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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (12): 3154-3161.doi: 10.3724/SP.J.1006.2023.31009

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

Creation of doubled haploid in wheat using distant hybridization and unreduced gamete genes

LIU Xiao-Juan1,2(), LIU Xin2, ZHANG Ming-Hu2, HAO Ming2, NING Shun-Zong2, YUAN Zhong-Wei2, HUANG Lin2, LIU Deng-Cai1,2, ZHANG Lian-Quan1,2,*()   

  1. 1State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China / Sichuan Agricultural University, Chengdu 611130, Sichuan, China
    2Triticeae Research Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
  • Received:2023-02-08 Accepted:2023-05-24 Online:2023-12-12 Published:2023-06-02
  • Contact: * E-mail: zhanglianquan1977@126.com
  • Supported by:
    National Natural Science Foundation of China(31671682);Key Research and Development Program of Sichuan Province, China(2021YFYZ0002)

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Abstract:

The union of unreduced female and male gametes leads to spontaneous chromosome doubling, which is not only an important way of the origin of polyploid species but also an essential tool for enhancing crop doubled haploid breeding efficiency. A major quantitative trait locus QTug.sau-3B responsible for unreduced gametes formation was discovered in tetraploid Triticum turgidum wheat and was further transferred to elite common wheat lines using synthetic hexaploid wheat as a bridge. This objective of this study is to make wheat/wheat F1 hybrids between the elite lines with this gene region and commercial cultivars and to evaluate the efficiency of QTug.sau-3B gene leading to spontaneous chromosome doubling. Wheat/wheat F1 hybrids and their parents were pollinated with the fresh pollen of Imperata cylindrica to produce wheat haploid through chromosome elimination of I. cylindrica. Doubled haploid (DH) were then developed by spontaneous chromosome doubling in haploids because of the union of unreduced female and male gametes. In this experiment, 5 F1 hybrid materials and 3 I. cylindrica materials were used for distant hybridization. 4610 florets were pollinated, 1965 seeds were produced, 244 embryos were obtained, and 50 wheat haploid plants were obtained. In different temperature treatments, the haploid plant numbered H31 set seeds at 25℃/18℃ (18 h/6 h) and 25℃/10℃ (18 h/6 h), and the self-fertilization rate was 4.35% and 2.41%, respectively. The results of this study provide a reference for the establishment of wheat haploid breeding technology of chromosome elimination based on wheat-I. cylindrica hybridization and automatic chromosome doubling based on unreduced gamete genes.

Key words: haploid breeding, Imperata cylindrica, unreduced gametes, embryo rescue

Table 1

Location of I. cylindrica and its heading time and flowering time in 2018"

编号
Code		 来源地
Location		 抽穗时间
Heading time (month/day)		 开花时间
Flowering time (month/day)
BMZ1616 四川宜宾翠屏 Cuiping, Yibin, Sichuan 4/23 4/28
BMZ1617 四川资阳乐至 Lezhi, Ziyang, Sichuan 4/26 4/29
BMZ1801 四川成都崇州 Chongzhou, Chengdu, Sichuan 4/26 4/29
BMZ1605 江苏宿迁沭阳 Shuyang, Suqian, Jiangsu 4/27 4/29
BMZ1602 福建宁德屏南 Pingnan, Ningde, Fujian 4/28 4/30
BMZ1612 山东临沂费县 Feixian, Linyi, Shandong 4/28 4/30
BMZ1604 广东揭阳东山 Dongshan, Jieyang, Guangdong 4/28 5/1
BMZ1610 湖北十堰丹江口 Danjiangkou, Shiyan, Hubei 4/29 5/1
BMZ1621 江苏徐州沛县Peixian, Xuzhou, Jiangsu 4/29 5/1
BMZ1606 贵州遵义习水 Xishui, Zunyi, Guizhou 5/6 5/8
BMZ1618 四川自贡富顺 Fushun, Zigong, Sichuan 5/6 5/8
BMZ1619 浙江温州永嘉 Yongjia, Wenzhou, Zhejiang 5/9 5/11

Table 2

Pseudo seed and haploid embryo formation frequency obtained from different hybrids F1 between wheat/wheat F1 and I. cylindrica in 2019"

