Allele-specific Marker Development of Pigm
By conducting multiple sequence alignment of the Pi2/9/Pigm locus in various cultivars including GM4 (Pigm), 75-1-127 (Pi9), C101A51 (Pi2), Nipponbare and 93-11, we discovered a single nucleotide polymorphism (SNP) located at the 5' side of the locus, which has the potential to distinguish between Pigm and non-Pigm allele. Confronting two-pair primers targeted the region containing the identified SNP were designed to develop a marker PIGM (Table S2). To assess the specificity of the PIGM marker, we analyzed 28 rice varieties. PCR results confirmed that only GM4 exhibited amplification of a 621bp fragment, while the remaining 27 varieties lacking the Pigm allele displayed a 448bp fragment (Figure 2a). Therefore, this functional allele-specific PCR-gel-based marker can be used for Pigm allele identification in rice breeding pipeline.
Introgression of Pigm by MAS
Pigm was transferred into an elite TGMS line LK638S by advanced-backcrossing combined with MAS. As shown in the schematic flow (Fig. 2), the donor GM4 harboring Pigm was used for backcrossing with the recurrent female parent LK638S. In the backcross generation, the F1 plants showing male-sterile and harboring heterozygous Pigm gene were ratooned and transferred in water incubation with controlled temperature to produce pollens for the backcrossing process. Male-sterile plants with homozygous Pigm were selected in BC3F2, BC8F2 and BC9F2 populations and ratooned in water incubation to obtain next generation seeds by selfing. This procedure was repeated to obtain advanced generations including BC3F12, BC8F8 and BC9F4. Blast resistance was evaluated in the Daweishan blast nursery (Liuyang county, Huan province, China), while phenotyping, including evaluation of fertility and agronomic performance, was conducted at each generation to select plants with desirable phenotypes. Finally, a total of 13 elite male sterile lines with homozygous Pigm were selected from the BC8F8 population, along with 4 lines from the BC9F4 population in 2018. In addition, 10 lines from BC3F12 were also included for further experimentation.
Genetic Linkage-drag between Blast Resistance and Photosensitivity
During the introgression of blast resistance gene Pigm by MAS, we observed that a delayed heading phenotype in the sterile plants carrying Pigm. Furthermore, we noticed that the majority of hybrids derived from the improved lines as female parents exhibited photoperiod sensitive, resulting in failure to head during rice-cropping season at Changsha (Hunan province, China). To figure out this particular phenomenon, we conducted genome sequencing of the region surrounding Pigm, and discovered that the CCT domain-containing protein gene Hd1 is situated approximately 181.5 kb away from Pigm. Hd1 is known to regulate the heading date in a photoperiod sensitivity manner (Yano et al. 2000). By comparing the Hd1 sequences of LK638S and GM4, we observed that LK638S carried a loss-of-function allele of Hd1, while GM4 possessed the wild type (functional) Hd1 allele. This indicates that the genetic linkage-drag between blast resistance and photoperiod sensitivity may be a result of the genetic linkage between Pigm and Hd1. To develop a desired photoperiod insensitive variety, it is indeed possible to eliminate this genetic linkage.
We designed functional molecular markers of Hd1 based on a 4-bp InDel variation in CCT domain for MAS selection (Fig.1; Table S2). In BC4F1 population, ~5000 plants were genotyped with the molecular markers of Pigm and Hd1, and nine recombinants with heterozygous Pigm and homozygous Hd1LK638S were identified. Top three recombinants exhibiting phenotypes more similar to LK638S were selected for further backcrossing with LK638S. The lines with Hd1638S-Pigm exhibited a significant reduction in days to heading (DTH) compared to those harbouring Hd1GM4-Pigm (Table 1).
