method was compared with other mapping methods for method optimization, which will provides basis for further exploration of candidate gene system and molecular marker-assisted design breeding. 【Method】A recombinant inbred line population consisting of 427 lines derived from a cross between Kefeng-1 and NN1138-2 was tested for its 100-seed weight under three environments. A total of 3 683 SNPLDBs (SNP linkage disequilibrium blocks) composed of 39 353 SNPs were applied to QTL mapping using three different mapping procedures, including the composite interval mapping (CIM) method, the mixed linear model (MLM-GWAS) method and the RTM-GWAS method, and the best mapping procedure was selected for the analysis of the 100-seed weight genetic system in NJRIKY population through comparing their detection power, including the detected number of QTLs and total phenotypic variation explained. 【Result】The 100-seed weight of Kefeng-1 and NN1138-2 were 9.0 g and 17.9 g, respectively, showing significant difference. The genotypic coefficient of variation and heritability of the trait were 12.4% and 85.4%, respectively. These results indicated that the population was suitable for genetic analysis of 100-seed weight trait. The RTM-GWAS procedure performed the best with the largest number of QTLs (57) explaining the most phenotypic variation (PVE=70.78%), while a total of 14 and 6 QTLs contributing 56.47% and 18.47% phenotypic variation were detected using CIM and MLM-GWAS, respectively. The 57 QTLs detected from the RTM-GWAS distributed on 19 chromosomes, of which 41 QTLs overlapped with 81 QTLs identified from 30 bi-parental populations in the literature. Furthermore, the PVE of 57 QTLs ranged from 0.03% to 7.57%, of which 16 QTLs were novel ones, including one large contribution major QTL Sw-09-2 (PVE>3%). Furthermore, 20 QTLs had significant interaction effect with environment. A total of 36 candidate genes were annotated from 37 QTLs through χ2 test between SNPLDB markers and SNPs harboring in the predicted genes, of which 4 candidate genes were from the large contribution QTLs and other 32 candidate genes were from the small contribution QTLs. These candidate genes were included in different biological processes, of which 13 candidate genes were grouped in seed development directly, and the remaining candidate genes were grouped into different functions, such as transport, transcriptional regulators, etc., indicating that these genes from different biological pathways regulate the expression of 100-seed weight trait in NJRIKY together. 【Conclusion】Among the three different mapping procedures, RTM-GWAS procedure is the most powerful one which can provide a relatively thorough detection of 100-seed weight QTLs in NJRIKY population, therefore, it is more suitable for QTL mapping in bi-parental population such as RIL population. The candidate genes with various functions jointly regulated the complex expression of 100-seed weight trait.

Key words: soybean [Glycine max (L.) Merr.], 100-seed weight, QTL (quantitative trait locus), recombinant inbred lines population, restricted two-stage multi-locus genome-wide association analysis

Fig. 1

Frequency distribution of 100-seed weight in NJRIKY under multiple environments Mean represents the aveage 100-seed weight of each line across three environments"

Table 1

Frequency distribution and descriptive statistics of 100-seed weight of NJRIKY under multiple environments"

环境
Environment
亲本 Parent (g) 重组自交系群体 RIL
科丰1号
Kefeng-1
南农1138-2
NN1138-2
平均值
Mean
最小值
Min.
最大值
Max.
遗传变异
系数GCV(%)
遗传率
h2(%)
12JP 8.5 15.7 11.4 7.5 16.0 13.5 93.3
13JP 8.8 14.9 11.6 7.6 20.6 15.5 92.5
13SF 9.8 23.2 13.9 9.2 19.5 15.1 93.9
平均 Mean 9.0 17.9 12.3 8.6 17.1 12.4 85.4

Table 2

Variance analysis of 100-seed weight under multiple environments in NJRIKY population"

