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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (1): 55-66.doi: 10.3724/SP.J.1006.2024.33011

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

Mapping and cloning of plant height gene PHR1 in maize

YANG Chen-Xi1(), ZHOU Wen-Qi1,2,*(), ZHOU Xiang-Yan1,*(), LIU Zhong-Xiang2, ZHOU Yu-Qian2, LIU Jie-Shan1, YANG Yan-Zhong2, HE Hai-Jun2, WANG Xiao-Juan2, LIAN Xiao-Rong2, LI Yong-Sheng2   

  1. 1College of Life Science and Technology, Gansu Agricultural University, Gansu 730070, Lanzhou, China
    2Crop Research Institute, Gansu Academy of Agriculture Sciences, Gansu 730070, Lanzhou, China
  • Received:2023-02-24 Accepted:2023-06-29 Online:2024-01-12 Published:2023-07-25
  • Contact: *E-mail: zhouwenqi850202@163.com; E-mail: zhouxy@gsau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32160490);National Natural Science Foundation of China(31960443);Major Project of Gansu Province(21ZD11NA005);Major Project of Gansu Province(21ZD10NF003);Biological Breeding Project of Gansu Academy of Agricultural Sciences(2022GAAS04);Youth Mentor Fund Project of Gansu Agricultural University(GSAU-QDFC-2021-14);Strategic Research and Consulting Project of Chinese Academy of Engineering(2021-DFZD-21-3)

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Abstract:

Plant height is an important index for the ideal maize breeding, which not only affects the mechanical harvest of maize, but also closely relates to the lodging resistance and the biological yield of maize. In this study, a plant height reducing mutant-1 (phr-1) was obtained by using low-dose fast neutron (4.19 Gy) irradiation to mutate the maize inbred line KWS39 at the low ear position. Phenotypic traits were investigated and analyzed in the field, and candidate genes were identified by mining and functional annotation of genes in the target region based on the B73 reference genome using the extreme trait pool sequencing analysis (BSA-seq) and the method of target region recombination exchange identification with F2 segregation population of phr-1×B73. These results indicated that there might be a variation site in Bin1.06 interval on chromosome 1, and the target region was precisely located between two markers, Umc1122 and Umc1583a, with a 600 kb interval by using the large segregation population and polymorphic markers. Brachytic2 (BR2), encoding a sugar protein regulating the polar transport of auxin in maize stems, is a known gene controlling plant height in this region. The sequencing of candidate genes revealed that phr-1 was a new allelic mutation of br2-1, with a 165 bp insertion in the fourth exon of the BR2 gene resulting in the amino acid at position 547 changing to a stop codon and premature protein translation termination. The mutation site and variation mode of phr-1 were completely different from those of br2-1 single base mutation site. The allelic hybridization experiment confirmed that the PHR1 candidate gene is a new allelic mutation of the BR2 gene. This study provides new germplasm resources for maize BR2 gene in plant height genetic improvement.

Key words: maize, plant height, ear height, the bulked segregant analysis, map-based localization, function analysis

Table 1

Primers used in this study"

引物名称
Primer name		 正向序列
Forward sequence (5′-3′)		 反向序列
Reverse sequence (5′-3′)
GAPDH CCATCATGCCACACAGAAAAC AGGAACACGGAAGGACATACCAG
BR2-Q1F/Q1R TGAGCAACTCCAGCTCTTAACCA GCGAACTGGACATCCTCAGATTA
BR2-Z1F/Z1R ATTCACGCAGAGCAGAAGAGC GCATAAGGTTGGACAGGGAAAG
BR2-H1F/H1R AGGTTGGGGAGCGCGGCCTGCAG ACCCATCCATCCATCCATTCCGTCCT
RT-Br2-F1/R1 ACGAGCATCAGGGAGAACC GCTCCCCAACCTGCGTGTCG

Fig. 1

Phenotype of KWS39 (CK) and phr-1 mutant plants A, B: the comparison of plant height between KWS39 and phr-1, with phr-1 being shorter than KWS39; Bar: 10 cm. C: statistical analysis of PH and EH using t-test. D: the comparison of internode lengths between KWS39 and phr-1. E: KWS39 and phr-1 both have 11 internodes; the internodes are indicated from root to tip by 1, 2, 3, …, 9, 10, 11; but the length between each internode in phr-1 is shorter than in the control. Scale: 10 cm."