Table 1 Blast resistance and heading date of selected improved lines and parents
Cultivar/breeding lines
|
Generation
|
Haplotype
|
DTH
|
BR
|
LK638S
|
Recurrent parent
|
Hd1638S
|
94
|
S
|
GM4
|
Donor parent
|
Hd1GM4-Pigm
|
87
|
R
|
X5-4-1021
|
BC2F13
|
Hd1GM4-Pigm
|
103
|
R
|
X5-4-768
|
BC2F13
|
Hd1GM4-Pigm
|
102
|
R
|
X7-7-1024
|
BC2F13
|
Hd1GM4-Pigm
|
101
|
R
|
X7-7-1031
|
BC2F13
|
Hd1GM4-Pigm
|
102
|
R
|
D23-924
|
BC2F13
|
Hd1GM4-Pigm
|
103
|
R
|
D23-933
|
BC2F13
|
Hd1GM4-Pigm
|
102
|
R
|
D5-123
|
BC3F12
|
Hd1GM4-Pigm
|
102
|
R
|
D5-141
|
BC3F12
|
Hd1GM4-Pigm
|
101
|
R
|
D5-205
|
BC3F12
|
Hd1GM4-Pigm
|
102
|
R
|
D5-269
|
BC3F12
|
Hd1GM4-Pigm
|
101
|
R
|
D5-333
|
BC3F12
|
Hd1GM4-Pigm
|
101
|
R
|
D5-397
|
BC3F12
|
Hd1GM4-Pigm
|
103
|
R
|
D5-425
|
BC3F12
|
Hd1GM4-Pigm
|
102
|
R
|
D5-461
|
BC3F12
|
Hd1GM4-Pigm
|
101
|
R
|
D5-478
|
BC3F12
|
Hd1GM4-Pigm
|
101
|
R
|
D5-487
|
BC3F12
|
Hd1GM4-Pigm
|
102
|
R
|
LP3-6-904-2
|
BC8F8
|
Hd1638S-Pigm
|
91
|
R
|
LP3-3-829-1
|
BC8F8
|
Hd1638S-Pigm
|
84
|
R
|
LP3-6-900
|
BC8F8
|
Hd1638S-Pigm
|
92
|
R
|
LP3-6-901-1
|
BC8F8
|
Hd1638S-Pigm
|
93
|
R
|
LP3-6-902-2
|
BC8F8
|
Hd1638S-Pigm
|
91
|
R
|
LP3-5-820
|
BC8F8
|
Hd1638S-Pigm
|
95
|
R
|
LP3-6-901-2
|
BC8F8
|
Hd1638S-Pigm
|
90
|
R
|
LP3-6-896-1
|
BC8F8
|
Hd1638S-Pigm
|
92
|
R
|
LP3-3-832-1
|
BC8F8
|
Hd1638S-Pigm
|
85
|
R
|
LP3-6-805-2
|
BC8F8
|
Hd1638S-Pigm
|
92
|
R
|
LP3-6-902-2
|
BC8F8
|
Hd1638S-Pigm
|
92
|
R
|
LP3-6-902-4
|
BC8F8
|
Hd1638S-Pigm
|
92
|
R
|
LP3-6-899-1
|
BC8F8
|
Hd1638S-Pigm
|
91
|
R
|
A145-5-1
|
BC9F4
|
Hd1638S-Pigm
|
91
|
R
|
A145-5-4
|
BC9F4
|
Hd1638S-Pigm
|
96
|
R
|
A145-5-6
|
BC9F4
|
Hd1638S-Pigm
|
93
|
R
|
A145-5-8
|
BC9F4
|
Hd1638S-Pigm
|
93
|
R
|
DTH, days to heading; BR, Blast resistance.
3.4. Evaluation of blast resistance in natural nursery and inoculation
The seedling blast resistance of the newly developed TGMS lines and two parents were evaluated in natural nursery and greenhouse conditions, respectively. The newly improved lines and donor GM4 showed stable and high-level seedling blast resistance without any typical lesions in the blast nursery trails for three consecutive years (2016-2018). In contrast, the parental line LK638S displayed susceptible to rice blast (Table 1; Fig. S1). In the greenhouse, we inoculated the improved lines and parents with 28 isolates collected from different regions of China, French, Guyana and South Korea. CO39 (susceptible control) and LK638S showed resistance frequency of 0% and 57.1%, respectively, indicating the presence of other blast resistance gene(s) in LK638S. The donor GM4 was immune to all of the 28 isolates. The 12 newly developed TGMS lines showed significantly enhanced blast resistance, with frequencies ranging from 92.9% to 96.4% (Table 2).
