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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (10): 1854-1862.doi: 10.3724/SP.J.1006.2021.04208

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

Identification of a cotton flower organ mutant 182-9 and cloning of candidate genes

YANG Qin-Li1(), YANG Duo-Feng1, DING Lin-Yun1, ZHANG Ting2, ZHANG Jun2, MEI Huan2, HUANG Chu-Jun2, GAO Yang1, YE Li1, GAO Meng-Tao1, YAN Sun-Yi2, ZHANG Tian-Zhen1,2, HU Yan1,2,*()   

  1. 1State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
    2College of Agricultural & Biotechnology, Zhejiang University, Hangzhou 310000, Zhejiang, China
  • Received:2020-09-10 Accepted:2021-01-13 Online:2021-10-12 Published:2021-03-11
  • Contact: HU Yan E-mail:739768392@qq.com;njauhuyan@njau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31970320);National Major Project for Developing New GM Crops(2016ZX08009-003)

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Abstract:

Cotton is an important economic crop and the main source of natural fiber in the world. The budding, flowering and bolling during cotton growth and development directly affect the yield and quality of cotton fiber that are the main economic traits of cotton. In this study, we found a flower organ mutant (named 182-9) in transgenic cottons, which displayed the floral organ petaloid feature. PCR and Southern blotting confirmed that the foreign T-DNA was integrated into the 182-9 genome with a single copy. Comparative analysis of the resequencing data revealed that the exogenic T-DNA was inserted in the 182-9 on chromosome A11: 59086840. The insertion site was further verified by PCR and southern blot. According to the gene annotation of cotton genome, there were three candidate genes of GH_A11G2251, GH_A11G2252, and GH_ A11G2253, near to the insertion site. GH_ A11G2251 encoded AP2 genes controlling the formation of sepals and petals in the ABC model of flower organs as previous report. qRT-PCR showed that there were significant differences in the expression level of GH_A11G2251 in petals, pistil and stamens of transgenic receptor W0 and mutant 182-9. Our study provided the basis for further study of molecular mechanism in cotton floral organ development.

Key words: cotton, transgenic, floral organ, mutant, gene cloning

Fig. 1

T-DNA structure of the transgenic vector"

Fig. 2

Phenotype comparison of floral organ between the mutant 182-9 and transgenic receptor W0 A: the whole flower organ and ovary of transgenic receptor W0. B: floral organ of the mutant 182-9. C: bract, sepal, petal, pistil stamen of transgenic receptor W0. D: bracts, sepals, and petaloid structure of the mutant 182-9. Bar: 1 cm."

Fig. 3

PCR detection of NPTII gene in transgenic mutant 182-9 M: DNA molecular-weight markers; W0: negative control; 1: transgenic line 182-9; 2: transgenic line 182-36; 3: transgenic line 182-91; 4: transgenic line 182-150; 5: transgenic line 182-172; 6: transgenic line 182-173; 7: transgenic line 182-187."

Fig. 4

Southern blotting of NPTII gene in mutant 182-9 genome M: DNA marker; 1: transgenic receptor W0; C: plasmid; 2: transgenic line 182-187; 3: transgenic line 182-36; 4: transgenic line 182-173; 5: transgenic line 182-9 (indicated by the arrow)."

Table 1

Evaluation of the resequencing data"

样品
Sample		 原始reads
Raw reads		 过滤后reads
Clean reads		 原始碱基数
Raw base (Gb)		 过滤后碱基数Clean base (Gb) Q30
(%)		 GC含量
GC content (%)
182-9 245,670,776 245,536,479 73.70 73.66 91.81 35.43
W0 236,081,132 235,926,310 70.82 70.78 89.16 35.51

Fig. 5

PCR detection of the T-DNA insertion site in mutant 182-9 A: diagram of PCR detection; B: the sequencing results of the amplified sequence from 182-9 (the red part is the cotton genome sequence, and the others are T-DNA sequences). M: DNA marker; W0: transgenic receptor; 182-9: transgenic line."

Fig. 6

Southern blot detection of T-DNA insertion site M: DNA marker; C: positive plasmid; 1: transgenic line 182-9; 2: transgenic receptor W0. The specific band of 182-9 is shown by an arrow."

Table 2

Function annotation of the three candidate genes"

基因ID
Gene ID		 拟南芥中同源基因
Homologous genes in Arabidopsis		 功能描述
Function annotation
GH_A11G2251 ANT AP2类乙烯反应转录因子
AP2-like ethylene-responsive transcription factor ANT
GH_A11G2252 At1g65750 假定核糖核酸酶H蛋白At1g65750
Putative ribonuclease H protein At1g65750
GH_A11G2253 NA NA

Fig. 7

Position of the candidate gene relative to the insertion site"

Fig. 8

Comparison of the amplified sequences of GH_A11G2251 in mutant 182-9 and W0"

Fig. 9

Comparison of the amplified sequences of GH_A11G2252 in mutant 182-9 and W0"

Fig. 10

Comparison of the amplified sequences of GH_A11G2253 in mutant 182-9 and W0"

Fig. 11

Relative expression levels of candidate genes in floral organs A: the schematic diagram of floral organs of W0 and 182-9; B: the expression levels of the candidate genes in mutant 182-9 and wild-type W0. * means significant difference at the 0.05 probability level."

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