Title: A genome-wide association study reveals that the glucosyltransferase OsIAGLU regulates root growth in rice
题目：全基因组关联研究显示葡萄糖基转移酶OsIAGLU调节水稻根系生长
通讯作者：王州飞
机构：华南农业大学
杂志：J EXP BOT
IF/分区：6.99/Q1
发表时间：2020.10
研究意义：
通过GWAS挖掘候选基因，明确OsIAGLU通过多种激素途径调控根系生长，使其成为未来水稻育种改良的潜在靶点。
M&M：
材料：178份水稻材料，（3个iaglu突变材料，86份粳稻，64份籼稻，其余为混合材料）
性状：发芽3、5、7天后的最长根长、侧根数、根冠数
方法：
1.GWAS （EMMAX）
2.候选基因挖掘，QTL （200kb区间内包含三个及以上的显著SNP当做一个QTL）
3.qRT-PCR
4.GUS染色
5.激素含量测定
结论：
1.GWAS定位根长相关QTL：表型上籼稻的根长显著大于粳稻的根长；通过EMMAX进行GWAS，当200kb区间内有3个及以上显著SNP，则为一个QTL，共检测到4个QTLs，包含93个SNP；其中两个QTL与前人报道一致，qRL11包含34个显著的SNP，是主要的QTL。
Fig. 1. Phenotypic diversity in rice root length and identification of QTLs by GWAS. (A) Examples of variation in root length among rice accessions at
5 d after germination. The scale bar is 10 mm. (B) Distribution of root length among the 178 accessions of the rice population studied. (C) Box-plots of
root length for indica (n=64) and japonica (n=86) accessions. (D) Manhattan plots and (E) quantile–quantile (Q-Q) plots for the whole population of rice
accessions. The red arrows indicate the four QTLs identified. (F) Details of the QTLs.
-
候选基因挖掘：在每个QTL中最显著的SNP前后各100kb进行候选基因挖掘，共找到67个候选基因，其中有50个是非同义突变，或者差异表达基因。
Fig. 2. Identification of candidate genes for the rice root-length QTL qRL11 as determined by integrated analyses of GWAS and RNA-seq data. (A) Identification of candidate genes in the different regions of qRL11. Negative log10-transformed P-values are plotted on the vertical axis, and dots above the red line show the significant SNPs in the QTL region. The annotated genes are indicated. (B) Variations in SNPs in the coding regions of the candidate genes of qRL11. (C) Heat-map showing differential expression of candidate genes for qRL11 in roots at 3, 5, and 7 d after germination. Three candidate genes (LOC_Os11g25860,LOC_Os11g25920, and LOC_Os11g25990) were identified with >2-fold changes. The comparisons are: WT, Nipponbare wild-type; S_3d, seeds at 3 d after germination; R_5d, roots at 5 d after germination; and R_7d roots at 7 d after germination. Red, up-regulation; green, down-regulation. -
OsIAGLU的自然等位基因变异有助于根系生长：对OsIAGLU上游2kb研究SNP多态性，找到两个单体型，Hap1有更长的根长，且主要出现在籼稻中，Hap2根长较短，主要出现在粳稻中；挑选Hap1中长根的籼稻品种和Hap2中的短根粳稻品种研究其OsIAGLU表达量，长根品种中OsIAGLU表达量显著低于短根品种，证明OsIAGLU的自然等位基因变异与水稻不同品种根系生长相关。
Fig. 3. Haplotypes of OsIAGLU associated with root length in rice. (A) Haplotypes of OsIAGLU identified in the coding sequence (CDS) and the region 2 kb upstream of the gene. (B) Box-plots of root lengths of accessions containing the different Hap 1 (n=85) and Hap 2 (n=79) haplotypes. (C) Images of roots from randomly selected accessions containing either Hap 1 or Hap 2. (D) Relative expression of OsIAGLU in the roots of the accessions shown in (C), as determined by quantitative RT-PCR. Expression is relative to that in the NSFTV3 accession, the value of which was set as 1. OsActin was used as the internal control Data are means (±SD), n=3. The significant difference between the two groups was determined using Student’s t-test -
破坏OsIAGLU会导致根系生长不良：三个粳稻OsIAGLU突变品种的根长比对照减少约30%，根冠数和侧根数也显著减少，表明OsIAGLU是水稻苗期根系前期生长的主要控制因子。
Fig. 4. Root growth in the Nipponbare wild-type and Osiaglu mutants. (A) Representative images of the roots of the wild-type (WT) and the three mutant lines at 5 d and 7 d after germination. (B) Dynamic changes in root length in the first 7 d after germination. (C) Representative images of crown roots at 9 d after germination, and (D) quantification of crown roots per plant. (E) Representative images of lateral roots at 9 d after germination, and (F) quantification of lateral roots per plant. Scale bars are 10 mm. Data are means (±SD), n=24. Significant differences compared with the WT were determined using Student’s t-test: **P<0.01. -