Table 2 Blast resistance reaction of selected improved lines and parents to 28 M. oryzae isolates
Isolate
|
Origin
|
GM4
|
LK638S
|
LP3-3-829-1
|
LP3-3-832-1
|
LP3-6-805-2
|
LP3-6-896-1
|
LP3-6-899-1
|
LP3-6-901-2
|
LP3-6-902-4
|
LP3-6-904-2
|
A145-5-1
|
A145-5-4
|
A145-5-6
|
A145-5-8
|
CO39
|
LTH
|
17-1-1
|
Chongqing, China ()
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-2-1
|
Chongqing, China
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-2-2
|
Chongqing, China
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-3-1
|
Chongqing, China
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-5-1
|
Chongqing, China
|
R
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
R
|
S
|
R
|
S
|
R
|
R
|
S
|
S
|
17-6-1
|
Chongqing, China
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-7-1
|
Chongqing, China
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-8-2
|
Hubei, China
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-8-7
|
Hubei, Chin
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-8-8
|
Hubei, China
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-9-1
|
Sichuan, China
|
R
|
S
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-9-3
|
Sichuan, China
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-9-7
|
Sichuan, China
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-9-8
|
Sichuan, China
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-12-4
|
Hunan, China
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-12-5
|
Hunan, China
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-13-5
|
Hunan, China
|
R
|
S
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-13-9
|
Hunan, China
|
R
|
S
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
17-14-1
|
Jiangxi, China
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-14-2
|
Jiangxi, China
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-14-3
|
Jiangxi, China
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-15-2
|
China (Guangdong)
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-15-4
|
China (Guangdong)
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-16-7
|
China (Guangdong)
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-19-1
|
Hunan, China
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
17-19-2
|
Hunan, China
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
GUY11
|
French Guyana
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
KJ201
|
South Korea
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
Resistance frequency (%)
|
100.0
|
57.1
|
96.4
|
92.9
|
96.4
|
92.9
|
96.4
|
92.9
|
96.4
|
96.4
|
96.4
|
92.9
|
96.4
|
96.4
|
0.0
|
0.0
|
R, resistant; S, susceptible
Assessment of Fertility-sterility Alternation Characterization and Agronomic Traits
The fertility-sterility alternation of the 12 improved TGMS lines and the recurrent parent LK638S was assessed in a water-filled concrete tank with controlled water temperature. The experiment was conducted in August 2019, with daily mean water temperatures (DWT) set at 21°C, 22°C, 23°C, 24°C, and 25°C. The results showed that both the improved TGMS lines and LK638S exhibited complete male sterility, with 100% pollen sterility observed when the daily mean water temperature (DWT) reached 23°C. Partial fertility was observed under 22°C (Table 3). The pollen fertility-sterility alternation pattern of the improved TGMS lines was similar to that of the recurrent parent LK638S, with the critical temperature point for fertility-sterility alternation occurring between 22°C and 23°C of DWT.
Table 3 Fertility-sterility alteration behavior of improved lines and the recurrent parent under five different temperature regimes
Cultivar/lines
|
Pollen sterility (%)
|
21℃/6d
|
22℃/6d
|
23℃/6d
|
24℃/6d
|
25℃/6d
|
LK638S
|
58.03
|
90.94
|
100.00
|
100.00
|
100.00
|
LP3-3-829-1
|
41.80
|
85.00
|
100.00
|
100.00
|
100.00
|
LP3-3-832-1
|
44.20
|
84.40
|
100.00
|
100.00
|
100.00
|
LP3-6-805-3
|
54.00
|
89.70
|
100.00
|
100.00
|
100.00
|
LP3-6-896-1
|
52.30
|
89.50
|
100.00
|
100.00
|
100.00
|
LP3-6-899-1
|
52.80
|
87.90
|
100.00
|
100.00
|
100.00
|
LP3-6-901-2
|
52.48
|
88.19
|
100.00
|
100.00
|
100.00
|
LP3-6-902-4
|
53.77
|
89.51
|
100.00
|
100.00
|
100.00
|
LP3-6-904-2
|
54.30
|
90.09
|
100.00
|
100.00
|
100.00
|
A145-5-1
|
54.80
|
89.23
|
100.00
|
100.00
|
100.00
|
A145-5-4
|
54.70
|
89.11
|
100.00
|
100.00
|
100.00
|
A145-5-6
|
55.17
|
89.07
|
100.00
|
100.00
|
100.00
|
A145-5-8
|
55.44
|
89.31
|
100.00
|
100.00
|
100.00
|
Twelve newly developed TGMS lines were also evaluated for their agronomic traits at Changsha (China) in the summer of 2019. The results showed no significant differences for all tested traits between newly developed TGMS lines and LK638S (Table 4).
