# US 10,117,411 B2 · Maize cytoplasmic male sterility (CMS) C-type restorer RF4 gene, molecular markers and their use

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- Number: US 10,117,411 B2
- Document ID: US10117411B2
- Status: Granted
- Kind: B2
- Application number: 13/244,049
- Filed: 2011-09-23
- Published: 2012-04-12
- Granted: 2018-11-06
- Earliest claimed: 2010-10-06
- Assignees: Dow AgroSciences LLC
- Applicants: Ruihua Ren; Bruce A. Nagel; Siva P. Kumpatla; Peizhong Zheng; Gary L. Cutter; Thomas W. Greene; Steven A. Thompson
- Inventors: Ruihua Ren; Bruce A. Nagel; Siva P. Kumpatla; Peizhong Zheng; Gary L. Cutter; Thomas W. Greene; Steven A. Thompson
- Web page: https://patentgrep.com/patent/US10117411B2

## CPC

- A01H 6/4684: {Zea mays [maize]}
- A01H 1/023: {Male sterility}
- A01H 5/10: Seeds
- C07K 14/415: from plants
- C12N 15/8289: {Male sterility}
- C12Q 1/6895: for plants, fungi or algae
- C12Q 2600/13: Plant traits
- C12Q 2600/156: Polymorphic or mutational markers
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

This disclosure concerns high-resolution mapping and candidate gene cloning of Rf4, a maize restorer of fertility gene that restores fertility to C-type cytoplasmic male sterility. The disclosure also relates to molecular markers that are tightly-linked to, or reside within, the Rf4 gene. In some embodiments, methods are provided whereby hybrid seeds may be produced from crosses of a male plant comprising nucleic acid molecular markers that are linked to or that reside within the Rf4 gene and a female plant carrying C-type CMS.

## Claims

### Claim 1

A maize plant comprising an Rf4 functional restorer gene for maize C-type cytoplasmic male sterility from restorer line XJH58,
wherein the plant comprises a marker nucleotide sequence selected from the group consisting of Marker ID Nos. 1-50:
B73 Chrom. 8
Position
Marker ID No.
Start
Stop
Marker
1
8885
8885
C
2
11684
11684
G
3
11883
11883
C
4
12831
12831
C
5
13168
13168
A
6
13171
13171
C
7
13179
13179
T
8
13191
13191
G
9
14191
14191
A
10
15309
15309
A
11
15639
15639
T
12
15735
15735
T
13
15744
15744
A
14
15790
15790
A
15
17395
17395
T
16
18749
18749
T
17
18796
18796
T
18
18798
18799
GC
19
19119
19119
A
20
22230
22230
T
21
22266
22266
C
22
41082
41082
C
23
41335
41335
C
24
41944
41944
T
25
44285
44285
C
26
44387
44387
T
27
45202
45202
—
28
46136
46136
A
29
46447
46447
G
30
46912
46912
T
31
46957
46957
A
32
46959
46959
T
33
46973
46973
C
34
46975
46975
A
35
46989
46989
G
36
47001
47001
C
37
47026
47026
A
38
65062
65063
—
39
65438
65438
GGT
40
66993
66993
A
41
79739
79739
A
42
82093
82093
T
43
85155
85155
A
44
85667
85667
A
45
85782
85784
—
46
86079
86079
A
47
86097
86097
TC
48
86113
86113
T
49
86247
86247
T
50
86653
86653
—,
and
wherein the plant comprises a marker nucleotide sequence selected from the group consisting of SEQ ID NOs:6-9, SEQ ID NO:105, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:115, SEQ ID NOs:118-120, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:173, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:183, SEQ ID NOs:189-191, SEQ ID NO:197, and the markers referred to as Marker ID Nos. 51-106:
B73 Chrom. 8
Position
Marker ID No.
Start
Stop
Marker
51
96248
96248
T
52
97087
97087
A
53
97177
97177
GCC
54
97337
97338
TT
55
97341
97341
G
56
97371
97371
G
57
97382
97382
GGCGTACTTGCGCGCGGAC
(SEQ ID NO: 212)
58
97405
97405
GTC
59
97411
97411
C
60
97421
97433
—
61
97452
97452
GTT
62
97673
97673
T
63
97827
97827
C
64
97962
97962
T
65
97964
97964
ATGCATTACTT (SEQ ID NO: 214)
66
97973
97973
GTGCTATACTACCTAACCTA
(SEQ ID NO: 215)
67
97989
97989
C
68
98188
98188
A
69
98323
98326
—
70
98335
98335
A
71
98350
98350
C
72
98359
98359
C
73
98367
98369
—
74
98393
98393
T
75
98403
98403
T
76
98426
98430
—
77
98468
98468
TGGTTTCT
78
98489
98489
G
79
98533
98533
C
80
98540
98540
A
81
98552
98552
T
82
98557
98566
CCCCTGAACC (SEQ ID NO: 216)
83
98660
98660
T
84
98679
98679
A
85
98735
98735
A
86
98811
98811
C
87
98857
98857
ATAT
88
98906
98906
C
89
98927
98927
A
90
98935
98935
G
91
99026
99026
T
92
99027
99027
G
93
99037
99037
—
94
99074
99074
C
95
99079
99079
A
96
99089
99089
G
97
99091
99091
T
98
99137
99137
ATATT
99
99298
99298
A
100
99418
99418
C
101
99443
99443
T
102
99447
99447
A
103
99491
99491
G
104
99622
99622
CTAATGGT
105
99925
99925
A
106
99962
99962
G.