变异来源 Source of variation 自由度 DF 均方 MS FF value PP value
环境 Environment 2 2308.47 101.66 <0.0001
重复(环境)Replication(Environment) 6 20.08 12.59 <0.0001
区组(环境×重复)Block(Environment×Replication) 180 1.34 1.78 <0.0001
家系 Line 426 23.66 6.81 <0.0001
家系×环境 Line×Environment 851 3.50 4.67 <0.0001
误差 Error 2245 0.75
合计 Total 3710

Table 3

The summary information of QTLs associated with 100-seed weight in NJRIKY population using different QTL mapping methods"

定位方法 Method 检测的QTL Detected QTLs 表型变异解释率 PVE (%) 已报道的QTL Reported QTLs
RTM-GWAS
Mapped QTL 57(19) 70.78 41(84)
LC major QTL 5(5) 23.30
SC major QTL 52(19) 47.48
QTL×Env. 20(13) 4.20
Unmapped QTL 14.52
CIM
Mapped QTL 14(8) 56.47 13(16)
MLM-GWAS
Mapped QTL 6(3) 18.47 6(18)

Fig. 2

Genome-wide distributions of 100-seed weight QTLs detected by three QTL mapping procedures a: Chromosome structure with heterochromatin regions in light red color (scale in Mb unit)"

Table 4

The 100-seed weight QTLs of NJRIKY population using CIM and MLM-GWAS procedure"

标记Marker LOD/-lgP 表型变异解释率PVE (%) SoyBase QTL
CIM
Gm04_BLOCK_15191938_15271503 3.67 2.94 5-2
Gm04_BLOCK_36700318_36900222 3.08 2.55 36-15
Gm04_BLOCK_39069193_39185415 2.93 2.37 36-15
Gm04_BLOCK_38868989_39068925 2.45 2.01 36-15
Gm04_BLOCK_29674388_29831299 1.96 1.59 20-2
Gm08_BLOCK_19121233_19314940 4.11 3.41 7-1,34-1,34-13,36-1,37-8
Gm09_BLOCK_35812609_35851884 6.81 5.76 40-4
Gm09_36859571 6.97 5.86 40-4
Gm10_BLOCK_49435374_49524464 4.44 3.69
Gm11_3955010 7.78 6.91 37-9
Gm11_BLOCK_5228756_5269867 12.39 11.03 21-1
Gm14_BLOCK_29877119_30044908 2.36 1.90 23-1,13-2
Gm15_BLOCK_29781764_29980832 3.69 2.95 6-1,36-12
Gm18_54211355 4.12 3.50 3-3
总计Total 56.47
MLM-GWAS
Gm04_BLOCK_23431433_23626782 3.72 3.36 20-2,5-2,36-15,45-3
Gm11_BLOCK_4611160_4662697 3.27 2.88 37-9
Gm11_BLOCK_5228756_5269867 4.03 3.69 37-9,21-1,22-2,49-2
Gm11_20972740 3.12 2.73 21-1,20-4,20-3,11-1,32-1,4-1,10-3,36-11
Gm18_BLOCK_16884478_16885174 3.40 3.03 2-5
Gm18_45987500 3.17 2.78 27-1
总计Total 18.47

Table 5

The QTLs associated with 100-seed weight in NJRIKY population"