Table 4 Agronomic performance of improved lines and the recurrent parent in Changsha of 2019
Cultivar/lines
|
DTH (d)
|
PL
|
PN
|
NGP
|
LK638S
|
91±3.1
|
24.8±1.0
|
8.5±1.3
|
211.3±13.6
|
LP3-3-829-1
|
89±2.8
|
22.8±1.3
|
8.4±0.9
|
189.9±11.1
|
LP3-3-832-1
|
88±4.1
|
23.0±1.2
|
8.3±0.8
|
189.0±12.8
|
LP3-6-805-3
|
93±1.0
|
26.3±1.3
|
9.1±0.9
|
215.3±12.1
|
LP3-6-896-1
|
92±2.5
|
26.4±0.8
|
9.0±1.1
|
214.2±10.8
|
LP3-6-899-1
|
91±3.3
|
26.8±1.5
|
9.2±1.2
|
225.4±14.9
|
LP3-6-901-2
|
92±1.6
|
26.6±0.8
|
8.4±0.6
|
226.9±14.1
|
LP3-6-902-4
|
91±2.2
|
26.6±0.7
|
9.3±0.8
|
213.7±12.8
|
LP3-6-904-2
|
92±1.8
|
26.0±0.9
|
8.3±0.9
|
209.4±9.5
|
A145-5-1
|
90±3.2
|
24.9±1.1
|
9.2±1.1
|
210.5±9.4
|
A145-5-4
|
91±1.9
|
25.0±1.3
|
8.4±0.9
|
211.1±11.7
|
A145-5-6
|
92±2.0
|
25.1±0.5
|
9.1±1.1
|
210.9±10.9
|
A145-5-8
|
91±2.3
|
24.8±1.1
|
8.3±1.3
|
211.4±9.9
|
DTH, days to heading; PL, panicle length; NGP, number of grains per panicle.
Genomic Evaluation of Improved TGMS Lines
A 1.1 K multiplex-PCR panel, based on the GBTS platform, was used for genomic background genotyping of the newly improved TGMS lines. Of the 1125 SNP markers covering 12 chromosomes, 264 SNPs showing polymorphism between the donor GM4 and recipient LK638S were used for genomic recovery analysis of improved TGMS lines. The newly improved TGMS lines showed a high genome recovery rate of the recurrent parent at 89.58% - 95.83% with the introgression harbouring Pigm of approximately 1.92-8.52 Mb (Table 5). Additionally, the genetic background of the newly improved TGMS lines were also evaluated with 48 SSR makers, which are commonly used for the rice varieties identification according to the Agricultural industry standard of the People's Republic of China (NY/T 1433-2014). The results revealed only 0-3 polymorphisms between the improved lines and the parent LK638S (Table 5).
For further verification, whole-genome resequencing data were generated for GM4, LK638S, LP3-6-899-1 and LP3-6-902-4 with an average minimum coverage of 26.0X, resulting in a total of 3,272,591 SNPs for background genotyping of LP3-6-899-1 and LP3-6-902-4. A graphical genotype map was constructed and assumed that the introgressed chromosome segment containing the Pigm locus of LP3-6-899-1 and LP3-6-902-4 were approximately 7.2 and 8.1 Mb, respectively, with a genome recovery rate of 92.07% and 91.84% for the recurrent parent (Fig. S2; Table 5).
Table 5 Genome recovery rate of 12 improved lines
Cultivar/lines
|
264 SNPs
|
No. of polymorphism markers among 48-SSR
|
~ 3272k SNPs
|
Genome ratio (%)
|
Introgression size with Pigm (Mb)
|
Genome ratio (%)
|
Introgression size with Pigm (Mb)
|
LK638S
|
100.00
|
|
|
|
|
LP3-3-829-1
|
89.58
|
1.92-8.50
|
2
|
|
|
LP3-3-832-1
|
90.91
|
1.92-8.50
|
1
|
|
|
LP3-6-805-3
|
94.70
|
1.38-8.25
|
2
|
|
|
LP3-6-896-1
|
92.42
|
1.38-8.25
|
3
|
|
|
LP3-6-899-1
|
93.18
|
1.38-8.25
|
1
|
92.07
|
7.2
|
LP3-6-901-2
|
93.18
|
1.38-8.25
|
2
|
|
|
LP3-6-902-4
|
92.42
|
1.38-8.25
|
2
|
91.84
|
8.1
|
LP3-6-904-2
|
93.94
|
1.38-8.25
|
1
|
|
|
A145-5-1
|
92.99
|
1.38-8.25
|
0
|
|
|
A145-5-4
|
92.42
|
1.38-8.25
|
0
|
|
|
A145-5-6
|
95.08
|
1.38-8.25
|
0
|
|
|
A145-5-8
|
95.83
|
1.38-8.25
|
0
|
|
|
Evaluation of Blast Resistance and Agronomic Traits of LZ36S Derived Hybrids
Based on the comprehensive evaluation of blast resistance, fertility-sterility alternation characterization, agronomic traits, combining ability and genetic background profiling of the newly improved TGMS lines, the improved line F3-6-902-4 was selected for further breeding application and named LZ36S. LZ36S and LK638S were used as female parents to produce hybrid F1 seeds with 13 male parents. All male parents, except for R1988, harbour Pi2 allele. All 13 hybrids of LK638S have been national certificated as new hybrid rice varieties and promoted extensively in Sothern-China. Such as, Longliangyou-Huazhan (LK638S/Huazhan), a hybrid rice variety with wide-adaptability, has been national approved in all the major rice cropping region of Southern-China and can be cropped as middle-season rice in the whole Yangtze River region and the Wuling mountain area, as well as late-season rice in south China. Field blast resistance of the 26 hybrids was assessed in the Daweishan blast nursery with high blast disease pressure in 2019. The LZ36S-dericed hybrids all displayed improved seedling and panicle blast resistance comparied to the corresponding control hybrid of LK638S with same male parents (Table 6; Fig. S1).
