Maize cytoplasmic male sterility (CMS) S-type restorer Rf3 gene, molecular markers and their use
The present disclosure provides a method for selecting a plant comprising a functional restorer gene for maize S-type cytoplasmic male sterility comprising the steps of (a) screening a population of plants for at least one marker nucleic acid, wherein the marker nucleic acid comprises an allele linked to the functional restorer gene for maize S-type cytoplasmic male sterility; (b) detecting the marker nucleic acid; (c) identifying a plant comprising the marker nucleic acid; and (d) selecting the plant comprising the marker nucleic acid, wherein the plant comprising the marker nucleic acid further comprises the functional restorer gene for maize S-type cytoplasmic male sterility. The present disclosure also provides methods for restoring fertility in a progeny of an S-type cytoplasmic male sterile plant and methods for transferring an Rf3 gene into a progeny plant.
1. A method for transferring Rf3 restorer gene for maize S-type cytoplasmic male sterility into one or more progeny maize plants, the method comprising:
(a) crossing a first parent maize plant and a second parent maize plant to produce F1 progeny maize plants, wherein at least the second parent maize plant comprises the Rf3 restorer gene;
(b) analyzing F1 progeny maize plants for the presence of genomic sequence comprising a SEQ ID NO:88 mutation in maize PPR2 gene;
(c) selecting one or more F1 progeny maize plants having the SEQ ID NO:88 mutation in maize PPR2 gene to obtain one or more Rf3 F1 progeny maize plants;
(d) backcrossing the selected one or more Rf3 F1 progeny maize plants with either the first parent maize plant or the second parent maize plant to produce F2 progeny maize plants;
(e) analyzing F2 progeny maize plants for the presence of the SEQ ID NO:88 mutation in maize PPR2 gene; and
(f) selecting one or more F2 progeny maize plants having the mutation to obtain one or more Rf3 F2 progeny maize plants having the SEQ ID NO:88 mutation in the PPR2 gene.
2. The method of claim 1 , wherein analyzing for the presence of the genomic sequence comprising SEQ ID NO:88 mutation in maize PPR2 gene comprises DNA sequencing.
3. The method of claim 1 , wherein analyzing the F1 and/or F2 progeny maize plants comprises detecting the presence of one or more nucleic acid markers, wherein the markers are selected from the group consisting of Mo17-14388, PZE-102180901, PZE-102180129, FG-1318, PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578, PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2, PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34, DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, DASCMS-SRf39 and combinations thereof.
4. The method of claim 1 , wherein analyzing the F1 and/or F2 progeny maize plants comprises the use of one or more nucleic acid primers or probes comprising a sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, and combinations thereof.
5. The method of claim 1 , wherein analyzing the F1 and/or F2 progeny maize plants comprises detecting the presence of one or more nucleic acid markers selected from the group consisting of CMSS03, CMSS10, CMSS15, CMSS34, DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, DASCMS-SRf39 and combinations thereof.
6. The method of claim 1 , wherein analyzing the F1 and/or F2 progeny maize plants comprises the use of one or more nucleic acid primers or probes comprising a sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, and combinations thereof.
7. The method of claim 1 , wherein analyzing the F1 and/or F2 progeny maize plants comprises the use of nucleic acid primers and probe comprising SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 88.
8. The method of claim 1 , wherein analyzing the F1 and F2 progeny maize plants comprises confirming the presence of the DASCMS-SRf39 nucleic acid marker in the F1 and F2 progeny maize plants.
9. The method of claim 8 , wherein the presence of the DASCMS-SRf39 nucleic acid marker is confirmed by nucleic acid sequencing.
10. The method of claim 1 , wherein the Rf3 F1 and/or Rf3 F2 progeny maize plants belongs to the Stiff Stalk heterotic group.
11. The method of claim 1 , wherein the Rf3 F2 progeny maize plants comprise Rf3 restorer gene sequence SEQ ID NO:92.
12. The method of claim 1 , wherein the Rf3 F2 progeny maize plant includes one or more male Rf3 plants and the method further comprises crossing the male Rf3 plants with one or more female plants having cytoplasmic male sterility to produce hybrid seed.
13. The method of claim 1 , wherein the method comprises:
isolating nucleic acid molecules from the F1 and/or F2 progeny maize plants;
contacting the isolated nucleic acid molecules with one or more nucleic acids selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, and combinations thereof; and
amplifying the isolated nucleic acid molecules and the oligonucleotides to produce an amplicon, wherein the amplicon comprises a detectable signal that is indicative of the presence of the functional Rf3 restorer gene for maize S-type cytoplasmic male sterility.
14. A method for transferring Rf3 restorer gene for maize S-type cytoplasmic male sterility into one or more maize plants, the method comprising introducing the nucleic acid marker DASCMS-SRf39 (SEQ ID NO:88) mutation into the PPR2 gene sequence of one or more maize plants by transformation, homologous recombination, mutagenesis, site-directed mutagenesis, or induced mutation.
15. The method of claim 14 , wherein the method comprises inducing the DASCMS-SRf39 (SEQ ID NO:88) mutation in native PPR2 gene sequence.
16. The method of claim 14 , wherein the heterologous PPR2 gene sequence is SEQ ID NO:92.