QTL 标记
Marker
-lgP 贡献率
R2 (%)
QTL 标记
Marker
-lgP 贡献率
R2 (%)
Sw-11-2 Gm11_BLOCK_5228756_5269867 56.22 7.57* Sw-05-1 Gm05_BLOCK_28628661_28825701 7.42 0.82*
Sw-09-2 Gm09_BLOCK_34358756_34538440 39.72 5.12* Sw-04-1 Gm04_BLOCK_10110270_10257050 6.78 0.74
Sw-04-7 Gm04_BLOCK_39928569_39957258 29.72 3.72* Sw-06-1 Gm06_BLOCK_5723355_5918489 6.58 0.71
Sw-08-1 Gm08_16468204 28.90 3.61 Sw-06-3 Gm06_30304358 6.52 0.71*
Sw-10-1 Gm10_BLOCK_41285429_41285653 26.41 3.27 Sw-15-2 Gm15_25471398 6.31 0.68
Sw-18-5 Gm18_59805347 24.23 2.98* Sw-16-5 Gm16_BLOCK_23266112_23462965 5.98 0.64
Sw-13-1 Gm13_31358574 23.33 2.86* Sw-02-1 Gm02_BLOCK_2190737_2384686 5.72 0.61*
Sw-17-1 Gm17_BLOCK_2938402_3123352 20.28 2.45 Sw-04-4 Gm04_BLOCK_19626939_19826251 5.51 0.59
Sw-04-9 Gm04_BLOCK_40868266_40917625 19.75 2.38 Sw-03-2 Gm03_BLOCK_41020781_41042956 5.12 0.54
Sw-16-1 Gm16_BLOCK_12959169_13153966 16.33 1.94 Sw-03-1 Gm03_3970385 4.52 0.47
Sw-01-2 Gm01_BLOCK_55629182_55799927 15.79 1.87* Sw-15-3 Gm15_36895087 4.48 0.46
Sw-18-1 Gm18_BLOCK_1922631_2035120 15.72 1.86* Sw-06-2 Gm06_20892550 4.34 0.45*
Sw-14-4 Gm14_BLOCK_44088379_44288183 15.18 1.79* Sw-04-2 Gm04_BLOCK_10815779_11015107 3.97 0.40
Sw-14-1 Gm14_1033115 14.10 1.65 Sw-10-3 Gm10_BLOCK_41542940_41691982 3.68 0.37
Sw-07-1 Gm07_BLOCK_1944869_1966911 12.09 1.40 Sw-01-1 Gm01_BLOCK_12639088_12838656 3.65 0.36
Sw-09-1 Gm09_BLOCK_264001_463636 11.95 1.38* Sw-12-2 Gm12_BLOCK_37972975_37975786 3.46 0.34*
Sw-15-1 Gm15_BLOCK_9079223_9250661 11.21 1.29* Sw-14-2 Gm14_BLOCK_13649138_13846641 3.19 0.31
Sw-04-8 Gm04_BLOCK_40184350_40334324 10.84 1.24 Sw-14-3 Gm14_BLOCK_42062012_42261888 2.98 0.29
Sw-18-2 Gm18_36907653 10.77 1.23 Sw-04-11 Gm04_BLOCK_47626591_47646641 2.94 0.28
Sw-10-2 Gm10_41392627 10.09 1.15 Sw-16-2 Gm16_15575581 2.92 0.28
Sw-08-2 Gm08_BLOCK_19121233_19314940 9.71 1.10 Sw-04-10 Gm04_41805915 2.65 0.25
Sw-09-3 Gm09_BLOCK_39036252_39210642 9.27 1.04 Sw-16-4 Gm16_BLOCK_21928405_22126811 2.54 0.24
Sw-11-1 Gm11_BLOCK_767896_842085 8.80 0.99 Sw-04-6 Gm04_BLOCK_26906733_27091677 2.54 0.24*
Sw-12-1 Gm12_BLOCK_35239969_35240158 8.77 0.98 Sw-18-4 Gm18_56862453 2.13 0.19
Sw-17-2 Gm17_BLOCK_7387475_7572446 8.11 0.90* Sw-04-3 Gm04_BLOCK_17249185_17373282 2.03 0.18
Sw-16-3 Gm16_BLOCK_17551378_17602014 8.03 0.89 Sw-05-2 Gm05_30058805 1.83 0.16*
Sw-18-3 Gm18_BLOCK_56236139_56414021 8.00 0.89 Sw-12-3 Gm12_BLOCK_38208501_38296133 1.61 0.13*
Sw-02-2 Gm02_BLOCK_48637411_48823121 7.59 0.84 Sw-20-1 Gm20_236372 1.52 0.13
Sw-04-5 Gm04_BLOCK_23431433_23626782 7.59 0.84* 共计Total 57 70.78

Fig. 3

Manhattan and quantile-quantile plots of GWAS for 100-seed weight in NJRIKY population"