Table 6 Varity information and blast resistance performance of the hybrids derived from LK638S and LZ36S with same male parent
Hybrid variety
|
Combination
|
Blast resistance gene at Pigm/Pi2 in female/male parent
|
Seedling blast resistance score
|
Panicle blast score
|
State Approval Variety No.
|
Approved promotion area
|
Annotation
|
LongliangyouHuazhan
|
LK638S/Huazhan
|
-/Pi2
|
4
|
5
|
No.2015026
|
MRMLY
|
Super rice recognized by Ministry of Agriculture and Rural Affairs of China in 2017; Green super rice recognized by green super-rice committee (China) in 2019; The top five largest promoted hybrid variety in 2018-2022 with annual promotion area 399.3 thousand hectares
|
No.2016045
|
MRWL
|
No.20170008
|
MRMLY; LRSC
|
ZhenliangyouHuazhan
|
LZ36S/Huazhan
|
Pigm/Pi2
|
2
|
3
|
-
|
|
|
Longliangyou1686
|
LK638S/R1686
|
-/Pi2
|
5
|
5
|
No.20186058
|
MRMLY
|
|
Zhenliangyou1686
|
LZ36S/R1686
|
Pigm/Pi2
|
2
|
3
|
-
|
|
|
Longliangyou1273
|
LK638S/R1273
|
-/Pi2
|
5
|
5
|
No.20186065
|
MRMLY
|
|
Zhenliangyou1273
|
LZ36S/R1273
|
Pigm/Pi2
|
3
|
3
|
-
|
|
|
Longliangyou1308
|
LK638S/R1308
|
-/Pi2
|
4
|
5
|
No.20176065
|
MRMLY
|
Super rice recognized by Ministry of Agriculture and Rural Affairs of China in 2019; The 35st and 63th largest promoted hybrid variety in 2021 and 2022, respectively, with annual 28.9 thousand hectares
|
No.20196182
|
LRSC
|
No.20206050
|
MRUY
|
Zhenliangyou1308
|
LZ36S/R1308
|
Pigm/Pi2
|
3
|
3
|
-
|
|
|
Longliangyou7810
|
LK638S/R7810
|
-/Pi2
|
4
|
5
|
No.20186009
|
MRUY; MRMLY
|
|
Zhenliangyou7810
|
LZ36S/R7810
|
Pigm/Pi2
|
3
|
1
|
-
|
|
|
Longliangyou1234
|
LK638S/R1234
|
-/Pi2
|
5
|
5
|
No.20186011
|
MRMLY
|
|
Zhenliangyou1234
|
LZ36S/R1234
|
Pigm/Pi2
|
3
|
3
|
-
|
|
|
Longliangyou1206
|
LK638S/R1206
|
-/Pi2
|
4
|
5
|
No.2016606
|
MRUY
|
|
No.20176009
|
MRMLY
|
|
Zhenliangyou1206
|
LZ36S/R1206
|
Pigm/Pi2
|
2
|
1
|
-
|
|
|
Longliangyou1212
|
LK638S/R1212
|
-/Pi2
|
5
|
5
|
No.20170022
|
MRMLY; MRWL
|
Super rice recognized by Ministry of Agriculture and Rural Affairs of China in 2019; The 42st and 40th largest promoted hybrid variety in 2021 and 2022, respectively, with annual 31.8 thousand hectares
|
No.20196013
|
MRUY; LRSC
|
Zhenliangyou1212
|
LZ36S/R1212
|
Pigm/Pi2
|
3
|
3
|
-
|
|
|
Longliangyou1377
|
LK638S/R1377
|
-/Pi2
|
4
|
5
|
No.20176007
|
MRMLY; LRSC
|
Super rice recognized by Ministry of Agriculture and Rural Affairs of China in 2019; The 17st and 24st largest promoted hybrid variety in 2021 and 2022, respectively, with annual 60.4 thousand hectares
|
Zhenliangyou1377
|
LZ36S/R1377
|
Pigm/Pi2
|
3
|
1
|
-
|
|
|
Longliangyou1988
|
LK638S/R1988
|
-/-
|
7
|
7
|
No.2016609
|
MRMLY
|
Super rice recognized by Ministry of Agriculture and Rural Affairs of China in 2018; The 33st and 45st largest promoted hybrid variety in 2021 and 2022, respectively, with annual 33.6 thousand hectares
|
No.20176010
|
MRUY; LRSC
|
Zhenliangyou1988
|
LZ36S/R1988
|
Pigm/-
|
4
|
5
|
-
|
|
|
Longliangyou534
|
LK638S/R534
|
-/ Pi2
|
4
|
5
|
No.2016603
|
LRSC
|
