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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (11): 2173-2183.doi: 10.3724/SP.J.1006.2021.02076

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

Creation and combining ability analysis of recessive genic sterile lines with a new ptc1 locus in rice

LI Jing-Lin(), LI Jia-Lin, LI Xin-Peng, AN Bao-Guang, ZENG Xiang, WU Yong-Zhong, HUANG Pei-Jing, LONG Tuan*()   

  1. Hainan Bolian Rice Gene Technology Co., Ltd., Haikou 570203, Hainan, China
  • Received:2020-11-15 Accepted:2021-03-19 Online:2021-11-12 Published:2021-04-06
  • Contact: LONG Tuan E-mail:250864463@qq.com;longtuan2001@aliyun.com
  • Supported by:
    Open Project of State Key Laboratory of Rice Biology Foundation of China Rice Research Institute(20190203)

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Abstract:

Cytoplasmic male sterile (CMS) lines and photoperiod/thermo-sensitive genic male sterile (PTGMS) lines are widely used as female parents in the commercial production of rice hybrid seeds. however, both systems have their intrinsic defects such as low usage of germplasm resources and unstable sterility. Recessive genic male sterile (GMS) lines, which overcome the problems of CMS and PTGMS lines, have played a key role in the development of next generation technologies for rice hybrid seed production. In this study, a GMS mutant ptc1-2 without pollen grains was identified from an irradiation-induced mutant library of 9311. Using a map-based cloning approach, a 257.37 kb deletion region was detected, which contained entire coding region of PTC1 on chromosome 9. PCR co-segregation analysis showed that the male sterility was completely associated with the ptc1-2 deletion region. The ptc1-2 deletion locus was then introgressed into a PTGMS line C815S and a CMS maintainer line Wufeng B by marker-assisted backcrossing. Corresponding GMS lines C815G and Wufeng G were obtained at BC3F3 generation, which showed the phenotypical similarity to the C815S and Wufeng B lines, respectively. Combining ability tests revealed that C815G and Wufeng G had the same combining ability as C815S and the CMS line Wufeng A in conventional field, respectively. These results indicated that ptc1-2 was a new allele of PTC1, which could be applied for breeding GMS lines and be the potential of GMS lines in rice hybrid seed production.

Key words: rice, male sterile, ptc1, map-based cloning, backcrossing, combining ability test

Fig. 1

Phenotypes of the ptc1-2 mutant and wild type plants A: plants of 9311 and ptc1-2 mutant at filling stage; B: panicles of 9311 and ptc1-2 mutant at ripening stage; C: floral organs of 9311 and ptc1-2 mutant; D: pollen grains of 9311; E: pollen grains of ptc1-2 mutant. Scale bars: 15 cm (A); 3 cm (B); 2 mm (C); 150 μm (D)."

Table 1

Partial list of the primers used in this study"

序号
No.		 标记/基因名称
Marker/gene name		 上游引物序列
Forward primer (5'-3')		 下游引物序列
Reverse primer (5'-3')		 扩增产物长度
Length of amplified products (bp)		 用途
Purpose
1 RM7039 GCACATTTGCCATTCTACCG GCCTTCCAGTGAGGTGACTC 168 定位 Mapping
2 RM257 CAGTTCCGAGCAAGAGTACTC GGATCGGACGTGGCATATG 147 定位 Mapping
3 D9.168 CAATGTTAAAAGTGATGGGGTC GGGAGAGAGTGAGAGAATAGAG 171 定位 Mapping
4 D3102 ATCTCGCAGTTTACATGCAG ACGGAAATAAAATTCAGTTGGT 450 定位 Mapping
5 D3676 ATACTTATTGCGTTTCATGGTCA CTTGAATGGACTCGACGCTA 462 定位 Mapping
6 D5146 ATACACCAATGCCTAGCGGAA CTCTCCCCTCGGTGACCAGA 492 定位 Mapping
7 D461 CTATGTCATCAACTGCGTGCC ATTCAGTTCGTTGCCAACTCG 454 定位 Mapping
8 D27560 ACATCAGGTTCATCACGGAGC GTCCCCACCTTCGTCCACT 601 定位 Mapping
9 D27420 CATTCCAGAGCATATTGAAACGA ATTTACATCCATGACCCGTCT 527 定位 Mapping
10 D27580 TGGTTCATCGCTCTTCTTCGTT CATGCTGCCTCCATACGGGAA 606 定位 Mapping
序号
No.		 标记/基因名称
Marker/gene name		 上游引物序列
Forward primer (5'-3')		 下游引物序列
Reverse primer (5'-3')		 扩增产物长度
Length of amplified products (bp)		 用途
Purpose
11 D27590 CTTTACCTATGTTCAAGCCTTCG CCTAACAAGGTCAGACGCATC 735 定位 Mapping
12 D27650 CTCTTGTTGTTCTTCATCGTCCT CATGCACCAGCAGCAACCA 451 定位 Mapping
13 D27820 ATGCGAGTCTATAAATTGCACCT GTCCATCAGCTCCGTCGAGA 468 定位 Mapping
14 1664SP1 GCACACTGCACGGCGACGTTTAGG — — 侧翼序列分离 Isolating flanking sequences
15 1664SP2 ACGATGGACTCCAGTCTGGCTGCCGTGGGAATTAGAGCAT — — 侧翼序列分离 Isolating flanking sequences
16 1664SP3 CCCTCCAGGAGATTGTCTAAAATTGACTTT — — 侧翼序列分离 Isolating flanking sequences
17 1664F1 CATCTCGCAGTTTACATGCAG — — ptc1-2标记 ptc1-2 markers
18 1664R1 — AGTCTACTCGAGCTACTACCG — ptc1-2标记 ptc1-2 markers
19 1664R2 — CCATCTGAAACTAGTACTCCCA — ptc1-2标记 ptc1-2 markers
20 Tms5 ATCCCACAAATCCTTTAGCA CCGTTATAGATAGACCCGAGA RsaI/329/414 tms5标记
tms5 markers

