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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (11): 1615-1627.doi: 10.3724/SP.J.1006.2019.91009

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

Transcription factor gene TaNAC67 involved in regulation spike length and spikelet number per spike in common wheat

ZHANG Hong-Juan1,2,LI Yu-Ying2,3,MIAO Li-Li2,WANG Jing-Yi2,LI Chao-Nan2,YANG De-Long1,*(),MAO Xin-Guo1,2,*(),JING Rui-Lian2   

  1. 1 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, Gansu, China
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Crop Germplasm and Utilization, Ministry of Agriculture, Beijing 100081, China
    3 College of Agriculture, Henan Agricultural University, Zhengzhou 450002, Henan, China
  • Received:2019-01-23 Accepted:2019-05-12 Online:2019-11-12 Published:2019-05-22
  • Contact: De-Long YANG,Xin-Guo MAO E-mail:yangdl@gsau.edu.cn;maoxinguo@caas.cn
  • Supported by:
    This study was supported by the National Key Research and Development Plan(2017YFD0300202);Gansu Agriculture Research System(GARS-01-04)

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Abstract:

NAC transcription factor is a plant specific superfamily and plays an essential role in regulating plant growth and development and stress response. Our previous research indicated that TaNAC67 involved in the response to various environmental stimuli and its overexpression resulted in enhanced tolerance to multiple abiotic stresses. To probe its roles in plant growth and development, a panel consisted of 36 wheat accessions with high diversity we need for polymorphism assays. The genomic sequences, covering the promoter region and gene coding region, were obtained by Sanger sequencing, and named as TaNAC67-6A, TaNAC67-6B, and TaNAC67-6D according to their genomic origins. For TaNAC67-6A, a SNP (A/G) at -1516 nt and a 126 bp InDel between -873 and -748 nt were identified in the promoter region, and one CAPS and one allele specific marker were developed, respectively. For TaNAC67-6B, two SNPs, at -2014 and -1916 nt were detected, and two dCAPS markers were developed accordingly. For TaNAC67-6D, two SNPs, one at -1795 nt in the promoter region, and the other at 357 nt in the gene coding region were identified, and one CAPS and one dCAPS marker were designed, respectively. To probe the relationship of molecular markers and potential agronomic traits, we introduced a population (PA) with 282 common wheat accessions to perform association assay. No association was identified between markers from TaNAC67-6A and 6B and agronomic traits. However, strong associations were identified between SNP-D-1and spike length (SL), and between SNP-D-2 and the total number of spikelet per spike (TNSS). Haplotype assays showed there were three major haplotypes in PA, i.e. Hap-6D-1, -2, -3. Furthermore, the SL of accessions with Hap-6D-3 was significant longer than that with the other two haplotypes under all 30 environments, and the TNSS was significant larger under 22 environments, thus Hap-6D-3 is an elite haplotype for both SL and TNSS, which was selected positively in the process of wheat breeding in China. Further transgenic experiments revealed that TaNAC67-overexpressing rice lines had lager panicles and more seeds relative to wild type, which is consistent with the association assay results. Therefore, TaNAC67 is a potential candidate gene in spike traits improvement, and TaNAC67-6D might be used in marker assistant molecular breeding in wheat.

Key words: wheat, transcription factor, single nucleotide polymorphism, functional marker, haplotype, association assay

Table 1

Primers used in experiments"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 退火温度
Annealing temperature (℃)		 用途
Purpose
NAC67AsF TGTTCCGTATTCAGTACCGGC 55 基因组特异引物
Genome specific primers
NAC67AsR ACACGTTGACCTTACGAATTACAAG
NAC67BsF TATCTCGTCCACTGTTCTCATCTTC 60
NAC67BsR GACTATTGAGAGGTCAGAAGAAGAATG
NAC67DsF GTTAAAGAGAAAGGGAGGAAGGGA 58
NAC67DsR CGTGAGAGTATTGAGAGCTCAGAAG
NAC67AseqF1 ACGTGTGCCCCTATATTTTTAC 测序引物
Sequencing primers
NAC67AseqF2 GACGGCTGAAACACCTAAAAC
NAC67BseqF1 CCGGGCGCGATGTG
NAC67BseqF2 TTAGACACCTAAAAAGTGTCATCG
NAC67DseqF1 GGCTGACAGTAATGATGTTTAGTG
NAC67DseqF2 GTGCCGTTTGGGTCAGG
NAC67ABDseqR CCCGCGGCGTGAAG
A-1516-F GCCTCCTTGTGACTTTGCGA 65 功能标记特异引物
Specific primers for
functional markers
A-1516-R CGCCGGATAATTACAGGTCAGTT
A-126-F GAAGCCTCATTAGGCCCAATT 65
A-126-R TCGACCGGCCGTAGCCT
B-2014-F GGTCTAAGCGGGGTACCGGACTGCA 61
B-2014-R CCTTCTTTGGTATGCCAGGAACTCA
B-1916-F TTGCACAACATGATCATTGAGAGCTA 61
B-1916-R TCACACATCGCGCCCGGC
D-1795-F AGACGATGTGAAGACGACCAAGA 61
D-1795-R ACTGTGGTTCGTGCAACCCTAG
D357-F CCCATCATCGCCGAGGTCGA 64
D357-R CTTCTTGATCCCGACGGTCCTG

