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作物学报 ›› 2022, Vol. 48 ›› Issue (7): 1601-1613.doi: 10.3724/SP.J.1006.2022.14130

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗HTD2基因的表达特征及基因多态性分析

李旭娟1(), 李纯佳1, 吴转娣1, 田春艳1, 胡鑫1, 丘立杭2, 吴建明2, 刘新龙1,*()   

  1. 1云南省农业科学院甘蔗研究所 / 云南省甘蔗遗传改良重点实验室, 云南开远 661699
    2广西农业科学院甘蔗研究所 / 广西甘蔗遗传改良重点实验室, 广西南宁 530007
  • 收稿日期:2021-07-20 接受日期:2021-10-19 出版日期:2022-07-12 网络出版日期:2021-11-02
  • 通讯作者: 刘新龙
  • 作者简介:E-mail: lixujuan2011@163.com
  • 基金资助:
    国家自然科学基金项目(31760412);国家重点研发计划项目(2019YFD1000500)

Expression characteristic and gene diversity analysis of ScHTD2 in sugarcane

LI Xu-Juan1(), LI Chun-Jia1, WU Zhuan-Di1, TIAN Chun-Yan1, HU Xin1, QIU Li-Hang2, WU Jian-Ming2, LIU Xin-Long1,*()   

  1. 1Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences / Yunnan Key Laboratory of Sugarcane Genetic Improvement, Kaiyuan 661699, Yunnan, China
    2Sugarcane Research Institute of Guangxi Academy of Agricultural Sciences / Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007, Guangxi, China
  • Received:2021-07-20 Accepted:2021-10-19 Published:2022-07-12 Published online:2021-11-02
  • Contact: LIU Xin-Long
  • Supported by:
    National Natural Science Foundation of China(31760412);National Key Research and Development Program of China(2019YFD1000500)

摘要:

分蘖是无性繁殖经济作物-甘蔗最重要的农艺性状之一, 挖掘分蘖关键基因用于甘蔗理想株型调控是增加品种产量的重要途径。本研究使用实时荧光定量PCR技术对前期从甘蔗中获得的与水稻分蘖关键基因HTD2高度同源的ScHTD2基因开展表达特征分析, 然后探讨其在甘蔗种质资源群体中的基因多态性情况, 筛选与分蘖性状相关的变异位点。结果显示, 该基因表达具有组织特异性, 在叶中表达最高; 腋芽萌动发育过程中, 在休眠芽中表达量最高, 随着蔗芽萌动, 开始显著下调表达, 负调控蔗芽的萌动发育; 植物激动素、生长素和独脚金内酯都能显著诱导该基因在萌动蔗芽和蔗苗分蘖芽中的表达, 结合激素处理的表型变化特征, 预示生长素和独脚金内酯诱导该基因的高表达会抑制萌动蔗芽的继续发育和延迟蔗苗的分蘖, 但植物激动素诱导的高表达并没有这种抑制效应。对从26份甘蔗种质资源中获得的520条HTD2基因组DNA克隆序列开展基因多态性分析表明, 在基因组结构上, 该基因具有2个外显子和1个内含子, 其中内含子区域变异最为丰富。从群体基因多态性看, 原始种亲本群体在外显子1和2区域表现出较高的核苷酸多样性, 而主栽品种群体在内含子区域表现出较高的核苷酸多样性。从编码区单倍型多样性来看, 原始种亲本群体间单倍型多样性最为丰富, 其次为主栽品种群体。基因选择性测验中, 原始种亲本群体受正向选择, 选择压力较大, 基因进化速度快, 而骨干亲本群体和主栽品种群体受负向选择, 向纯化方向发展。编码区单倍型演化分析表明, Hap3和Hap4处于单倍型演化的辐射中心, 属于较为原始的类型。基因编码区变异位点与分蘖率相关性检测揭示该基因有23个SNP位点和5个InDel位点不同变异类型剂量与甘蔗种质资源分蘖率呈显著相关, 变异类型剂量效应将是未来甘蔗分子辅助育种重要的关注点。研究为进一步深入解析甘蔗分蘖调控关键基因的功能作用和开发功能性标记奠定了重要基础。