小麦材料
Wheat material		 授粉小花数
Pollinated florets		 结实数
Pseudo seeds (%)		 得胚数
Embryo formation (%)		 获得植株
Plantlet rate (%)
L14-6/CM602 F1 1588 701 (44.14)c 169 (24.11)a 26 (15.38)b
L14-6/SM114 F1 868 439 (50.58)b 61 (13.9)b 21 (34.43)a
L13-81/SM114 F1 963 395 (41.02)c 9 (2.28)c 0 (0)
L13-471/CM96 F1 973 299 (30.73)d 5 (1.67)c 3 (60)a
L13-316/SM830 F1 218 131 (60.09)a 0 (0) 0 (0)
总计Total 4610 1965 (42.62) 244 (12.42) 50 (20.49)

Table 3

Frequencies of seed formation obtained from crosses of wheat with different I. cylindrica in 2019"

白茅编号
I. cylindrica		 授粉小花数
Pollinated florets		 结实数
Pseudo seeds (%)		 得胚数
Embryo formation (%)		 获得植株
Plantlet rate (%)
BMZ1801 430 146 (33.95)b 30 (20.55)a 8 (26.67)a
BMZ1617 1418 514 (36.25)b 28 (5.45)c 5 (17.86)a
BMZ1616 2762 1305 (47.25)a 186 (14.25)b 37 (19.89)a

Fig. 1

Chromosome observation of root tip number of haploid plant H31 obtained from L14-6/CM602//I. cylindrica F1 (a, 2n = 21), chromosome observation of pollen mother cells in metaphase I (b, 2n = 21), and the detection on flow cytometry (c) Bar: 10 μm."

Fig. 2

Chromosome observation of pollen mother cells of haploid plant H31 obtained from L14-6/CM602//I. cylindrica F1 at meiosis stage a: late prophase I; b: restitutive nucle; c: anaphase II; d: dyad. Bar: 10 μm."

Fig. 3

PCR amplification of Xgpw1146 in 50 haploid plants and their parent lines M: marker; L: Langdon; A: AS2255; 1: L13-471; 2: CM96; 3-5: haploid plants H1-H3; 6: L14-6; 7: SM114; 8-28: haploid plants H4-H24; 29: L14-6; 30: CM602; 31-56: haploid plants H25-H50; 57: L14-6; 58: CM602. Red words mark the same amplification products of Xgpw1146, the red arrow shows the haploid plant H31."

Table 4

Detection of QTug.sau-3B using Xgpw1146 in different haploid plants"

含QTug.sau-3B单株编号
Code of haploid plants with QTug.sau-3B		 单倍体株数(编号)
Number of haploid plants
(Plant ID)		 系谱
Pedigree		 QTug.sau-3B来源
Origin of QTug.sau-3B
H1 3 (H1-H3) L13-471/CM96//I. cylindrica F1 AS2255
H7, H8, H10, H11, H12, H13, H14, H16, H19, H20, H23, H24 21 (H4-H24) L14-6/SM114//I. cylindrica F1 Langdon
H25, H26, H27, H28, H31, H33, H35, H36, H38, H39, H41, H45, H47, H48, H49, H50 26 (H25-H50) L14-6/CM602//I. cylindrica F1 Langdon

Table 5

Selfed seedset rate of haploid plants H31 (pedigree: L14-6/CM602//I. cylindrica F1) in different temperatures and light treatments"

单倍体编号
Haploid number		 处理时期
Treatment stage		 处理温度和光照时间
Temperature (℃)/time (h)		 自交小花数
Selfed florets		 自交结实数
Selfed seeds		 自交结实率
Selfed seed set rate (%)
H31-1 孕穗期Booting stage 25/18, 18/6 92 4 4.35
H31-2 孕穗期Booting stage 25/15, 18/6 89 0 0
H31-3 孕穗期Booting stage 25/10, 18/6 83 2 2.41

Fig. 4

Chromosomes in root tip cells of four doubled haploid plants derived from H31 (2n = 42) a: DH-H31-1; b: DH-H31-2; c: DH-H31-3; d: DH-H31-4; e: the plant morphology in the field. Bar: 10 μm."

Table 6

Agronomic characters of four double haploid plants H31"

材料
Material		 株高
Plant height (cm)		 分蘖数
No. of tillers		 穗长
Spike length (cm)		 小穗数
Spikelets		 小花数
Florets		 穗粒数
Seeds		 自交结实率
Self-fertilization rate (%)
DH-H31-1 114.0 4 12.0 15 30 27 90.00
DH-H31-2 118.5 4 12.5 14 28 26 86.67
DH-H31-3 132.5 5 13.5 17 34 29 96.67
DH-H31-4 115.0 3 13.0 15 30 24 80.00
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