Fig. 1

TaNAC67-6A sequence polymorphism (A) and allele specific marker SNP-A-1 (B), and CAPS marker SNP-A-2 (C) Fig. C: if the nucleotide at -1516 nt is A, the PCR product cannot be digested; if it is G, the PCR products can be digested. M: 100 bp DNA ladder."

Fig. 2

TaNAC67-6B sequence polymorphism (A), dCAPS marker SNP-B-1 (B), and SNP-B-2 (C) Fig. B: if the genotype at -2014 nt is C the PCR products can be digested; if it is T the PCR products cannot be digested. Fig. C: if the genotype at-1916 nt is T, the PCR products cannot be digested, if it is C the PCR products can be digested. M: 100 bp DNA ladder."

Fig. 3

TaNAC67-6D sequence polymorphisms (A) and CAPS marker SNP-D-1 (B) and dCAPS marker SNP-B-2 (C) Fig. B: if the genotype at -1795 nt is G, the PCR products can be digested; if it is T the PCR products cannot be digested. Fig. C: if the genotype at 357 nt is T the PCR products cannot be digested, if it is C the PCR products can be digested. M: 100 bp DNA ladder."

Table 2

Associations between SNP-D-1, SNP-D-2 of TaNPC67-6D and agronomic traits"

年份
Year		 地点
Site		 环境条件
Environment		 性状Trait (P-value)
SNP-D-1-SL SNP-D-2-TNSS
2010 SY DS 2.43E-04*** n.s.
DS+HS 0.00327*** n.s.
WW 0.00346*** 0.02375*
WW+HS 0.01165* 0.00126***
2011 SY DS+HS 0.00162*** n.s.
DS 3.63E-04*** n.s.
WW+HS 1.72E-05*** n.s.
WW 5.24E-04*** 0.03478*
2012 SY DS+HS 0.00192*** 0.00506**
DS 0.00159*** 0.0313*
WW+HS 0.02646* n.s.
WW 0.00363*** 0.03707*
2013 CP DS 0.00129*** 0.01055*
WW 0.00168*** n.s.
2015 SY DS+HS 2.81E-04*** 0.00336***
DS 0.00121*** 0.01369*
WW+HS 0.00268*** n.s.
WW 0.02357* n.s.
2016 SY DS+HS 0.00895** n.s.
DS 0.00996** 0.01447*
WW+HS 9.65E-05*** 0.00877**
WW 0.00788** 4.15E-04***
CP WW 0.0022*** 0.00386***
DS 0.00598** 0.03743*
2017 SY DS+HS 5.86E-04*** 0.02236*
DS 0.01736* 3.51E-05***
WW+HS 9.82E-04*** 0.0054**
WW 0.00184*** 0.01334*
CP DS 0.00153*** 0.02641*
WW 0.00296*** 0.00599***

Fig. 4

Phenotypic comparisons of two genotypes for SNP-D-1 and SNP-D-2 under 30 environmental conditions Comparisons of spike length (A) and total number of spikelet per spike (B) for two genotypes of SNP-D-1 (A) and SNP-D-2 (B) under 30 environmental conditions. *, **, *** represent significant at the 0.05, 0.01, 0.001 probability levels, respectively."

Fig. 5

Phenotypic comparisons of three Haplotypes for TaNAC67-6D under 30 environmental conditions Comparisons of spike length (A) and total number of spikelet per spike (B) for three haplotypes of TaNAC67-6D under 30 environments. Bars superscripted by different letters are significantly different at the 0.05 probability level."

Fig. 6

Geographical distributions of the three haplotypes for TaNAC67-6D in the ten wheat zones in China Haplotype distributions of TaNAC67-6D in landrace (A) and modern variety (B) populations. I: Northern Winter Wheat Zone; II: Yellow and 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."

Fig. 7

Comparisons of panicle traits between transgenic rice and WT control Comparisons of panicle traits between transgenic rice (L1, L2) and WT in panicle phenotype (A), panicle length (B), panicle branch (C), grain number per panicle (D), sterile grain number per panicle (E), and thousand grain weight (TGW) (F). ** Significant at the 0.01 probability level."

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