关键词: 甘蔗, HTD2, 基因表达, 基因多态性, 变异位点

Abstract:

Tillering is one of the most important agronomic traits for sugarcane which is a vegetatively propagated economic crop. Mining these genes related to tillering trait and utilizing them form an ideal architecture are important to effectively increase cane yield of sugarcane varieties. In this study, qRT-PCR technology was used to explore the expression characteristics of a homologue of HTD2 related to rice tillering, which was named ScHTD2 previously obtained from sugarcane. The polymorphism of ScHTD2 gene in sugarcane germplasm resource population was analyzed, and the correlation between variation sites and tillering trait were also detected. The results showed that relative expression of this gene was tissue specific and highest in leaves. During the germination and development of axillary, the relative expression was the highest in the dormant buds. With the germination of sugar buds, the relative expression level first was significantly down-regulated, and the germination and development of sugar buds were negatively regulated. Treatments of phytokinin, auxin and strigolactone can significantly induce its expression in germinating axillary buds and tiller buds of seedling. By combining with the phenotypic changes after hormone treatment, the high expression of this gene induced auxin and strigolactone could inhibit the continued development of germinating axillary buds and delay the occurrence of tillers, but this inhibitory effect did not occur in the high expression of this gene induced by phytokines. Gene polymorphism analysis on 520 cloned sequences of HTD2 genomic DNA from 26 sugarcane germplasm resources showed that the genome sequence structure of this gene contained two exons and one intron, and the variation was the most abundant in the intron region, followed by the exon 1 region. Much higher nucleotide diversity in intron region was detected in main sugarcane variety population, while the origin species parent population had the higher nucleotide diversity in exon 1 and 2 regions. In terms of haplotype diversity in the coding region, the origin species parent population possessed the most abundant haplotype diversity, followed by the main variety population. In the genetic selectivity test, the original species parent population was subjected to positive selection, with high selection pressure and rapid gene evolution, while the backbone parents population and the main variety populations are subject to negative selection toward purification. Two haplotypes of code region located at radiating center of haplotype evolution map, indicating that they belonged to primitive types. The dose effect of mutation type from 23 SNP sites and 5 InDel sites in the code region was significantly related to the tillering rate of sugarcane germplasm resources, so more attention should be paid to the dose effect of mutation types in sugarcane molecular assisted-breeding plan in future. The present study lays a foundation for further analyzing the biological meaning of tillering related to key genes and the development of functional markers in sugarcane.

Key words: sugarcane, HTD2, gene expression, gene diversity, variation site

表1

甘蔗种质资源名称和类型"

编号No. 样品名称
Sample name
类型
Type
描述
Description
1 云南75-1-2 Yunnan 75-1-2 甘蔗原始种亲本
Sugarcane origin species parents
八倍体割手密 Octaploid clones of Saccharum spontaneum
2 云南6号 Yunnan 6 十倍体割手密 Decaploid clones of Saccharum spontaneum
3 黑车里本 Black cheribon 热带种 Saccharum officinarum
4 HATUNI 印度种 Saccharum barberi
5 51NG63 大茎野生种 Saccharum robustum
6 友巴 Uba 中国种 Saccharum sinense
7 F134 甘蔗骨干亲本
Sugarcane backbone parents
已育成品种数38个 38 varieties have been bred using this parent
8 CO419 已育成品种数25个 25 varieties have been bred using this parent
9 CP72-1210 已育成品种数17个 17 varieties have been bred using this parent
10 川糖57-416 Chuantang 57-416 已育成品种数15个 15 varieties have been bred using this parent
11 NCo310 已育成品种数13个 13 varieties have been bred using this parent
12 F108 已育成品种数12个 12 varieties have been bred using this parent
13 华南56-12 Huanan 56-12 已育成品种数10个 10 varieties have been bred using this parent
14 崖城71-374 Yacheng 71-374 已育成品种数9个 9 varieties have been bred using this parent
15 CP28-11 已育成品种数8个 8 varieties have been bred using this parent
16 桂糖11号 Guitang 11 已育成品种数6个 6 varieties have been bred using this parent
17 ROC10 甘蔗主栽品种
Sugarcane main varieties
主产蔗区大面积种植
Large-scale planting in the main sugarcane production areas
18 ROC16
19 ROC20
20 ROC22
21 ROC25
22 桂糖21号 Guitang 21
23 桂柳05-136 Guiliu 05-136
24 粤糖00-236 Yuetang 00-236
25 粤糖93-159 Yuetang 93-159
26 粤糖86-368 Yuetang 86-368