Fig. 2

Phenotype of KWS39 (CK) and phr-1 mutant plants A: there was no significant difference in plant height and root length between KWS39 and phr-1 after five days of germination. B: the inverted third leaf, the inverted second leaf, and the flag leaf from left to right. C: both KWS39 and phr-1 have normal root development with phr-1 having a more developed root system. D: phenotypic traits of self-pollinated ears in phr-1 and KWS39. E: there was no significant change in grain size and weight between KWS39 and phr-1. Bar: 10 cm (A-D)."

Table 2

Analysis of agronomic traits between KWS39 and phr-1 (mean±SD)"

类型
Type		 性状
Trait		 野生型KWS39
Wild type KWS39		 突变体phr-1
Mutant phr-1		 P-value N
株型Plant architecture 株高Plant height (cm) 182.8±11.0 93.3±2.3** 3.88E-09 15
穗位高Ear height (cm) 66.4±17.3 23.2±1.7** 0.000106 15
雄穗Tassel 雄穗分支数 Tassel branch number 5.1±1.4 4.7±1.6 0.582953 15
雄穗长Tassel length (cm) 31.65±2.3 27.68±1.9* 0.031581 15
雌穗Ear 穗长Ear length (cm) 13.81±1.3 13.08±0.5 0.447437 15
穗粗Ear diameter 3.97±0.1 3.7±0.2 0.094702 15
穗行数 Kernel row number 14.4±0.8 13.4±0.9 0.052177 15
行粒数 Grains row number 24.1±4.1 22.6±4.6 0.057321 15
秃尖长Bald tip length (cm) 1.5±0.6 1.31±0.4 0.078416 15
叶型Blade profile 倒3叶长Inverted 3 leaves long (cm) 47.25±3.8 44.3±3.6* 0.045242 15
倒3叶宽Inverted 3 leaves wide (cm) 6.61±0.7 6.36±0.6 0.486148 15

Table 3

Chi-square test of F2 population"

群体
Population		 观察值Observations 期望值Expectations 卡方检验
Chi-square
野生型Wild type 突变体Mutant 野生型Wild type 突变体Mutant
phr-1×B73 F2 762 238 760 240 0.88

Fig. 3

Phenotype of KWS39, phr-1, F1 hybrid plant, and dwarf mutant in F2 plant Scale: 10 cm."

Fig. 4

Linkage map of phr-1 candidate genes A: the black and gray dots represent ΔSNP-index. B: the blue line is the fitted line of ΔSNP-index after windowing. C: the orange-yellow line is the 95% confidence line. D: the red line is the 99% confidence line."

Table 4

Crossing-over value of preliminary localization markers and phenotypes"

标记名称
Molecular marker		 交换频率
Crossing-over value
umc2235 0.38
umc1035 0.29
umc2560 0.17
umc2569 0.15
umc1122 0.08
umc1583a 0.11
umc1278 0.21
umc2387 0.36

Fig. 5

Sequence analysis of BR2 transcript between KWS39 and phr-1"

Fig. 6

Generic identification of mutant F1 representative type is consistent A: plant phenotypes of B73 and the mutants br2-1, phr-1 and (bra2-1×ph-1) F1; B: plant phenotypes of B73 and KWS39, phr-1 and (bra2-1×phr-1) F1. Bar: 10 cm."

Fig. 7

Relative expression pattern of BR2 genes in four maize inbred line leaves The data are tested by t-test; ** indicates significant difference at P < 0.01."

Table 5

Genetic code and function annotation of BR2 in different species"

物种
Species		 同源基因
Orthologous gene		 功能预测
Putative function
玉米Zea mays GRMZM2G315375 PGP1
拟南芥Arabidopsis thaliana AT1G10680 P-glycoprotein 10
水稻Oryza sativa LOC_Os02g46680 Multidrug resistance protein, putative, expressed
白杨树Populus trichocarpa POPTR_0001s02200 PGP2; ATPase, coupled to transmembrane movement of substances
高粱Sorghum bicolor Sb04g023730 P-glycoprotein 1
葡萄Vitis vinifera GSVIVG0009946001 Multidrug resistance protein 1, 2

Fig. 8

Sequence alignment of animo acid sequence of ZmBR2 with its homologues and phylogenetic analysis A: sequence alignment of animo acid sequence of ZmBR2 with its homologues; B: homology tree of ZmBR2; C: phylogenetic tree of ZmBR2."

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