### Claim 2 (depends on 1)

The maize plant of claim 1, wherein the plant comprises a marker nucleotide sequence selected from the group consisting of SEQ ID NO:8 and Marker ID Nos. 1-3, 10, 11, 20, 21, 25-27, 39, 40, 44-49, 51-53, 55, 56, 59, 61-64, 68, 74, 78, and 83.

### Claim 3 (depends on 1)

The maize plant of claim 1, wherein the plant comprises a marker nucleotide sequence selected from the group consisting of SEQ ID NO:105; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:115; SEQ ID NOs:118-120; SEQ ID NO:123; SEQ ID NO:126 and SEQ ID NO:134.

### Claim 4

A hybrid maize plant that is an F1 progeny plant having XJH58 and a second plant as parents, the progeny plant comprising a marker nucleotide sequence selected from the group consisting of SEQ ID NOs:6-9, SEQ ID NO:105, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:115, SEQ ID NOs:118-120, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:173, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:183, SEQ ID NOs:189-191, SEQ ID NO:197, and the markers referred to as Marker ID Nos. 1-106:
B73 Chrom. 8
Position
Marker ID No.
Start
Stop
Marker
1
8885
8885
C
2
11684
11684
G
3
11883
11883
C
4
12831
12831
C
5
13168
13168
A
6
13171
13171
C
7
13179
13179
T
8
13191
13191
G
9
14191
14191
A
10
15309
15309
A
11
15639
15639
T
12
15735
15735
T
13
15744
15744
A
14
15790
15790
A
15
17395
17395
T
16
18749
18749
T
17
18796
18796
T
18
18798
18799
GC
19
19119
19119
A
20
22230
22230
T
21
22266
22266
C
22
41082
41082
C
23
41335
41335
C
24
41944
41944
T
25
44285
44285
C
26
44387
44387
T
27
45202
45202
—
28
46136
46136
A
29
46447
46447
G
30
46912
46912
T
31
46957
46957
A
32
46959
46959
T
33
46973
46973
C
34
46975
46975
A
35
46989
46989
G
36
47001
47001
C
37
47026
47026
A
38
65062
65063
—
39
65438
65438
GGT
40
66993
66993
A
41
79739
79739
A
42
82093
82093
T
43
85155
85155
A
44
85667
85667
A
45
85782
85784
—
46
86079
86079
A
47
86097
86097
TC
48
86113
86113
T
49
86247
86247
T
50
86653
86653
—
51
96248
96248
T
52
97087
97087
A
53
97177
97177
GCC
54
97337
97338
TT
55
97341
97341
G
56
97371
97371
G
57
97382
97382
GGCGTACTTGCGCGCGGAC
(SEQ ID NO: 212)
58
97405
97405
GTC
59
97411
97411
C
60
97421
97433
—
61
97452
97452
GTT
62
97673
97673
T
63
97827
97827
C
64
97962
97962
T
65
97964
97964
ATGCATTACTT (SEQ ID NO: 214)
66
97973
97973
GTGCTATACTACCTAACCTA
(SEQ ID NO: 215)
67
97989
97989
C
68
98188
98188
A
69
98323
98326
—
70
98335
98335
A
71
98350
98350
C
72
98359
98359
C
73
98367
98369
—
74
98393
98393
T
75
98403
98403
T
76
98426
98430
—
77
98468
98468
TGGTTTCT
78
98489
98489
G
79
98533
98533
C
80
98540
98540
A
81
98552
98552
T
82
98557
98566
CCCCTGAACC (SEQ ID NO: 216)
83
98660
98660
T
84
98679
98679
A
85
98735
98735
A
86
98811
98811
C
87
98857
98857
ATAT
88
98906
98906
C
89
98927
98927
A
90
98935
98935
G
91
99026
99026
T
92
99027
99027
G
93
99037
99037
—
94
99074
99074
C
95
99079
99079
A
96
99089
99089
G
97
99091
99091
T
98
99137
99137
ATATT
99
99298
99298
A
100
99418
99418
C
101
99443
99443
T
102
99447
99447
A
103
99491
99491
G
104
99622
99622
CTAATGGT
105
99925
99925
A
106
99962
99962
G.