图1

ScHTD2基因组序列PCR扩增产物电泳图"

图2

ScHTD2基因在甘蔗品种不同组织部位的相对表达量 误差线为每组处理的标准误差(n = 3)。柱上不同小写字母代表在0.05水平差异显著。"

图3

ScHTD2基因在蔗芽发育不同阶段及外源植物激素处理下的相对表达 A: 蔗芽发育不同阶段的相对表达量; B: 外源植物激素处理萌动蔗芽后的相对表达量; C: 外源植物激素处理蔗苗后分蘖芽中的相对表达量; D: 外源植物激素处理蔗苗后叶中的相对表达量。*和**分别表示在0.05和0.01水平差异显著。 "

表2

甘蔗3个群体HTD2基因编码区和内含子序列长度"

群体类型
Population
总序列长度
Total length of sequence
外显子1长度
Length of exon 1
内含子长度
Length of intron
外显子2长度
Length of exon 2
原始种亲本Origin species parents 1228 503 292 433
骨干亲本Backbone parents 1254 504 309 441
主栽品种Main varieties 1263 516 315 432

图4

甘蔗3个群体HTD2基因外显子和内含子区域核苷酸多样性(Pi)分布图 A: 原始种亲本群体; B: 骨干亲本群体; C: 主栽品种群体。"

表3

甘蔗3个群体HTD2基因外显子和内含子区域序列多态性"

序列区域
Sequence region
群体名称
Population
克隆序列数
Number of
sequenced clones
序列长度
Length of
sequence (bp)
SNP位点数
Number of SNP
InDel位点数
Number of InDel
核苷酸多样性
Nucleotide diversity (Pi)
外显子1
Exon 1
原始种亲本Origin species parents 120 503 29 8 0.0065
骨干亲本Backbone parents 200 504 46 5 0.0043
主栽品种Main varieties 200 516 43 7 0.0046
内含子
Intron
原始种亲本Origin species parents 120 292 32 39 0.0121
骨干亲本Backbone parents 200 309 42 51 0.0142
主栽品种Main varieties 200 315 39 55 0.0171
外显子2
Exon 2
原始种亲本Origin species parents 120 433 14 3 0.0033
骨干亲本Backbone parents 200 441 20 13 0.0012
主栽品种Main varieties 200 432 24 1 0.0016

图5

甘蔗3个群体HTD2基因外显子和内含子区域主要InDel位点类型"

表4

甘蔗3个群体HTD2基因编码区同义非同义位点数和Ka/Ks值"

群体类型
Population
编码区长度
Length of code region (bp)
同义突变位点数
Number of synonymous mutation sites
非同义突变位点数
Number of non-synonymous mutation sites
Ka/Ks
原始种亲本Origin species parents 936 7 34 1.7473
骨干亲本Backbone parents 945 25 38 0.2258
主栽品种Main varieties 945 27 41 0.2141

表5

甘蔗3个群体HTD2基因编码区单倍型多态性"

群体类型
Population
克隆序列数
Number of
sequenced clones
单倍型数量
Number of haplotypes
单倍型多样性
Haplotype diversity (Hd)
核苷酸多样性
Nucleotide diversity (Pi)
平均核苷酸差异数Average number of
nucleotide difference (k)
原始种亲本Origin species parents 120 32 0.8394 0.0050 4.3510
骨干亲本Backbone parents 200 34 0.6610 0.0028 2.4970
主栽品种Main varieties 200 43 0.7981 0.0032 2.9640

图6

甘蔗3个群体HTD2基因编码区单倍型网络演化图"

表6

甘蔗HTD2编码区主要单倍型在3个群体中的分布情况"