The top ten largest promoted hybrid variety in 2018-2022 with annual promotion area 310.7 thousand hectares
|
No.20170001
|
MRUY; MRMLY
|
Zhenliangyou534
|
LZ36S/R534
|
Pigm/Pi2
|
2
|
1
|
-
|
|
|
Longliangyou5438
|
LK638S/R5438
|
-/Pi2
|
5
|
5
|
No.20196050
|
MRML
|
Super rice recognized by Ministry of Agriculture and Rural Affairs of China in 2022
|
No.20210012
|
ERSC
|
Zhenliangyou5438
|
LZ36S/R5438
|
Pigm/Pi2
|
3
|
3
|
No.20206138
|
MRMLY
|
Promoted 5.1 thousand hectares in 2022
|
No.20220010
|
MRWL
|
Longliangyou8612
|
LK638S/R8612
|
-/Pi2
|
4
|
5
|
No.20190010
|
MRMLY
|
Super rice recognized by Ministry of Agriculture and Rural Affairs of China in 2023; The 32st and 14th largest promoted hybrid variety in 2021 and 2022, respectively, with annual 62.4 thousand hectares
|
Zhenliangyou8612
|
LZ36S/R8612
|
Pigm/Pi2
|
2
|
3
|
No.20206139
|
MRMLY
|
|
MRMLY, middle-season rice in the middle and lower reaches of the Yangtze River; MRUY, middle-season rice in the uper reaches of the Yangtze River; LRSC, late-season rice in South China; ERSC, Early-season rice in South China; MRWL, Middle-season rice in Wuling mountainous area. The variety promoting data was from National Agricultural Technology Extension Center.
Yield performance of the hybrids of LZ36S and LK638S was field-trailed at the Guanshan experimental station under blast-control condition and the Daweishan blast nursery under open-field blast infection condition in 2019. The results showed that the average yield of the LZ36S-derived hybrids was 10.95 t/ha under blast-control condition, slightly lower than the yield (11.00 t/ha) of LK638S-derived hybrids. However, under open-field blast infection condition, the yield performance of the LZ36S-derived hybrids was significantly improved, the average yield increased by 15.53% compared with that of LK638S-derived hybrids. We analysed six agronomic traits (DTM, PH, PNH, NGP, SF and TGW) of the hybrids trailed in the Guanshan experimental station. Compared with LK638S-derived hybrids, LZ36S-derived hybrids exhibited decrease in DTM, PNH, and NGH by an average of -0.13%, -3.46%, and -3.53% respectively. While, PH, SF and TGW increased 0.88%, 0.06% and 0.64%, respectively (Table 7). These results indicated that the hybrids with the more major blast resistance gene allele (Pigm) possessed higher stable production capacity and no or minimal penalty on yield, although the hybrids also harbored another major broad-spectrum blast resistance allele of Pi2. This also highlights the effectiveness of utilizing double alleles of one favorable R gene present in both parental lines through hybrid rice system. From 2020 to 2023, a total of sixteen LZ36S-derived hybrid varieties, all exhibiting MR or R blast resistance ability, along with favorable yield and grain quality performance, have been national or/and provincial approved and certificated (Table 8; Table S3). In 2023, the promoting area of LZ36S-derived hybrid varieties is expected to 200 thousand hectares, and Zhenlaingyou8612 (LZ36S/R8612) exceeded 80,000 hectares.
