WO2013036595A1

WO2013036595A1 - A stable dw3 allele for sorghum and a molecular marker to facilitate selection

Info

Publication number: WO2013036595A1
Application number: PCT/US2012/053898
Authority: WO
Inventors: Mitchell R. Tuinstra; Gurmukh S. Johal
Original assignee: Purdue Research Foundation
Priority date: 2011-09-06
Filing date: 2012-09-06
Publication date: 2013-03-14

Abstract

The identification of stable dw₃ alleles and development of molecular DNA markers to facilitate selection of these alleles in development of new cultivars provides a simple genetic solution to the problem of tall height mutants in commercial sorghum [Sorghum bicolor (L.) Moench] parent lines and hybrids.

Description

A STABLE dw₃ ALLELE FOR SORGHUM AND A MOLECULAR MARKER TO

FACILITATE SELECTION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 61/531 ,359, filed September 6, 201 1 , entitled "A stable dw₃ allele for sorghum and a molecular marker to facilitate selection," the entirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

The identification of genetically stable dw₃ alleles and development of a molecular DNA marker system to facilitate selection of these alleles in development of new cultivars provide a simple genetic solution to the problem of height mutants in commercial sorghum [Sorghum bicolor (L.) Moench] cultivars and hybrid seed production.

BACKGROUND OF THE INVENTION

This application is an extension of the research and Intellectual Property associated with U.S. Patent Application No. 13/047,594 filed on March 14, 201 1 , the entirety of which is incorporated by reference. The identification of a genetically stable dw₃ allele in the line Tx2737 and a DNA marker system may be used to facilitate selection of this allele in development of new cultivars. This disclosure describes additional accessions with the stable dw₃ allele and other stable dw₃ alleles with unique haplotypes.

Dwarfism plays a key role in adapting crops to high-input production systems and contributes to improved lodging resistance and fertilizer efficiency. The Dw₃ gene of sorghum represents a polar auxin transporter p- glycoprotein. The recessive dw₃ allele is caused by an 882 base-pair tandem duplication in exon five that is unstable since the duplication can be lost through unequal crossing over during meiosis. Tall revertant plants termed "height mutants" and are undesirable in seed and grain production fields.

Sorghum plant height is a quantitative trait controlled by four major genes (Dw₁:Dw₂:Dw₃:Dw4). Nearly all of the grain sorghum grown in the developed world is produced using semi-dwarf cultivars, These semi-dwarf cultivars commonly are called "3-dwarf" sorghums since they utilize recessive dwarfing alleles at three of the four major dwarfing genes (c/w_?; Dw₂: dw₃, dw₄).

Dw₃ is the only height gene that has been cloned in sorghum. The wild-type allele for Dw₃ encodes an auxin efflux transporter involved in stem internode elongation. The recessive allele of this gene (dw₃) is used to reduce plant height in nearly all commercial grain sorghum cultivars. This allele was originally identified and characterized in the early nineteen hundreds. It is noted that the dw₃ mutation produced a useful dwarf phenotype, but also noted that the dw₃ allele was unstable and reverted to wild-type Dw₃ at a frequency of approximately 1 in 600 plants. These revertant plants are generally termed "height mutants". Figure 1 illustrates tall dw₃ revertants in a commercial sorghum production field:

Since commercial sorghum hybrids often are grown at plant populations of 20,000 to 80,000 plants acre^"1 , one can expect approximately 10 to 100 height mutants per acre depending on the genetic background of the hybrid. Farmers dislike height mutants because these off-types are unsightly in commercial grain production fields. Commercial seed producers do not like height mutants because of the effort and cost required to remove these plants from seed production fields. These management efforts increase the "cost-of-goods".

Once the Dw₃ gene was cloned and its DNA sequence decoded, comparisons of the mutant and wild-type alleles indicated that the recessive dw₃ allele does not produce a functional protein due to a direct intragenic duplication of 882 bp DNA in exon 5. Comparisons of DNA sequences of dw₃ with the reverted Dw₃ allele height mutants demonstrated that the instability of the dw₃ allele was the result of unequal crossing-over between the tandemly duplicated regions that produced one wild-type allele and another allele with 3 tandem repeats.

SUMMARY OF THE INVENTION

The present invention identifies and characterizes stable alleles of the dw₃ locus. DNA markers can be used to move these putative-stable alleles into elite germplasm. One haplotype defined by a 6 base-pair deletion was evaluated in two different accessions and both accessions expressed a completely stable dwarf phenotype. An accession with a haplotype defined by an 82 base-pair deletion and another accession with a haplotype defined by a 6 base-pair duplication were also evaluated and no height mutants were observed in either accession. The identification of additional stable dw₃ alleles will provide useful genetic resources for addressing the problem of height mutants in sorghum. The DNA marker system as described in U.S. Patent Application No. 13/047,594 or any other markers linked to these alleles facilitate selection of the stable dwarf phenotype in plant breeding programs to address the problem of height mutants in sorghum. According to the present invention, stable dw₃ alleles of the sorghum Dw₃ gene comprise KS19 (nucleotide 2014_2019deletion), SC1 24 (nucleotide 1389_1470 deletion), and SC134 (nucleotide 1723_1724 duplication). The wild type Dw3

