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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (12): 3046-3054.doi: 10.3724/SP.J.1006.2024.41014

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

Genetic dissection of the albino hull mutations in barley (Hordeum vulgare L.)

SUN Man1,2(), AN Chao-Dan2, GAO Guang-Qi2, GUO Jie1, YANG Ping2, JIANG Cong-Cong2,*()   

  1. 1College of Agriculture, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China
    2Key Laboratory of Grain Crop Genetic Resources Evaluation and Utilization, Ministry of Agriculture and Rural Affairs / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2024-03-01 Accepted:2024-06-20 Online:2024-12-12 Published:2024-07-15
  • Contact: *E-mail: jiangcongcong@caas.cn

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Abstract:

Lemma and palea are the outermost organs on each floret of barley (Hordeum vulgare L.), where the majority of spike photosynthesis occurs, supplying carbohydrates to the developing grains. The barley albino hull (alh) mutants, obtained from EMS mutagenesis, showed albinistic lemma and palea, as well as albinistic pulvini, stem nodes, and stem bases, while the leaves and awns remain green. In this study, multiple allelic alh mutants were identified, and segregating populations were generated accordingly. Genetic analysis indicated that a single recessive gene is responsible for the alh phenotype. Through re-sequencing of the candidate gene in multiple alh mutants, and co-segregation tests using competitive allele-specific PCR (KASP) markers, loss-of-function mutations in the gene HvGLK2 were shown to account for the alh phenotype. Each of the three independent mutations identified in this study is distinct from previously reported albino lemma variants such as alm1 or ebu-a. HvGLK2 encodes a Golden 2-like (GLK2) transcription factor, belonging to the GARP subfamily of MYB transcription factors, and has a paralog designated GLK1 in most monocot and dicot species. The temporal and spatial expression patterns showed that HvGLK2 is abundantly transcribed in senescent leaves, lemmas, and rachises. This study highlights the importance of HvGLK2 in chlorophyll synthesis in various organs, including lemma, palea, and stem nodes of barley plants. Moreover, it provides valuable materials for further studies aimed at evaluating the contribution of spike photosynthesis to the eventual grain yield.

Key words: barley, hull, albino mutant, HvGLK2, loss-of-function

Fig. 1

Morphology of the wild-type HTX and the albino hull mutants A: whole spike of wild-type HTX (left) and the homozygous albino hull (alh.a) mutant identified from family T-M3-4073 (right); B: zooming in the middle section of spike from wild-type HTX (left) and alh.a mutant (right); C: stem base of wild-type HTX (left) and alh.a mutant (right); D: stem segments of wild-type HTX (left) and alh.a mutant (right); E: leaf lobe of wild-type HTX (left) and alh.a mutant (right); F: structural anatomy of central spikelet of wild-type HTX (left) and alh.a mutant (right) at the grain filling stage; G: fully matured grain of wild-type HTX (left) and alh.a mutant (right), which at the bottom row had hull been removed, respectively. Scale bars in plots are all 1 cm."

Fig. 2

Comparison of the thousand-grain weight of wild-type HTX and the alh.a mutant **, significant at P < 0.01."

Table 1

Phenotypic statistics in each of the ten T-M4-4073 families"

M4家系
M4 family		 野生型单株数目
Number of wild-type plants		 稃壳白化单株数目
Number of alh plants		 M3单株基因型
Deduced M3 genotype		 卡方测验
χ2-test (χ20.05=3.841)
T-M4-4073-1 0 12 M Na
T-M4-4073-2 12 0 WT Na
T-M4-4073-3 0 12 M Na
T-M4-4073-4 51 17 Het χ2(1:3)=0
T-M4-4073-5 52 19 Het χ2(1:3)=0.082
T-M4-4073-6 12 0 WT Na
T-M4-4073-7 7 5 Het Na
T-M4-4073-8 12 0 WT Na
T-M4-4073-9 12 0 WT Na
T-M4-4073-10 6 6 Het Na

Fig. 3

Diagram of the HvGLK2 gene structure (A), and alignment of its encoded amino acid sequence in wild-type HTX and multiple loss-of-function alh mutants (B)"

Table 2

Primers for fragmental amplification of the causal gene HvGLK2 of alh mutants"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 退火温度
Melting temperature
(℃)		 产物大小
Product size (bp)		 用途
Purpose
GLK2_5UTR_F1 TGCCATCGTCAAGTCAGCTG 60.12 746 覆盖exon1的片段扩增
Amplification of fragments covering exon1
GLK2_IN1_R1 GTGAGAGAATGCAGGGCAGTAC 58.95
GLK2_IN1_F1 CAGTGAGGCGCTCGATTTAG 58.62 521 覆盖exon2的片段扩增
Amplification of fragments covering exon2
GLK2_IN2_R1 GCGTACGTCCGTGTGAATTT 58.45
GLK2_IN2_F1 TATATGAGATTGCATTTGTGGTCG 59.82 1456 覆盖exon3-6的片段扩增
Amplification of fragments covering exon3-6
GLK2_3UTR_R1 TGCACGTAGGTACAGACGGAC 58.43
3H135348068-F CTCCATCTACCCATCGATCAAGA 60.75 178 检测基因型与表型关联性的KASP标记
KASP markers were used to detect genotype-phenotype associations
3H135348068-RA GAAGGTCGGAGTCAACGGATTGGCCGGACTAGGGCTTTA 61.11
3H135348068-RG GAAGGTGACCAAGTTCATGCTGGCCGGACTAGGGCTTTG 60.33

Fig. 4

Co-segregation analysis between the KASP marker that targets the loss-of-function mutation in alh.a and the albino hull phenotype"

Fig. 5

Spatial and temporal patterns of the HvGLK2 transcription during the barley lifecycle FPKM: Fragments Per Kilobase of exon model per Million mapped fragments. ROO1: roots from seedlings (10 cm shoot stage); LEA: shoots from seedlings (10 cm shoot stage); INF: young developing inflorescences (5 mm); INF2: developing inflorescences (1.0-1.5 cm); NOD: developing tillers, 3rd internode (42 DAP); CAR5: developing grain (5 DAP); CAR15: developing grain (15 DAP); ETI: etiolated seedling, dark cond. (10 DAP); LEM: inflorescences, lemma (42 DAP); LOD: inflorescences, lodicule (42 DAP); EPI: epidermal strips (28 DAP); RAC: inflorescences, rachis (35 DAP); ROO2: roots (28 DAP); SEN: senescing leaves (56 DAP)."

Fig. 6

The phylogenetic tree of GLK genes in various plant species Phylogenetic tree was constructed based on the GLK2 and GLK1 amino acid sequences in Hordeum vulgare (Hv), Oryza sativa (Os), Zea mays (Zm), Triticum aestivum (Ta), Sorghum bicolor (Sb), Setaria italica (Si), Arabidopsis thaliana (At), Gossypium raimondii (Gr), Glycine max (Gm), and Solanum lycopersicum (Sl)."

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