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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (5): 690-699.doi: 10.3724/SP.J.1006.2020.93051

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

Genetic analysis and molecular characterization of tasselseed mutant ts12 in maize

Li-Ping QIN,Er-Fei DONG,Yang BAI,Lian ZHOU,Lan-Yang REN,Ren-Feng ZHANG,Chao-Xian LIU(),Yi-Lin CAI()   

  1. Maize Research Institute, Southwest University / Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
  • Received:2019-09-16 Accepted:2020-01-15 Online:2020-05-12 Published:2020-01-23
  • Contact: Chao-Xian LIU,Yi-Lin CAI E-mail:cauxian@163.com;caiyilin1789@163.com

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Abstract:

Sex determination is closely associated with development of maize tassel and ear. Function study of the genes underlying maize sex determination is critical for elucidating its regulation network. One sex determination mutant with feminized tassel, named tasselseed12 (ts12), was created through B73 pollen treatment with chemical agent ethyl methyl sulfonate (EMS). The feminized tassel spikelets were observed under scanning electron microscope when the immature tassels were 13 mm long. Using map-based cloning strategy, the ts12 was mapped to an interval about 290 kb, flanked by markers LM4 and RM5, which harbored nine annotated genes including the reported sex determination gene Tasselseed2 (Ts2). The Ts2 coding sequence from ts12 mutant revealed the 196th base guanine was substituted by adenine, leading to the substitution of amino acids from glycine to arginine, which probably resulted in the tasselseed phenotype. An allelic test crossing ts2 with ts12 showed that all the F1, F2 plants could produce feminized tassels with lots of silks, which indicated ts12 was a new allelic mutant of ts2. The exogenous jasmonic acid (JA, 1 mmol L -1) could recover the normal phenotype of most of spikelets. The expression analysis of Ts12 in wild-type plants showed a high expression level in immature tassels, and moderate one in immature ears and leaves. However, its expression was dramatically decreased in immature tassels and ears of ts12. The mutation in conserved region and the reduced expression of Ts2 probably are the causes of tasselseed phenotype.

Key words: ts12, ts2, tasselseed, fine mapping, allelism test

Supplementary table 1

Primer sequences for ts12 mapping, candidate gene cloning and expression analysis"

引物名称
Primer name		 正向引物序列
Forward primer sequence (5′-3′)		 反向引物序列
Reverse primer sequence (5′-3′)		 标记类型
Genetic marker types
LM1 CACAGCAAGAGTACAGCATCAG TGATCCCAGTTCTCGATGTAGC SSR
LM2 GGAAATGTTTGACGTGACCTGC CACTTGGCAAACCAATCCAACG SSR
LM3 AATAGCGTGCTCCCGTGTTATC CTGTCGTTGCTTGCTTCTTCCA SSR
LM4 AATGGGATAAAGACCGGATTCT ATAAGCGTTGTAGGACAGGAGC InDel
RM1 TCGGAGAGGACACGGTTTAGTA CATGAACATTGGCGAAGCTACC SSR
RM2 GCTCCTGTGTGCAACTTGTTAG GCATATGAATCGGTGTTCGGTG SSR
RM3 CTGTTCCTTCGCTTTCACAGAA AGTAGCTCTCTAGTTGTGTCCC SSR
RM4 GCTGATTTAAGATATAAAGTGCCTCCC CCTGCTGGACCGGAGTAAAG InDel
RM5 AGAACGGTGATTTTTGTCTGGT GTTTTGCATGCTCATGTAGACG InDel
Ts2-JD ACAGCAGAGTAGAGTAGCACAC GCCATGGGAATGGGATATTTGG
Ts2-qPCR GTGGAGAAGATGGAGGAGGTGGT ATTGATTCACAAGCCGATGAGGTT
Actin TCACCCTGTGCTGCTGACCG GAACCGTGTGGCTCACACCA

Fig. 1

Phenotype of ts12 A and B: mature tassel of the wild type and ts12; C: the normal spikelets that could produce pollens on ts12 tassels. D and E: immature tassel of the wild type (12 mm) and ts12 (13 mm) under scanning electron microscope. WT: wild type; G: gynoecium; ST: stamen; C: carpel. "

Table 1

Segregation ratio test for ts12"

群体组配
Population construction		 F1植株雄穗表型
Phenotype of F1 plants		 F2分离群体 F2 segregation population F2植株总数
Total number of F2 plants		 χ2
野生型 WT ts12
ts12 × Mo17 正常 Normal 115 35 150 0.22#

Fig. 2

Screening and confirmation of SSR markers umc1169 linked with ts12 MT: DNA pool of ts12; WT: DNA pool of wild-type plants. "

Fig. 3

Fine mapping of ts12 The numbers above the solid back line between SSR markers represent physical distance. The numbers in parenthesis represent the exact position of the marker on chromosome. The red numbers under SSR markers represent recombination events. "

Table 2

Gene annotation of ts12 fine mapping region"

基因 ID Gene ID 基因注释 Gene annotation
Zm00001d028796 Meristematic receptor-like kinase
Zm00001d028797 Laccase-13
Zm00001d028798 Hypothetical protein
Zm00001d028799 CASP-like protein 4B1
Zm00001d028801 26S proteasome regulatory subunit 4 homolog A
Zm00001d028802 Hypothetical protein
Zm00001d028803 Protein SCAR2
Zm00001d028804 U2 small nuclear ribonucleoprotein B
Zm00001d028806 Sex determination protein Tasselseed2

Fig. 4

CDS alignment of Ts2 genes from B73 and ts12 mutant。。 The parts underlined in red color represent the conserved domain A of Ts2. The green rectangle indicates the mutation site of the amino acid substitution in Ts2. 。。"

Fig. 5

Sequence alignment of Ts2 partial fragment from ts12 and six inbred lines 。。 The blue rectangle indicates the single nucleotide mutation site of the Ts2. 。。"

Fig. 6

Allelism test of ts12 with ts2 A: the feminized tassel of ts2 mutant; B: the mutant phenotype in the offspring of allelism test of ts12 and ts2. "

Fig. 7

Phenotype of ts12 treated by JA and tassel seeds statistics A: untreated ts12 tassel phenotype; B: 0.05% ethanol-treated ts12 tassel phenotype; C: JA-treated ts12 tassel phenotype; D: number of tassel seeds per ts12 mutant. The numbers on top of the column represent the standard deviation. ** indicates extremely significant difference of tassel seeds number between JA-treated ts12 and untreated and 0.05% ethanol-treated ts12 plants (P < 0.01). "

Fig. 8

Expression analysis of Ts2 gene ** indicates extremely significant difference of expression in specific tissues between Ts2 and WT (P < 0.01). "

Supplementary fig. 1

Sequence analysis of conservative domain A of short-chain alcohol dehydrogenases SbTs2: Ts2 ortholog from sorghum; SiTs2: Ts2 ortholog from Setaria italic; OsTs2: Ts2 ortholog from rice; BdTs2: Ts2 ortholog from Brachypodium distachyon; ATSDR2: Ts2 ortholog from Arabidopsis thaliana; BHD: a short-chain alcohol dehydrogenase from rat; 17βHSD1: a short-chain alcohol dehydrogenase from human; NACMAN: a short-chain alcohol dehydrogenase from Flavobacterium; 3βHSD: a short-chain alcohol dehydrogenase from Pseudomonas testosteroni; FABG: a short-chain alcohol dehydrogenase from Escherichia coli. "

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