More specifically, the present invention relates to the identification and characterization of stable alleles of the dw₃ locus. Novel and potentially-stable alleles of dw3 were identified using a PCR assay to amplify a fragment containing the 882 base-pair tandem duplication that is diagnostic for the unstable dw₃ allele in a sorghum diversity panel containing 300 dwarf accessions. Leaf tissue was harvested from 14-d old seedlings of each accession. These samples were ground in liquid nitrogen and the ground tissue was transferred to a 2 ml collection tube and 500 μΙ_ of 65°C CTAB buffer that contained 0.17% of BME added to each tube. Samples were incubated at 65°C for 30 minutes. 500 μΙ_ of chloroform : octanol (24:1 ) pH 8.0 were added to the tubes, mixed well for 15 minutes at room temperature and centrifuged at 10,000 rpm for 10 minutes. The aqueous phase was transferred to a new tube and the DNA in the aqueous phase was precipitated by adding an equal volume of isopropyl alcohol and 1 /10 of the total volume of 3M NaOAc pH 5.0. DNA samples were centrifuged at 12,000 rpm for 10 minutes. The DNA pellets were washed with 1 ml of 70% ethyl alcohol and air dried. The DNA pellets were dissolved in 100 μΙ_ of double distilled water.

These DNA samples were genotyped using PCR to identify dw₃ alleles that did not contain the 882 base-pair tandem duplication. Several primer pairs were developed that flanked the duplicated region. Primers 4F and 5R were found to be efficient in amplifying this region.

The forward primer was coded Dw3_exon5/4F:

SEQ ID NO. 1 : CGT CCT GCA GAA GAT GTT CAT GAA GG

The reverse primer was coded Dw3_exon5/5R:

SEQ ID NO. 2: GTG CGC CAC CAC GAT GGT GGT GC

These primers were used in touchdown PCR reactions with 5% DMSO to amplify the duplicated region of exon 5 of the dw₃ gene in sorghum. Genotypes with the dw3 duplication should produce a 1960 bp product and Dw₃ genotypes without the duplication should produce a 1078 bp product as shown by Figure 2:

Specifically, Figure 2 illustrates duplicate PCR reactions for El Mota (Dw₃), Tx430 (dw₃), and Tx2737 (dw₃ stable-1) showing the absence of the duplication in El Mota and Tx2737 and the presence of the duplication in Tx430. SEQ ID NO: 3 provides a sequence for a region of exon 5 of the Dw₃ allele (wild type) from El Mota. These experiments showed that 180 of the 300 accessions had the 882 base-pair tandem duplication that defines the unstable dw₃ allele.

The PCR fragments from the 1 17 accessions that did not have the 882 base- pair tandem duplication were sequenced to evaluate variations in DNA sequence. These studies showed that 71 of the 1 17 accessions had a DNA sequence identical to the Dw₃ allele. Among the remaining accessions, numerous unique haplotypes distinct from either the Dw₃ or dw₃ allele were identified including three unique deletions, one insertion, and numerous SNPs.

Haplotypes defined by deletions or insertions were of particular interest as potential sources of a stable dw₃ phenotype as shown by Table 1 below. The haplotype position is referenced to the Sorghum bicolor gene Sb07g023730 after removing the 882 bp duplication. The S. bicolor gene transcript Sb07g023730.1 is available from the Phytozome Project at www.phytozome.org.

a The haplotype position is referenced to Sb07g023730.1 after removing the 882 bp duplication

A haplotype defined by a 6 base-pair deletion (2014_201 9 deletion) was found in Tx2737, KS1 9, and SC325. This is the same allele described in U.S. Patent Application No. 1 3/047,594. Growout experiments of Tx2737 in Puerto Rico— 201 0 and Indiana— 201 1 and KS1 9 in Indiana— 201 1 demonstrated that this allele was completely stable as shown in Table 2 below: Table 2: Genetic stability of new dw₃ haplotypes of sorghum measured as frequency of height mutants in growout experiments

Accession Haplotype³ Growout Observed Mutation

Location Mutations Frequency

Tx2737 2014-2019 PR-2010 0 in 51 ,540 0

deletion plants

IN-201 1 0 in 2,172 0

plants

KS19 2014-2019 IN-201 1 0 in 2,148 0

deletion plants

SC124 1389-1470 IN-201 1 0 in 2,808 0

deletion plants

SC134 1723-1724 IN-201 1 0 in 2,432 0

insertion plants

Tx430 - Sb07g023730.1 IN-201 1 5 in 1 ,496 0.334 %

Control plants

Another haplotype defined by an 82 base-pair deletion (1 389_1470 deletion) was found in SC124 (see Table 1 ). Growout experiments of SC1 24 in Indiana — 201 1 demonstrated that this allele was completely stable with no observed height mutants (see Table 2). A haplotype defined by a 6 base-pair insertion (1723_1724 insertion) was found in SC134, SC1017, SC1038, SC1 154, and SC1 155 (see Table 1 ). Growout experiments of SC134 in Indiana— 201 1 demonstrated that this allele was completely stable with no observed height mutants (see Table 2). These results suggest that the 882 base-pair tandem duplication of the original dw₃ is responsible for the instability of this allele.