主要单倍型
Main haplotypes
原始亲本种
Origin species parents
骨干亲本
Backbone parents
主栽品种
Main varieties
Hap3 51NG63, 黑车里本,
HATUNI, 友巴
51NG63, Black cheribon, HATUNI, Uba
川糖57-416, Co419, CP28-11,
C72-1210, F108, 桂糖11号, 华南56-12, NCo310, 崖城71-374
Chuantang 57-416, Co419, CP28-11, CP72-1210, F108, F134, Guitang 11,
Huanan 56-12, Yacheng 71-374
ROC25, 桂柳05-136, ROC10, ROC16, ROC20, ROC22, 粤糖00-236, 粤糖93-159, 桂糖21号, 粤糖86-368
ROC25, Guiliu 05-136, ROC10, ROC16, ROC20, ROC22, Yuetang 00-236, Yuetang 93-159, Guitang 21, Yuetang 86-368
Hap4 51NG63, 黑车里本, HATUNI, 友巴, 云南6号
51NG63, Black cheribon, HATUNI, Uba, Yunnan 6
Co419, CP28-11, F108, 桂糖11号,
华南56-12, NCo310, 崖城71-374
Co419, CP28-11, F108, Huanan 56-12, NCo310, Yacheng 71-374
ROC25, 桂柳05-136, ROC10, ROC16, ROC20, ROC22, 桂糖21号, 粤糖00-236, 粤糖93-159, 粤糖86-368
ROC25, Guiliu 05-136, ROC10, ROC16, ROC20, ROC22, Guitang 21, Yuetang 00-236, Yuetang 93-159, Yuetang 86-368
Hap18 友巴, 云南6号
Uba, Yunnan 6
F108 桂糖21号 Guitang 21
Hap37 HATUNI, 友巴
HATUNI, Uba
桂柳05-136 Guiliu 05-136
Hap39 HATUNI 桂柳05-136 Guiliu 05-136
Hap27 云南6号 Yunnan 6 桂糖11号 Guitang 11
Hap36 HATUNI, 云南75-1-2 HATUNI, Yunnan 75-1-2 桂柳05-136 Guiliu 05-136
Hap30 云南6号 Yunnan 6 桂糖21号 Guitang 21

表7

甘蔗种质资源HTD2编码区序列SNP位点变异碱基类型与分蘖率Pearson相关性分析"

序号
No.
SNP名称
SNP name
碱基类型
Base type
相关系数
Correlation coefficient
序号
No.
SNP名称
SNP name
碱基类型
Base type
相关系数
Correlation coefficient
1 SNP19 C 0.429* 14 SNP356 C -0.405*
T -0.429* A 0.405*
2 SNP21 A -0.442* 15 SNP392 C -0.429*
3 SNP39 G -0.444* A 0.429*
A 0.444* 16 SNP422 C 0.423*
4 SNP66 C -0.402* A -0.423*
5 SNP67 G -0.432* 17 SNP449 C 0.497**
6 SNP77 G -0.395* A -0.497**
7 SNP110 C 0.429* 18 SNP582 C -0.455*
G -0.429* A 0.455*
8 SNP111 C -0.429* 19 SNP606 G 0.429*
T 0.429* 20 SNP662 C -0.429*
9 SNP130 G 0.429* T 0.429*
10 SNP154 G -0.432* 21 SNP700 C -0.429*
11 SNP156 G -0.432* G 0.429*
12 SNP182 C -0.404* 22 SNP785 C -0.444*
T 0.429* T 0.444*
13 SNP213 T 0.484* 23 SNP843 C 0.536**
G -0.475* T -0.536**

表8

甘蔗种质资源HTD2编码区InDel位点与分蘖率Pearson相关性分析"

InDel名称
InDel name
相关系数
Correlation coefficient
特征描述
Characteristic description
InDel名称
InDel name
相关系数
Correlation coefficient
特征描述
Characteristic description
InDel 19 0.429* 4碱基插入
4 bases insert
InDel 150 0.429* 12碱基插入
12 bases insert
InDel 23 -0.429* 13碱基缺失
13 bases deletion
InDel 647 -0.429* 单碱基缺失
Single base deletion
InDel 54 0.429* 24碱基插入
24 bases insert
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