GENETIC DIVERSITY AND GENOME WIDE MAPPING OF STRESS INDUCED SECONDARY METABOLITES IN SORGHUM (Sorghum bicolor (L.) Moench)

Open Access
- Author:
- Elango, Dinakaran
- Graduate Program:
- Agronomy
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 14, 2018
- Committee Members:
- Surinder Chopra, Dissertation Advisor/Co-Advisor
Gregory Wayne Roth, Committee Chair/Co-Chair
Majid R Foolad, Committee Member
Lavanya Reddivari, Committee Member
Yinong Yang, Outside Member
Surinder Chopra, Committee Chair/Co-Chair - Keywords:
- GWAS
Sorghum anthracnose
Frost stress
Epi-cuticular wax
Brown mid-rib
Forage sorghum yield and quality
Flavonoid secondary metabolite
3-deoxyanthocyanidin
Anti-oxidant activity - Abstract:
- Sorghum is the fifth most important cereal crop and an excellent model system to study the role of secondary metabolites in response to biotic and abiotic stresses. Sorghum produces unique and rare flavonoid phytoalexins in the poaceace family. Phytoalexins are low molecular weight, stress inducible, secondary metabolites with activity against multiple biotic and abiotic stressors. Sorghum phytoalexin class contains 3-deoxyanthocyanidins (3-DAs), which consists of luteolinidin and apigeninidin and their methyl and acyl derivatives. Sorghum 3-DAs play a key role in maintaining plant health by restricting anthracnose fungal pathogen proliferation and also imparting resistance against certain insect pests. The occurrence of these inducible 3-DAs are low in nature and genotypes differ greatly for their ability to synthesize of these compounds in response to biotic and abiotic stresses. So, it is imperative to evaluate the large genetic pool of sorghum accessions for these novel induced phytoalexins and profile them for further genetic improvement. It is known that anthracnose resistant sorghum genotypes synthesize more phytoalexins at a faster rate at the infection site than the susceptible genotypes. We have used two sorghum mapping populations with dense SNPs (Single Nucleotide Polymorphisms) to map genes controlling the accumulation of 3-DAs, epi-cuticular wax (EW), and anti-oxidant activities in sorghum. Mapping populations consist of Sorghum Association Panel (SAP; 377 accessions) and an International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) sorghum mini-core panel (242 accessions). Colletotrichum sublineola is an aggressive hemi-biotrophic fungal pathogen that causes anthracnose leaf blight and stem rot in sorghum. In severe conditions, it cause substantial yield loss up to 45%. Often major gene resistance fails due to varying degree of virulent pathotypes across sorghum growing locations and availability of other host species for pathogen survival. One mechanism to control anthracnose leaf blight in sorghum is to identify genetic factors associated with anti-fungal production. It is known that Colletotrichum sublineola induce the production of anti-fungal compounds known as 3-deoxyanthocyanidins (3-DAs) up on the fungal ingress in sorghum. These compounds are initially colorless and move to the site of infection. After 36 hours, they turn into brick red color and kill the invading fungus and the cell. This mechanism prevents the fungal pathogen spread from one area to other, thus effectively preventing the disease spread. Our goal of this investigation was to identify the genetic factors associated with 3-DA bio-synthesis and regulation in sorghum. A genome wide association study (GWAS) was performed using the sorghum association panel (SAP), ICRISAT (International Crops Research Institute for the Semi-Arid Tropics) mini-core (MNC) panel, and nested association mapping (NAM) panel. Community resources of 265,487 SNP (Single Nucleotide Polymorphism) markers for SAP and MNC panel, and 5108 SNP markers for NAM panel were used in this study. Three biological replicates of SAP and MNC leaf samples were collected at V7 stage for fungus infection, whereas 21 days seedlings were used for NAM population screening for anthracnose disease. The quantitative measurement of 3-DAs was carried out using spectrophotometer (λmax 480 nm) and confirmation of a sub-set by HPLC (High Performance Liquid Chromatography) with known 3-DA standards. Significant variability for 3-DAs among the genotypes observed (0.02 (IS 1219) to 2.99 (IS 11619)) with a mean of 0.52 (abs/mg). GWAS identified 11 candidate genes (p-value ≤7.73E-06) for 3-DA bio-synthesis and regulation in sorghum which include Sb04g013160 (Guanylate-binding protein), Sb01g044000 (Sugar transporter), Sb02g034830 (BCS1 AAA type ATPase), Sb04g010280 (MYB-like DNA-binding domain), Sb09g004660 (Peroxidase) and others. Our results provide basis for anthracnose disease improvement through marker assisted