Taking Sorghum to New Heights: Identification of Genes Controlling Height Variation in Sorghum

Sorghum is an important cereal crop worldwide though it is particularly important in semi-arid regions. It is grown for many uses including food, feed, forage, sugar, and bioenergy. In its native Africa, sorghum is 3-4 meters in height. However, in the U.S. shorter plants were selected for grain production to reduce lodging and to facilitate mechanical harvesting. In the 1950s, researchers determined that this variation in height was controlled by four major genes they termed the dwarfing (Dw1-Dw4) genes. In 2003, Dw3 was identified as an ABCB efflux transporter of the plant hormone auxin. The locations of Dw1 and Dw2 have also been determined though the underlying genes remain to be elucidated. Dw1 was found to be on chromosome 9 at ~57 Mbp and Dw2 is located at ~42 Mbp on chromosome 6. The location of Dw4 has not been definitively determined though locations of ~6 Mbp on chromosome 6 and ~67 Mbp on chromosome 4 have both been suggested. In the work described in this dissertation, I determined that the gene that underlies Dw1 is Sobic.009G229800, a highly conserved gene of unknown function. Furthermore, Dw1 is found to interact with a QTL on chromosome 7. Dw2 was determined to be Sobic.006G067700 a kinase whose closest homolog in Arabidopsis is KCBP INTERACTING PROTEIN KINASE (KIPK). KIPK is a member of the AGC protein kinase family subgroup AGCVIII, which includes several kinases involved in the regulation of auxin transport. Lastly, I attempted to locate Dw4 through crosses with two different broomcorns. Surprisingly, no QTL matching the description of Dw4 was found. Overall this work increased our understanding of the genetic control of height in sorghum, as well as revealing some exciting possible new regulators of growth

Identification of Dw1, a Regulator of Sorghum Stem Internode Length

Sorghum is an important C4 grain and grass crop used for food, feed, forage, sugar, and biofuels. In its native Africa, sorghum landraces often grow to approximately 3-4 meters in height. Following introduction into the U.S., shorter, early flowering varieties were identified and used for production of grain. Quinby and Karper identified allelic variation at four loci designated Dw1-Dw4 that regulated plant height by altering the length of stem internodes. The current study used a map-based cloning strategy to identify the gene corresponding to Dw1. Hegari (Dw1dw2Dw3dw4) and 80M (dw1dw2Dw3dw4) were crossed and F2 and HIF derived populations used for QTL mapping. Genetic analysis identified four QTL for internode length in this population, Dw1 on SBI-09, Dw2 on SBI-06, and QTL located on SBI-01 and SBI-07. The QTL on SBI-07 was ~3 Mbp upstream of Dw3 and interacted with Dw1. Dw1 was also found to contribute to the variation in stem weight in the population. Dw1 was fine mapped to an interval of ~33 kbp using HIFs segregating only for Dw1. A polymorphism in an exon of Sobic.009G229800 created a stop codon that truncated the encoded protein in 80M (dw1). This polymorphism was not present in Hegari (Dw1) and no other polymorphisms in the delimited Dw1 locus altered coding regions. The recessive dw1 allele found in 80M was traced to Dwarf Yellow Milo, the progenitor of grain sorghum genotypes identified as dw1. Dw1 encodes a putative membrane protein of unknown function that is highly conserved in plants

Relative expression of <i>Dw1</i> in stem internodes.

<p>RNA was extracted from a full length internode (Mature), the lower half of an elongating internode, and the upper half of an elongating internode for each parental genotype (n = 3 each). Relative expression was determined by qRT-PCR using the ΔΔCt method with 18S rRNA as the normalizer and the sample from 80M mature tissue as the calibrator.</p

Distribution of <i>Dw1</i> Coding Sequence Variants in Sorghum Genotypes.

<p>The polymorphism number corresponds to the number in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151271#pone.0151271.t004" target="_blank">Table 4</a>.</p

A schematic of the region of SBI-09 encoding <i>Dw1</i>.

<p>The top bar shows the <i>Dw1</i> locus delimited by QTL mapping in the F₂. The region was refined in the F₃ population (n = 75 for each of six families) using the DG markers labeled in the diagram. The numbers below the bar are the number of recombinants (both bars). Note that all members of one of the families (237) had a breakpoint in between Fse5 and the end of the region shown. The lower bar represents the delimited <i>Dw1</i> locus defined by mapping in the F₃ generation with SNP markers labeled. Dark purple shows the location of <i>Dw1</i> based on fine mapping. SNP markers are named with the last six digits of the gene name of the gene the SNP is in or near. Fse4 is included for perspective though it was not scored in the F₄.</p

QTL for Average Internode Length Identified in the Entire Population of Hegari x 80M F₂.

<p>QTL for Average Internode Length Identified in the Entire Population of Hegari x 80M F₂.</p

Stem internode length QTL identified in a population from Hegari x 80M.

<p>F₂ plants from a cross of Hegari and 80M (n = 218) were grown in the greenhouse and the length of each internode was measured. The average internode length was used to map QTL. (A) The resulting graph shows four QTL, including <i>Dw1</i> and <i>Dw2</i>. The x-axis is the genetic map and the y-axis is the LOD score. The boxes above each trait identify the <i>Dw</i> loci, if any, the percentage of the variation explained by the QTL, and the location of the peak LOD value. (B) Photograph of Hegari (left) and 80M. (C) Photograph of F₅ plants that are <i>Dw1Dw1</i> (left), <i>Dw1dw1</i> (center), and <i>dw1dw1</i> (right) in otherwise uniform genetic backgrounds at the other loci that affect internode length.</p

Polymorphisms Distinguishing 80M and Hegari in Genes in the Delimited <i>Dw1</i> Locus.

<p>Polymorphisms Distinguishing 80M and Hegari in Genes in the Delimited <i>Dw1</i> Locus.</p

Genes in the Delimited <i>Dw1</i> Locus.

<p>Genes in the Delimited <i>Dw1</i> Locus.</p

Sequence Variants in Exons of Sobic.009G229800 in Diverse Sorghum Genotypes.

<p>Sequence Variants in Exons of Sobic.009G229800 in Diverse Sorghum Genotypes.</p