### Claim 5 (depends on 4)

The F1 hybrid maize plant of claim 4, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO:8 and Marker ID Nos. 1-3, 10, 11, 20, 21, 25-27, 39, 40, 44-49, 51-53, 55, 56, 59, 61-64, 68, 74, 78, and 83.

### Claim 6 (depends on 4)

The F1 hybrid maize plant of claim 4, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO:105; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:115; SEQ ID NOs:118-120; SEQ ID NO:123; SEQ ID NO:126 and SEQ ID NO:134.

### Claim 7 (depends on 4)

The F1 hybrid maize plant of claim 4, comprising some but not all of the nucleotide sequences selected from the group consisting of SEQ ID NOs:6-9; SEQ ID NO:105; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:115; SEQ ID NOs:118-120; SEQ ID NO:123; SEQ ID NO:126; SEQ ID NO:134; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:138; SEQ ID NO:144; SEQ ID NO:149; SEQ ID NO:151; SEQ ID NO:160; SEQ ID NO:163; SEQ ID NO:164; SEQ ID NO:167; SEQ ID NO:173; SEQ ID NO:177; SEQ ID NO:178; SEQ ID NO:183; SEQ ID NOs:189-191; SEQ ID NO:197; and the markers referred to as polymorphism ID nos. 1-106 in Table 3.

### Claim 8 (depends on 1)

The maize plant of claim 1, wherein the maize plant is an inbred maize plant.

### Claim 9 (depends on 8)

The inbred maize plant of claim 8, wherein the plant comprises a marker nucleotide sequence selected from the group consisting of SEQ ID NO:8 and Marker ID Nos. 1-3, 10, 11, 20, 21, 25-27, 39, 40, 44-49, 51-53, 55, 56, 59, 61-64, 68, 74, 78, and 83.

### Claim 10 (depends on 8)

The inbred maize plant of claim 8, wherein the plant comprises a marker nucleotide sequence selected from the group consisting of SEQ ID NO:105; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:115; SEQ ID NOs:118-120; SEQ ID NO:123; SEQ ID NO:126 and SEQ ID NO:134.

### Claim 11 (depends on 1)

A population consisting of at least 126 maize plants of claim 1, wherein the population does not comprise a semi-fertile maize plant.

