Identification and characterization of a new stable dwarf3 (dw3s) allele in sorghum

Ivan Dario Barrero-Farfan, Purdue University

Abstract

Plant height is an important plant architecture trait that influences radiation interception, biomass production and mechanized harvesting. Plant height is determined by the number and length of the internodes. Sorghum height is controlled by four dwarfing genes that contribute in a quantitative way to plant height. Dwarf sorghum cultivars have been exploited agronomically since the early 1920s. One of the dwarfing genes that was incorporated into U.S. germplasm by sorghum breeders is dwarf3 (dw3 ), which encodes for an ATP-binding cassette (ABC) transporter involved in auxin export. The nonfunctional dw3 allele is the result of a 882-bp tandem duplication in exon 5 that disrupts protein function and the plant’s ability to establish an auxin gradient in the intermediate internodes. This allele is unstable and tends to revert to wild type at a frequency that ranges from 0.166 to 0.3% (Karper, 1932). Multani et al. (2003) reported that unequal crossing over events that occur during meiotic recombination are the cause of dw3 instability. Dw3 revertants increase hybrid seed production costs. Height mutants resulting from unequal crossing over events are unsightly in commercial grain production fields and, therefore, negatively impact sorghum hybrid seed acceptance by farmers. A genetic fix to this problem is to identify a stable dw3 allele in the natural sorghum gene pool or identify de novo mutant alleles generated by nucleotide substitutions occurring during meiotic recombination. In this study, a high throughput DNA extraction protocol was adapted and PCR conditions for amplifying the target sequence in exon 5 of dw3 were optimized. These protocols were used in an attempt to screen thousands of sorghum seedlings to identify a new stable dw3 allele generated by meiotic recombination error during unequal crossing over. These efforts were followed by an analysis and characterization of existing dw3 alleles in the natural gene pool. Nine Dw3 revertants were found out of 3864 seedlings screened. These revertants were confirmed twice by PCR and the alleles characterized by DNA sequencing. These Dw3 revertants were characterized for differences in plant height in the greenhouse. Segregation analysis was conducted using a primer set specific for the dw3 allele. All of the Dw3 revertants exhibited a tall phenotype indicating that the gene was fully functional. Screening for meiotic revertants was suspended because of the failure to identify any Dw3 revertants exhibiting nucleotide substitutions. The failure to recover any stable dw3 allele generated during unequal crossing over is explained by the high fidelity of the recombination process regulated by DNA polymerase proofreading and the mismatch repair (MMR) mechanism. Based on this result, it is suggested a larger number of plants may need to be screened to identify a new allele generated by recombination error. The next strategy focused on characterization of dw3 alleles in the natural gene pool. A sorghum line was shown to have a new dw3 allele that did not contain the tandem 882-bp duplication in exon 5. Further characterization of this allele demonstrated a six-bp deletion encoding Q1275 and R1276 located in the C-terminal ABC transporter signature motif (LSGGQ) that disrupts protein function. The ABC transporter signature motif is a highly conserved motif in the ABC transporters that is involved in sensing the gamma phosphate in MgATP, protein conformational changes and substrate transport. Field studies demonstrated that this deletion generated a stable dw3 allele that did not produce any height mutants in more than 50000 plants evaluated. The new, stable dw3 allele was termed dw3s for dw3-stable and it will provide an opportunity to fix the dw3 instability in current sorghum germplasm.

Degree

M.S.

Advisors

Tuinstra, Purdue University.

Subject Area

Agronomy|Plant sciences

COinS