Table 7 Field trial evaluation of hybrids derived from LK638S and LZ36S
Combination
|
Guanshan
|
|
Daweishan
|
|
Mean yield over two environments (t/ha)
|
GY (t/ha)
|
DTM (d)
|
PH (cm)
|
PNH
|
NGP
|
SF (%)
|
TGW (g)
|
|
GY (t/ha)
|
|
LK638S/Huazhan
|
10.90
|
121
|
122
|
17.1
|
203
|
85.4
|
25.9
|
|
10.03
|
|
10.47
|
LZ36S/Huazhan
|
12.31
|
120
|
118
|
17.1
|
194
|
87.8
|
25.6
|
|
11.59
|
|
11.95
|
LK638S/R1206
|
9.96
|
121
|
126
|
15.6
|
191
|
91.3
|
25.8
|
|
9.98
|
|
9.97
|
LZ36S/R1206
|
9.73
|
120
|
125
|
15.6
|
188
|
90.0
|
25.9
|
|
10.75
|
|
10.24
|
LK638S/R1212
|
13.26
|
126
|
118
|
16.4
|
177
|
89.9
|
26.4
|
|
8.96
|
|
11.11
|
LZ36S/R1212
|
13.28
|
125
|
120
|
15.8
|
166
|
90.0
|
26.1
|
|
10.56
|
|
11.92
|
LK638S/R1234
|
12.01
|
121
|
124
|
15.8
|
204
|
88.6
|
26.7
|
|
9.49
|
|
10.75
|
LZ36S/R1234
|
11.46
|
121
|
122
|
15.2
|
198
|
89.5
|
26.5
|
|
10.97
|
|
11.22
|
LK638S/R1273
|
12.40
|
121
|
128
|
16.4
|
185
|
89.6
|
29.4
|
|
9.59
|
|
11.00
|
LZ36S/R1273
|
10.96
|
122
|
122
|
15.7
|
164
|
89.3
|
29.2
|
|
11.21
|
|
11.09
|
LK638S/R1308
|
10.91
|
122
|
132
|
15.4
|
199
|
85.6
|
28.6
|
|
9.27
|
|
10.09
|
LZ36S/R1308
|
9.99
|
122
|
133
|
14.6
|
199
|
86.6
|
30.0
|
|
10.73
|
|
10.36
|
LK638S/R1377
|
12.07
|
124
|
118
|
15.8
|
217
|
82.1
|
25.2
|
|
8.77
|
|
10.42
|
LZ36S/R1377
|
11.79
|
124
|
123
|
15.6
|
188
|
82.2
|
24.5
|
|
9.99
|
|
10.89
|
LK638S/R1686
|
11.38
|
121
|
123
|
15.0
|
195
|
87.4
|
27.3
|
|
10.41
|
|
10.90
|
LZ36S/R1686
|
10.70
|
120
|
123
|
15.4
|
185
|
85.7
|
27.9
|
|
11.26
|
|
10.98
|
LK638S/R1988
|
9.86
|
121
|
113
|
16.0
|
183
|
89.0
|
27.5
|
|
7.25
|
|
8.56
|
LZ36S/R1988
|
11.25
|
123
|
121
|
16.5
|
172
|
89.6
|
27.4
|
|
10.35
|
|
10.80
|
LK638S/R534
|
10.98
|
122
|
122
|
16.5
|
202
|
87.8
|
25.1
|
|
8.99
|
|
9.99
|
LZ36S/R534
|
10.29
|
123
|
121
|
14.6
|
189
|
85.4
|
24.0
|
|
10.10
|
|
10.20
|
LK638S/R5438
|
10.07
|
123
|
123
|
14.2
|
194
|
82.0
|
27.1
|
|
9.22
|
|
9.65
|
LZ36S/R5438
|
10.82
|
122
|
125
|
13.9
|
197
|
81.3
|
28.3
|
|
11.25
|
|
11.04
|
LK638S/R7810
|
9.15
|
123
|
113
|
14.7
|
168
|
78.1
|
27.0
|
|
9.00
|
|
9.08
|
LZ36S/R7810
|
9.95
|
122
|
118
|
13.0
|
201
|
83.3
|
29.2
|
|
10.44
|
|
10.20
|
LK638S/R8612
|
10.10
|
121
|
124
|
15.4
|
212
|
81.7
|
28.5
|
|
10.77
|
|
10.44
|
LZ36S/R8612
|
10.35
|
121
|
129
|
14.2
|
200
|
78.3
|
27.9
|
|
11.43
|
|
10.89
|
Mean derived from LK638S (MK)
|
11.00
|
122.08
|
122
|
15.7
|
195
|
86.0
|
27.0
|
|
9.36
|
|
10.18
|
Mean derived from LZ36S (MZ)
|
10.95
|
121.92
|
123
|
15.2
|
188
|
86.1
|
27.1
|
|
10.82
|
|
10.90
|
(MK-MZ)/MK (%)
|
-0.46
|
-0.13
|
0.88
|
-3.46
|
-3.53
|
0.06
|
0.64
|
|
15.53
|
|
7.07
|
GY, grain yield; DTM, days to maturity; PH, plant height; PNH, panicle number per hill; NGP, number of grains per panicle; SF, spikelet fertility; TGW, thousand-grains weight
Table 8 Varity information, blast resistance and grain yield performance of the hybrids of LZ36S in state and provincial regional test trail of new varieties
Hybrid varity
|
Combination
|
State or Provincial Approval Variety No.
