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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (2): 403-413.doi: 10.3724/SP.J.1006.2024.31016

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

Cloning of TabHLH112-2B gene and development of its functional marker associated with the number of spikelet per spike in wheat

FAN Zi-Pei1,2(), LI Long2, SHI Yu-Gang1, SUN Dai-Zhen1,*(), LI Chao-Nan2,*(), JING Rui-Lian2   

  1. 1College of Agriculture, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2023-02-27 Accepted:2023-05-24 Online:2024-02-12 Published:2023-06-20
  • Contact: *E-mail: sdz64@126.com;lichaonan@caas.cn
  • Supported by:
    National Key Research and Development Program of China(2022YFD1200201);China Agriculture Research System of MOF and MARA(Wheat, CARS-03-5)

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Abstract:

The bHLH (basic Helix-Loop-Helix) transcription factor plays an important role in plant growth and development. In this study, wheat gene TabHLH112-2B was cloned, which consists of seven exons and six introns, encoding 444 amino acids, and has a typical HLH conserved domain at 315-364 amino acids. The tissue expression pattern analysis showed that TabHLH112-2B was expressed in all tissues at seedling stage, jointing stage, heading stage, and flowering stages. Among them, the relative expression levels in leaves and roots were higher. The cis-acting element analysis showed that the promoter region of TabHLH112-2B contained a variety of cis-acting elements related to plant hormone responses, stress responses, and meristem development. The qRT-PCR exhibited that the relative expression level of TabHLH112-2B was responsive to plant hormones (such as ABA, IAA, MeJA) and abiotic stresses (such as drought, salt, low and high temperatures). Two SNPs were detected in its promoter region by genomic sequence polymorphism, which were classified into two haplotypes. A molecular marker was developed based on SNP-682, and association analysis showed that the marker was significantly correlated with the number of spikelet per spike in various environments such as drought and high temperature. Hap-2B-2 was a favorable haplotype with more spikelets per spike. These results of this study provide the valuable genetic resources and technical support for molecular marker-assisted breeding of wheat varieties with high yield and wide adaptability.

Key words: wheat, TabHLH112-2B, molecular marker, association analysis, the number of spikelet per spike, favorable haplotype

Table 1

Websites for software used in bioinformatics analysis"

软件
Software		 用途
Function		 网址
Website
SMART 结构域分析
Domain analysis		 http://smart.embl.de/
ProtParam 蛋白理化性质分析
Physical and chemical properties		 https://web.expasy.org/protparam/
ExPASy 蛋白亲疏水性分析
Hydrophilicity analysis		 https://web.expasy.org/protscale/SWISS-MODEL
SignalP-5.0 蛋白信号肽预测
Signal peptide prediction		 https://services.healthtech.dtu.dk/service.php?SignalP-5.0
NetPhos-3.1 磷酸化位点分析
Phosphorylation site analysis		 https://services.healthtech.dtu.dk/service.php?NetPhos-3.1
SOPMA 蛋白二级结构预测
Protein secondary structure		 https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
phyre2 蛋白三级结构预测
Protein tertiary structure		 http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index
PlantCARE 顺式作用元件分析
Cis-element analysis		 https://bioinformatics.psb.ugent.be/webtools/plantcare/html/

Table 2

Primers used in this study"

引物名称
Primer name		 上游引物序列
Forward sequence (5'-3')		 下游引物序列
Reverse sequence (5'-3')
TabHLH112-2B ACACTACCCCTCATCTTAGCTCC CTATGACATAATTCAGCCCTCTCA
TabHLH112-pro-2B GAGGCGACTATAGTACACGCAAA CGAAGGGTAAACTGATCCAGC
TabHLH112-2B-qRT CGGCTGAGCAGACTAGCGT AGACGAGCTGTTGGAGTGCC
TaTUB CGTGCTGTCTTTGTAGATCTCG GACCAGTGCAGTTGTCTGAAAG
TabHLH112-2B-BamH I GACAATCGAGTTAAAGAAATTAGGA TCTGTATGAATCTCTGACCGCA

Fig. 1

Predictive analysis of TabHLH112-2B gene sequence and protein structure A: gene structure; B: protein domains; C: protein secondary structure; D: protein tertiary structure."