Table 2

Genetic analysis of the ptc1-2 mutant"

组合
Cross		 F1结实率
Seed-setting rate of F1
(%)		 F2 χ2(1:3) χ20.05
野生型植株数
No. of wild-type plants		 突变体植株数
No. of mutant plants
ptc1-2/9311 89.2 312 92 0.953 3.841
ptc1-2/明恢63 ptc1-2/Minghui 63 88.1 351 107 0.570 3.841

Fig. 2

Map-based cloning of ptc1-2 A: the ptc1-2 locus was first mapped to an interval between RM7039 and RM257 on the long arm of chromosome 9; B: ptc1-2 was fine mapped to an interval of 573 base pairs between D1380 and D3676; C: the ptc1-2 locus consisted of a 259,370 base-pair deletion from position 15,324,556 base-pair to position 15,583,926 base-pair. The deletion fragment contained the entire coding region of PTC1."

Fig. 3

Isolation of sequences flanking the ptc1-2 locus and co-segregation analysis A: the second round of TAIL-PCR results for isolation of genomic sequences flanking the ptc1-2 locus; B: co-segregation analysis of the ptc1-2 locus with the male sterile phenotype. All PCR products of F2 sterile plants were 546 bp in length. The size of PCR products of F2 fertile plants was either 811 bp, or 546 bp, and 811 bp. M: DNA 2000 bp marker."

Table 3

Agronomic traits of C815S, C815G, Wufeng A, and Wufeng G"

性状
Trait		 C815S C815G 五丰A
Wufeng A		 五丰G
Wufeng G
抽穗期Heading date (d) 82.60±0.47 81.60±0.47 72.00±0.81 71.60±0.47
株高Plant height (cm) 67.50±1.75 70.60±3.13 68.60±1.02 72.30±2.59
总分蘖数Total tiller number 8.00±0.81 8.30±0.47 8.00±0.81 8.30±0.47
穗长Panicle length (cm) 23.43±0.16 22.96±0.09 23.26±0.12 23.83±0.20
穗粒数No.of spikelets per panicle 169.30±4.10 167.60±3.39 174.00±4.08 180.00±2.44
粒长Grain length (mm) 8.64±0.13 8.84±0.08 8.88±0.04 8.84±0.10
粒宽Grain width (mm) 2.76±0.10 2.60±0.06 2.78±0.10 2.82±0.07

Fig. 4

Phenotypes of C815G and Wufeng G A: phenotypes of C815S and C815G plants at heading stage; B: spikelets of C815S and C815G; C: floral organs of C815S and C815G; D: pollen grains of C815S; E: pollen grains of C815G; F: phenotypes of Wufeng A and Wufeng G plants at heading stage; G: spikelets of Wufeng A and Wufeng G; H: floral organs of Wufeng A and Wufeng G; I: pollen grains of Wufeng A; J: pollen grains of Wufeng G. Scale bars: 10 cm (A, F); 0.5 cm (B, C, G, H); 200 μm (D, E, I, J)."

