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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (7): 1601-1613.doi: 10.3724/SP.J.1006.2022.14130

• OCROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2022-07-12 Published:2021-11-02
  • Contact: LIU Xin-Long E-mail:lixujuan2011@163.com;lxlgood868@163.com
  • Supported by:
    National Natural Science Foundation of China(31760412);National Key Research and Development Program of China(2019YFD1000500)

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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

Table 1

Name and type of sugarcane germplasm resources in this study"

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

Fig. 1

Electrophoresis results of PCR amplification products forScHTD2 genomic sequence"

Fig. 2

Relative expression of ScHTD2 in different tissues of sugarcane variety The error bar represents the standard error of each treating group (n = 3). Different lowercase letters above the bar mean the significant difference at P < 0.05."

Fig. 3

Relative expression of ScHTD2 in different developmental stage of bud and exogenous plant hormone treatment A: the relative expression in different developmental stage of bud; B: the relative expression in stirring bud after exogenous plant hormone treatment; C: the relative expression in tillering bud of seedling after exogenous plant hormone treatment; D: the relative expression in leaf of seedling after exogenous plant hormone treatment. * and ** mean significant difference at P < 0.05 and P < 0.01, respectively."

Table 2

Sequence length of code and intron region of HTD2 in three sugarcane populations (bp)"

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

Fig. 4

Distributing map of nucleotide diversity (Pi) in exon and intron region nucleotide diversity of HTD2 for three sugarcane populationsHTD2 for three sugarcane populations A: origin species parents’ population; B: backbone parents’ population; C: main varieties population."

Table 3

Polymorphism of exon and intron regions of HTD2 for three sugarcane populations"

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

Fig. 5

Main types of InDel loci in exon and intron regions of HTD2 gene for three sugarcane populations"

Table 4

Ka/Ks and number of synonymous and nonsynonymous in code region of HTD2 for three sugarcane populations"

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

Table 5

Haplotype diversity of code region of HTD2 for three sugarcane populations"

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

Fig. 6

Haplotype network of code region of HTD2 in three sugarcane populations"

Table 6

Distribution of main haplotypes of code region of HTD2 in three sugarcane populations"

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

Table 7

Pearson correlation analysis between variation base type of SNPs of code region of HTD2 and tillering rate for sugarcane germplams resources"

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**

Table 8

Pearson correlation analysis between InDels of code region of HTD2 and tillering rate for sugarcane germplasm resources"

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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