|
Approved promotion area
|
Blast resistance
|
GY
|
GY(t/ha)
|
±CK (%)
|
ZhenlaingyouTaisi
|
LZ36S/Taisi
|
20216014
|
MRMLY
|
MR
|
8.95
|
0.35
|
ZhenlaingyouYuzhan
|
LZ36S/Yuzhan
|
20216142
|
MRMLY
|
MR
|
9.58
|
3.44
|
20226038
|
MRUY
|
MR
|
9.47
|
-0.30
|
20226038
|
LRSC
|
MR
|
6.99
|
1.40
|
20216005
|
MRHN
|
MR
|
9.35
|
4.20
|
ZhenlaingyouNongzhan
|
LZ36S/Nongzhan
|
20216004
|
MRHN
|
MR
|
9.24
|
3.10
|
Zhenlaingyou1468
|
LZ36S/R1468
|
20216002
|
MRHN
|
MR
|
9.44
|
3.40
|
Zhenlaingyou8612
|
LZ36S/R8612
|
20206139
|
MRMLY
|
MR
|
10.03
|
5.95
|
Zhenlaingyou5438
|
LZ36S/R5438
|
20206138
|
MRMLY
|
MR
|
9.80
|
5.97
|
20220010
|
MRWL
|
R
|
8.88
|
0.20
|
Zhenlaingyou3703
|
LZ36S/R3703
|
20206010
|
MRHN
|
MR
|
9.76
|
6.00
|
Zhenlaingyou5688
|
LZ36S/R5688
|
20216135
|
MRMLY
|
MS
|
9.88
|
3.38
|
ZhenliangyJinsi
|
LZ36S/Jinsi
|
20226096
|
MRMLY
|
MR
|
9.17
|
2.30
|
Zhenliangy6076
|
LZ36S/R6076
|
20226095
|
MRMLY
|
MR
|
9.10
|
1.80
|
Zhenliangy5281
|
LZ36S/R5281
|
20220019
|
MRMLY
|
MR
|
9.51
|
2.60
|
Zhenliangy1988
|
LZ36S/R1988
|
20220021
|
MRHN
|
MR
|
9.61
|
6.60
|
Zhenliangy4811
|
LZ36S/R4811
|
20220019
|
MRHN
|
MR
|
9.06
|
2.50
|
Zhenliangy2646
|
LZ36S/R2646
|
20226002
|
MRHN
|
MR
|
9.00
|
-0.20
|
Zhenliangy3485
|
LZ36S/R3485
|
20226003
|
MRHN
|
MR
|
8.89
|
-0.30
|
ZhenlaingyouHuabao
|
LZ36S/Huabao
|
20220022
|
MRHN
|
MR
|
9.48
|
4.90
|
Mean
|
9.26
|
2.86
|
MRMLY, middle-season rice in the middle and lower reaches of the Yangtze River; MRUY, middle-season rice in the upper reaches of the Yangtze River; LRSC, late-season rice in South China; MRWL, Middle-season rice in Wuling mountainous area; MRHN, middle-season rice in Hunan province; MR, moderate resistance; R, resistance; GY, grain yield; CK, trial check