Table 4

Phenotypes of hybrids using C815G or Wufeng G as maternal parents"

性状
Trait		 C815S/+ C815G/+ 五丰A/+
Wufeng A/+		 五丰G/+
Wufeng G/+
抽穗期Heading date (d) 84.60±4.84 84.30±4.98 81.45±5.84 82.13±5.78
株高Plant height (cm) 103.80±5.43 103.50±5.47 113.29±4.88 108.92±5.47*
总分蘖数Total tiller number 12.69±1.55 12.96±1.03 11.13±1.22 11.42±1.21
穗长Panicle length (cm) 23.15±0.62 23.23±0.89 23.18±1.12 23.29±0.97
穗粒数No.of spikelets per panicle 186.61±16.36 186.45±15.42 201.75±24.63 196.96±18.33
结实率Seed-setting rate (%) 83.90±4.70 83.92±3.72 87.83±5.21 88.38±4.79
粒长Grain length (mm) 8.94±0.36 8.94±0.40 8.88±0.25 8.90±0.27
粒宽Grain width (mm) 2.56±0.20 2.56±0.19 2.58±0.13 2.59±0.15
千粒重1000-grain weight (g) 24.43±1.91 24.35±1.99 24.92±2.04 24.97±2.00
单株产量Grain yield per plant (g) 52.44±9.03 53.93±9.27 54.05±9.24 51.75±5.04

Table 5

Variance analysis of combining ability of C815S and C815G in fields"

性状
Trait		 变因Variance
区组
Block		 组合Combination 父本
Male parent		 母本
Female parent		 父本×母本
Male × female		 误差
Error
自由度DF 3 29 14 1 14 87
抽穗期Heading date 16.03** 99.92** 126.39 2.7 80.4** 1.4
株高Plant height 128.76** 122.89** 208.11** 2.7** 46.25 26.8
总分蘖数Total tiller number 5.34 7.23** 10.2* 2.13 4.64* 2.47
穗长Panicle length 1.14 2.43** 2.58 0.18 2.43** 0.56
穗粒数No. of spikelets perpanicle 462.34* 1046.13** 1018.12 0.71 1148.8** 142.64
结实率Seed-setting rate 0.005* 0.004** 0.003 0.001 0.004** 0.001
粒长Grain length 0.03 0.59** 0.54 0.0007 0.69** 0.03
粒宽Grain width 0.02 0.16** 0.15 0.0001 0.18** 0.01
千粒重1000-grain weight 0.24 15.82** 21.19* 0.23 11.56** 0.3
单株产量Grain yield per plant 1279.36** 348.89** 486.62* 66.11 231.37** 37.54

Table 6

Variance analysis of combining ability of Wufeng A and Wufeng G in fields"

性状
Trait		 变因Variance
区组
Block		 组合Combination 父本
Male parent		 母本
Female parent		 父本×母本
Male × female		 误差
Error
自由度DF 3 29 14 1 14 87
抽穗期Heading date 39.56** 140.12** 170.42 14.01 118.83** 3.24
株高Plant height 395.38** 130.89** 178.40** 571.60** 51.89** 50.23
总分蘖数Total tiller number 9.95** 6.23** 6.85 2.64 5.86** 2.11
穗长Panicle length 5.07** 4.55** 4.83 0.37 4.57** 0.78
穗粒数No. of spikelets per panicle 702.16** 1973.93** 2169.24 686.89 1870.55** 136.50
结实率Seed-setting rate 0.003** 0.01** 0.01 0.001 0.01** 0.001
粒长Grain length 0.59** 0.29** 0.29 0.01 0.30** 0.07
粒宽Grain width 0.10** 0.08** 0.05 0.01 0.13 0.02
千粒重1000-grain weight 1.14 16.88** 21.29 0.08 13.68** 0.57
单株产量Grain yield per plant 500.55** 234.86** 256.85 158.86 218.30** 70.25

Table 7

Relative effect values of general combining ability in fields"

性状
Trait		 C815S C815G 五丰A
Wufeng A		 五丰G
Wufeng G
抽穗期Heading date 0.1776 -0.1776 -0.4177 0.4177
株高Plant height 0.1447 -0.1447 1.9643 -1.9643
总分蘖数Total tiller number -1.0399 1.0399 -0.2967 0.2967
穗长Panicle length -0.1653 0.1653 -0.2403 0.2403
穗粒数No. of spikelets per panicle 0.0411 -0.0411 1.2001 -1.2001
结实率Seed-setting rate 0.0795 -0.0795 -0.3093 0.3093
粒长Grain length -0.0280 0.0280 -0.0984 0.0984
粒宽Grain width 0.0391 -0.0391 -0.2902 0.2902
千粒重1000-grain weight 0.1784 -0.1784 -0.1012 0.1012
单株产量Grain yield per plant -1.4929 1.4929 2.1750 -2.1750
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