OsIAGLU的表达模式：qRT-PCR表明OsIAGLU在根中的表达量远远高于茎；GUS显示，OsIAGLU主要在根系生长前期，根冠，以及侧根高表达；OsIAGLU通过多种激素共同调节根系生长。
Fig. 5. Expression patterns of OsIAGLU in shoots and roots of rice. (A) Relative expression in the shoots and roots, as determined by quantitative RT-PCR. (B) The different regions into which the roots were divided (scale bar is 10 mm) and (C) the relative expression of OsIAGLU in those regions. Expression is relative to that in the shoots (A) and to that in region R1 (C), the values of which were set as 1. OsActin was used as the internal control. Data are means (±SD), n=3. (D–J) Histochemical staining for GUS activity in plants transformed with the OsIAGLU promoter-GUS fusion construct. (D–F) Primary roots, (G, H) crown roots, and (I, J) lateral roots. The GUS staining is indicated by the arrows. -
OsIAGLU影响根系中多种激素：对osiaglu-2和WT进行RNA-seq，共找到差异表达基因5452个，且主要都为激素相关基因；突变材料中ABA, IAA,JA等多种激素含量升高，而CTK显著减少，表明OsIAGLU可以激活ABA\IAA和JA。
Fig. 6. Association between OsIAGLU and hormone contents in the roots of rice at 5 d after germination. (A) Numbers of significantly differentially expressed genes (DEGs; P<0.01) between the Nipponbare wild-type (WT) and the Osiaglu-2 mutant (MT) in roots. (B) Pie-chart showing the number of DEGs associated with different hormones. (C–K) The contents of hormones in the WT and three Osiaglu mutant lines: (C) abscisic acid (ABA), (D, E) auxins (IAA and MeIAA), (F–H) cytokinins (IP, cZ, and tZ), and (I–K) jasmonic acids (JA, JA-Ile, and MeJA). Data are means (±SD), n=3. Significant differences compared with the WT were determined using Student’s t-test: *P<0.05; **P<0.01; n.s., not significant. -
OsIAGLU通过多种激素通路调控根系生长：对突变体施用外源激素，表明随激素浓度升高，根系变短；综合结果表明在萌发后苗早期，OsIAGLU对根系生长的调节可能主要通过调节游离IAA、JA和ABA的浓度，而不是CTK的浓度。
Fig. 7. Effects of hormones on root elongation in Nipponbare rice at 5 d after germination. Representative images and box-plots are shown of the effects of exogenous applications of different concentrations of (A, B) abscisic acid, (C, D) jasmonic acid, (E, F) auxin, and (G, H) cytokinin. Scale bars are 10 mm. Data represent n=24 replicates. Significant differences compared with the WT were determined using Student’s t-test: **P<0.01; n.s., not significant.
Fig. 8. Effects of hormones on root elongation in Nipponbare wild-type (WT) rice and Osiaglu mutants at 5 d after germination. Representative images and box-plots are shown of the effects of treating the roots with (A, B) water (control), (C, D) auxin (10 μM), (E, F) abscisic acid (0.5 μM), -
OsIAGLU影响根生长相关基因的表达：36个已报道过与水稻根系生长相关的基因，在WT和突变体中比较其表达量，找到9个差异基因，其中5个IAA和CTK相关基因在突变体中表达量更高，2个IAA、CTK相关基因和1个多激素相关基因在突变体中表达量更低，推测OsIAGLU通过调控这几个基因的表达从而调控水稻根系的生长。
Fig. 9. OsIAGLU alters the expression of genes associated with root development and growth in rice. Heat-map showing differential expressions of genes that have previously been reported to be involved in rice root growth between the Nipponbare wild-type and the Osiaglu-2 mutant, as determined using RNA-seq. The comparisons are: WT, wild-type; MT, mutant; S_3d, seeds at 3 d after germination; R_5d, roots at 5 d after germination; and R_7d, roots at 7 d after germination. Red, up-regulation; green, down-regulation.
亮点：
1.没有构建LD block ,将显著SNP转换为QTL进行候选基因挖掘；
2.构建突变体确定候选基因功能；
3.不再聚焦于某一个基因，而是从宏观解释调控作用。
原文链接：
https://academic.oup.com/jxb/article-abstract/72/4/1119/5948529?redirectedFrom=fulltext