Table 6

The candidate gene system of 100-seed weight annotated from the detected QTLs using RTM-GWAS procedure"

QTL 解释率
R2 (%)
候选基因
Candidate gene
SNP数目
No. of SNPs
GO注释
GO description
Sw-01-2 1.87 Glyma01g45220 6 胚胎发育Embryo Development
Sw-02-1 0.61 Glyma02g02860 1 PSII相关的光收集复合物II过程PSII associated light-harvesting complex II process
Sw-02-2 0.84 Glyma02g43960 2 种子休眠时胚胎发育终止Embryo development ending in seed dormancy
Sw-03-1 0.47 Glyma03g04020 1 多糖分解过程Polysaccharide catabolic process
Sw-03-2 0.54 Glyma03g33360 1 麦芽糖代谢过程Maltose metabolic process
Sw-04-1 0.74 Glyma04g11582 1 转运Transport
Sw-04-7 3.72 Glyma04g34070 1 响应镉离子Response to cadmium- ion
Sw-04-9 2.38 Glyma04g34660 1 脂肪酸代谢过程Fatty acid catabolic process
Sw-04-10 0.25 Glyma04g35511 1 转录的调节,依赖DNA Regulation of transcription, DNA-dependent
Sw-04-11 0.28 Glyma04g41810 2 蛋白质糖基化Protein glycosylation
Sw-05-1 0.82 Glyma05g23100 1
Sw-06-1 0.71 Glyma06g07880 2 种子休眠时胚胎发育终止Embryo development ending in seed dormancy
Sw-07-1 1.40 Glyma07g02940 3 ATP分解过程ATP catabolic process
Sw-08-1 3.61 Glyma08g21620 3 分生组织生长调节Regulation of meristem growth
Sw-08-2 1.10 Glyma08g24950 1 细胞生长Cell growth
Sw-09-1 1.38 Glyma09g00430 4 响应脱落酸Response to abscisic acid stimulus
Sw-09-3 1.04 Glyma09g32540 2 毒素分解过程Toxin catabolic process
Sw-10-1, 3.27 Glyma10g32920 2 种子发育Seed development
Sw-10-2 1.15 2
Sw-10-3 0.37 Glyma10g33160 1 花器官的形成Floral organ formation
Sw-11-1 0.99 Glyma11g01405 4 鸟苷四磷酸代谢过程Guanosine tetraphosphate metabolic process
Sw-11-2 7.57 Glyma11g07430 1 葡糖醛酸木聚糖代谢过程Glucuronoxylan metabolic process
Sw-12-1 0.98 Glyma12g31670 2 细胞过程规则Abaxial cell fate specification
Sw-12-3 0.13 Glyma12g35020 4 转录正调控Positive regulation of transcription
Sw-13-1 2.86 Glyma13g28260 1
Sw-14-1 1.65 Glyma14g01790 1 多糖生物合成过程Polysaccharide biosynthetic process
Sw-14-3 0.29 Glyma14g33820 1 花发育调节作用Regulation of flower development
Sw-14-4 1.79 Glyma14g35260 1 核转录mRNA分解代谢过程Nuclear-transcribed mRNA catabolic process
Sw-15-1 1.29 Glyma15g12410 4 蛋白质糖基化Protein glycosylation
Sw-16-5 0.64 Glyma16g20730 1 过渡金属离子转运Transition metal ion transport
Sw-17-1 2.45 Glyma17g04360 1
Sw-17-2 0.90 Glyma17g09961 3
Sw-18-1 1.86 Glyma18g02940 1 转录调控Regulation of transcription
Sw-18-3 0.89 Glyma18g46517 2
Sw-18-4 0.19 Glyma18g47240 1 表皮细胞分裂调节Regulation of epidermal cell division
Sw-18-5 2.98 Glyma18g50760 3 多糖生物合成过程Polysaccharide biosynthetic process
Sw-20-1 0.13 Glyma20g00565 4
共计Total 54.12 36
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