selection and genomic selection for plant secondary metabolites that act as fungicides in sorghum. Sorghum exhibits poor tolerance to cold and frost. A number of flavonoid secondary metabolites are induced during the plant–stress cross talk, and they play a major role in imparting stress tolerance to plants. Our objective of this study is to quantify the flavonoid induction before and after frost stress, and determine its anti-oxidant activity in the global sorghum panel and also identify the key candidate genes involved in the process through genome wide association mapping. GWAS identified 51 genes for DPPH (anti-oxidant activity) and 20 genes for total phenolic content (TP) before frost. Whereas, 19 genes were identified 3-deoxyanthocyanidins (3-DAs) and 6 genes for TPC under after frost stress. Most of the identified genes are involved in plant defense pathways for biotic and abiotic resistance. The probable candidates for after stress were peroxidase (sb01g041770), vesicle fusing ATPase (sb01g041930), leucine rich repeats (sb08g001430), flavonol reductase (sb01g025770), cytosine deaminase (sb02g041770), and anthocyanidin reductase (sb02g038520) for 3-DAs; and leucine rich repeat (Sb08g023030) and UDP-glucosyl transferase (Sb06g021900) for TP. Anti-fungal gene (Sb06g014340) was the most probable candidate for DPPH before frost stress. The identified candidate genes can be used in breeding climate resilient sorghum. Sorghum accumulates epi-cuticular wax (EW) or bloom in plant surfaces such as leaves, sheaths, and culm. EW reduces the non-transpiration water loss and protects the plant from severe drought stress and also imparts resistance against various insect pests. We present here results from the analysis of epi-cuticular wax (EW) content of 387 diverse sorghum accessions and its genome-wide association study (GWAS). EW content in sorghum leaves ranged from 0.1 mg cm-2 to 29.7 mg cm-2 with a mean value of 5.1 mg cm-2. GWAS using 265,487 single nucleotide polymorphisms (SNPs) identified thirty-seven putative genes that were associated (p < 9.89E-06) with EW biosynthesis and transport in sorghum; out of these, Sobic.002G310400 (3-Oxoacyl-[acyl-carrier-protein (ACP)] synthase III C terminal), Sobic.004G154200 (Ankyrin repeats), and Sobic.003G004500 (bHLH-MYC and R2R3-MYB transcription factors N-terminal) have been shown to be involved in EW biosynthesis; Sobic.001G447300 (ABC transporter), Sobic.004G089400 (Lipid exporter ABCA1 and related proteins, ABC superfamily), Sobic.002G311200 (Multidrug resistance protein), Sobic.001G447200 (Inositol 1, 3, 4-trisphosphate 5/6-kinase), and Sobic.005G217500 (Cytochrome p450) are involved in EW regulation or transport in sorghum. GWAS results from this study demonstrate the potential for genetic manipulation of EW content in sorghum and other grain cereals for better adaptation to biotic and abiotic stresses. Brown mid-rib (BMR) forage sorghum (Sorghum bicolor (L.) Moench) silage is a reasonable alternative to corn silage for areas with limited soil moisture. Traditional forage sorghum varieties are tall and prone to lodging with lower forage quality. Brachtyic dwarf BMR forage lines are shorter, lodging resistant and have higher forage quality. Newer, earlier hybrids have expanded the potential adaptation of forage sorghums to more northern areas. A two-year study was conducted during the 2014 and 2015 growing seasons using newly available brachytic dwarf BMR forage hybrids to determine the effects of different seeding rates and N (nitrogen) fertilization rates on forage dry matter (DM) yield and quality for two hybrids. The experimental design was split-split-plot with four replications. In each replication, main plots were two hybrids (AF7202 and AF7401), subplots were two seeding rates (198,000 and 296,400 seeds ha-1), and sub-subplots were two N rates (123 and 168 kg ha-1). DM yield and forage quality parameters were measured for each treatment. We observed significant varietal differences for all the parameters except neutral detergent fiber digestibility (NDFD) in 2015. The early maturity line, AF7202, had higher yields, higher starch and net energy for lactation (NEL) levels than AF7401. The dwarf line, AF7401, had higher crude protein (CP) and NDFD than AF7202. AF7202 was more responsive to the higher N rate than AF7401. CP was increased with increased N for both varieties. Other forage quality traits were unaffected by N rates. Neither variety responded to an increase in seeding rate. This study showed that the earlier brachytic dwarf forage sorghums such as AF7202, managed with recommended seeding rates and possibly higher N rates have good potential for high forage yield and quality in central PA.