### Claim 12

A maize plant comprising an Rf4 functional restorer gene for maize C-type cytoplasmic male sterility from restorer line XJH58,
wherein the plant comprises a genetic marker selected from the group consisting of Marker ID Nos. 1-50:
B73 Chrom. 8
Position
Marker ID No.
Start
Stop
Marker
1
8885
8885
C
2
11684
11684
G
3
11883
11883
C
4
12831
12831
C
5
13168
13168
A
6
13171
13171
C
7
13179
13179
T
8
13191
13191
G
9
14191
14191
A
10
15309
15309
A
11
15639
15639
T
12
15735
15735
T
13
15744
15744
A
14
15790
15790
A
15
17395
17395
T
16
18749
18749
T
17
18796
18796
T
18
18798
18799
GC
19
19119
19119
A
20
22230
22230
T
21
22266
22266
C
22
41082
41082
C
23
41335
41335
C
24
41944
41944
T
25
44285
44285
C
26
44387
44387
T
27
45202
45202
—
28
46136
46136
A
29
46447
46447
G
30
46912
46912
T
31
46957
46957
A
32
46959
46959
T
33
46973
46973
C
34
46975
46975
A
35
46989
46989
G
36
47001
47001
C
37
47026
47026
A
38
65062
65063
—
39
65438
65438
GGT
40
66993
66993
A
41
79739
79739
A
42
82093
82093
T
43
85155
85155
A
44
85667
85667
A
45
85782
85784
—
46
86079
86079
A
47
86097
86097
TC
48
86113
86113
T
49
86247
86247
T
50
86653
86653
—,
and
wherein the plant comprises a genetic marker selected from the group consisting of the markers referred to as Marker ID Nos. 51-106:
B73 Chrom. 8
Position
Marker ID No.
Start
Stop
Marker
51
96248
96248
T
52
97087
97087
A
53
97177
97177
GCC
54
97337
97338
TT
55
97341
97341
G
56
97371
97371
G
57
97382
97382
GGCGTACTTGCGCGCGGAC
(SEQ ID NO: 212)
58
97405
97405
GTC
59
97411
97411
C
60
97421
97433
—
61
97452
97452
GTT
62
97673
97673
T
63
97827
97827
C
64
97962
97962
T
65
97964
97964
ATGCATTACTT (SEQ ID NO: 214)
66
97973
97973
GTGCTATACTACCTAACCTA
(SEQ ID NO: 215)
67
97989
97989
C
68
98188
98188
A
69
98323
98326
—
70
98335
98335
A
71
98350
98350
C
72
98359
98359
C
73
98367
98369
—
74
98393
98393
T
75
98403
98403
T
76
98426
98430
—
77
98468
98468
TGGTTTCT
78
98489
98489
G
79
98533
98533
C
80
98540
98540
A
81
98552
98552
T
82
98557
98566
CCCCTGAACC (SEQ ID NO: 216)
83
98660
98660
T
84
98679
98679
A
85
98735
98735
A
86
98811
98811
C
87
98857
98857
ATAT
88
98906
98906
C
89
98927
98927
A
90
98935
98935
G
91
99026
99026
T
92
99027
99027
G
93
99037
99037
—
94
99074
99074
C
95
99079
99079
A
96
99089
99089
G
97
99091
99091
T
98
99137
99137
ATATT
99
99298
99298
A
100
99418
99418
C
101
99443
99443
T
102
99447
99447
A
103
99491
99491
G
104
99622
99622
CTAATGGT
105
99925
99925
A
106
99962
99962
G.

### Claim 13 (depends on 12)

The maize plant of claim 12, wherein the plant comprises a genetic marker selected from the group consisting of Marker ID Nos. 73-106.

### Claim 14 (depends on 13)

The maize plant of claim 13, wherein the maize plant is an inbred maize plant.

### Claim 15 (depends on 13)

The maize plant of claim 13, wherein the maize plant is a hybrid maize plant that is a progeny plant having XJH58 and a second plant as parents.

### Claim 16 (depends on 13)

A population consisting of at least 126 maize plants of claim 13, wherein the population does not comprise a semi-fertile maize plant.

### Claim 17 (depends on 12)

The maize plant of claim 12, wherein the plant comprises in its genome a polynucleotide at least 95% identical to SEQ ID NO:217.

### Claim 18 (depends on 17)

The maize plant of claim 17, wherein the plant comprises in its genome a polynucleotide at least 99% identical to SEQ ID NO:217.

### Claim 19 (depends on 18)

The maize plant of claim 18, wherein the plant comprises in its genome the polynucleotide of SEQ ID NO:217.