Another deletion mutant haplotype was found in SC991 and is defined by a 15 base-pair deletion (1901_1915 deletion) (see Table 1 ). This haplotype has not been evaluated in growout experiments but probably represents another stable dw₃ allele. The PCR primers described above can be used to differentiate these stable dw₃ alleles from the unstable dw₃ allele. The stable alleles produce 996-1084 bp PCR products and the unstable allele produces a 1959 bp product (see Table 1 ). These markers provide an important tool in the development of stable dwarf inbreds and breeding populations because plants with these stable alleles cannot be differentiated from plants with the unstable allele (dw₃) by visual inspection of individual plants. However, a quick screen of plant DNA using the PCR protocol described above will indicate which plants carry the stable allele.

The present invention and its attributes and advantages will be further understood and appreciated with reference to the accompanying drawings.

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid molecule comprising a fragment of a nucleotide sequence of a sorghum dw3 gene, wherein the fragment comprises a mutation selected from the group consisting of an 82 base pair deletion (SC124), a 6 base pair insertion (SC134), and a 15 base pair deletion (SC991 ).

2. The isolated nucleic acid molecule of claim 1 , wherein the 82 base pair deletion (SC124) is present in a region represented by nucleic acid positions 1389 to 1470 of the sorghum dw3 gene after removing an 882 base pair duplication.

3. The isolated nucleic acid molecule of claim 2, wherein the mutation is stable.

4. The isolated nucleic acid molecule of claim 1 , wherein the 6 base pair insertion is present in a region represented by nucleic acid positions 1723 to 1724 of the sorghum dw3 gene after removing an 882 base pair duplication.

5. The isolated nucleic acid molecule of claim 4, wherein the mutation is stable.

6. The isolated nucleic acid molecule of claim 1 , wherein the 15 base pair deletion is present in a region represented by nucleic acid positions 1901 to 1915 of the sorghum dw3 gene after removing an 882 base pair duplication.

7. The isolated nucleic acid molecule of claim 6, wherein the mutation is stable.

8. A sorghum plant comprising a fragment of a nucleotide sequence of a sorghum dw3 gene, wherein the fragment comprises a mutation selected from the group consisting of an 82 base pair deletion (SC124), a 6 base pair insertion (SC134), and a 15 base pair deletion (SC991 ).

9. The sorghum plant of claim 8, wherein the plant is a dwarf variety.

10. The sorghum plant of claim 8, wherein the mutation is the 82 base pair deletion present in a region represented by nucleic acid positions 1389 to 1470 of the sorghum dw3 gene after removing an 882 base pair duplication.

1 1. The sorghum plant of claim 8, wherein the mutation is the 6 base pair insertion present in a region represented by nucleic acid positions 1723 to 1724 of the sorghum dw3 gene after removing an 882 base pair duplication.

12. The sorghum plant of claim 8, wherein the mutation is the 15 base pair deletion present in a region represented by nucleic acid positions 1901 to 1915 of the sorghum dw3 gene after removing an 882 base pair duplication.

13. A method of screening for a stable dwarf mutant, the method comprising:

extracting DNA from a plant;

analyzing the DNA using polymerase chain reaction; and

determining whether a mutation selected from the group consisting of an 82 base pair deletion (SC124), a 6 base pair insertion (SC134), and a 15 base pair deletion (SC991 ) is present.

14. The method according to claim 13, wherein the mutation is the 82 base pair deletion present in a region represented by nucleic acid positions 1389 to 1470 of the sorghum dw3 gene after removing an 882 base pair duplication.

15. The method according to claim 13, wherein the mutation is the 6 base pair insertion present in a region represented by nucleic acid positions 1723 to 1724 of the sorghum dw3 gene after removing an 882 base pair duplication.

16. The method according to claim 13, wherein the mutation is the 15 base pair deletion present in a region represented by nucleic acid positions 1901 to 1915 of the sorghum dw3 gene after removing an 882 base pair duplication.

17. The method according to claim 13, wherein a nucleotide sequence comprising SEQ ID NO: 1 is used as a forward primer.

18. The method according to claim 13, wherein a nucleotide sequence comprising SEQ ID NO: 2 is used a reverse primer.

19. The method according to claim 13, wherein the PCR results in an amplified product having a length significantly different from a 1960 base pair product produced when an 882 base pair duplication is present.

20. The method according to claim 13, further comprising sequencing an amplified PCR product.