Fig. 2

Prediction of TabHLH112-2B protein properties and phosphorylation sites A: the amino acid composition of the protein. A: alanine; R: arginine; N: asparagine; D: aspartic acid; C: cysteine; Q: glutamine; E: glutamic acid; G: glycine; H: histidine; I: isoleucine; L: leucine; K: lysine; M: methionine; F: phenylalanine; P: proline; S: serine; T: threonine; W: tryptophan; Y: tyrosine; V: valine. B: the affinity/hydrophobicity analysis of protein. C: phosphorylation site prediction of proteins. D: the amino acid sites with phosphorylated predictive values higher than 0.99."

Fig. 3

Tissue expression pattern of TabHLH112-2B genes at different developmental stages in wheat The relative expression pattern of TabHLH112-2B genes in different tissues at seedling stage (A), jointing stage (B), heading stage (C), and flowering stage (D). L: leaf; R: root; RB: root base; S: spike; YL: young leaf; P: peduncle; PN: penultimate node; AN: antepenultimate node; FL: flag leaf. Error bar represents mean±SD. Different lowercase letters indicate significant difference at P < 0.05."

Fig. 4

Relative expression pattern of TabHLH112-2B genes in wheat seedling roots under different hormone treatments A: cis-acting element prediction in TabHLH112-2B promoter. TabHLH112-2B expression levels in roots of two-week-old seedlings. B: 50 μmol L-1 ABA. C: 0.1 mmol L-1 IAA. D: 0.1 μmol L-1 MeJA treatment. Error bar represents mean±SD. *: P < 0.05; **: P < 0.01; ***: P < 0.001."

Fig. 5

Relative expression patterns of TabHLH112-2B genes in seedling stage under abiotic stress conditions in wheat The relative expression level of TabHLH112-2B genes in 16.1% PEG (A), 250 mmol L-1 NaCl (B), the low temperature stress (C), and the high temperature stress (D). Error bar represents mean±SD. *: P < 0.05; **: P < 0.01; ***: P < 0.001."

Fig. 6

Sequence polymorphism and dCAPS molecular marker of TabHLH112-2B A: the schematic diagram of SNPs and molecular marker in the promoter region of TabHLH112-2B, red box indicates digestion site, red letter indicates variation site, and red dot indicates mismatched base. B: the agarose gel electrophores is the image of two haplotypes. M: 100 bp DNA ladder; C: Hap-2B-1; T: Hap-2B-2."

Table 3

Association analysis between haplotype of TabHLH112-2B and spikelet number per spike"

环境
Environment		 P值
P-value		 环境
Environment		 P值
P-value
E1 0.011228526 E9 0.000000386
E2 0.113354414 E10 0.000000934
E3 0.048992919 E11 0.008263578
E4 0.031388574 E12 0.098658098
E5 0.002171438 E13 0.016524218
E6 0.000019440 E14 0.000638932
E7 0.000003438 E15 0.000032654
E8 0.000227778 E16 0.000022470

Fig. 7

Analysis of association between haplotype of TabHLH112-2B and spikelet number per spike Abbreviations of E1-E16 are same as those given in Table 3. Error bar represents mean ± SD. *: P < 0.05; **: P < 0.01; ***: P < 0.001."

Fig. 8

Application of TabHLH112-2B favorable haplotype in Chinese wheat breeding history Distribution frequency of two haplotypes in landraces (A) and modern cultivars (B) collected from 10 Chinese wheat production zones. I: Northern Winter Wheat Zone; II: Yellow-Huai River Valleys Facultative Wheat Zone; III: Middle and Low Yangtze Valleys Autumn-Sown Spring Wheat Zone; IV: Southwestern Autumn-Sown Spring Wheat Zone; V: Southern Autumn-Sown Spring Wheat Zone; VI: Northeastern Spring Wheat Zone; VII: Northern Spring Wheat Zone; VIII: Northwestern Spring Wheat Zone; IX: Qinghai-Tibetan Plateau Spring-Winter Wheat Zone; X: Xinjiang Winter-Spring Wheat Zone. C: the number of spikelet per spike of cultivars released in different decades in two growing seasons, in 2005-2006 (2005) and 2010-2011 (2010). D: the frequency of two haplotypes in cultivars released in different decades."

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