Evaluation of sorghum germplasm for new sources of resistance to greenbugs

Continuous improvement in crop defence is dependent on the availability of diverse genetic resources and judicious use of effective sources of resistance. At present, over 40,000 sorghum germplasm accessions have been captured and conserved in the USDA National Plant Germplasm System (NPGS, 2010). This collection represents the great wealth of sorghum genetics resources and is invaluable to both sorghum research and various crop improvement programmes. However, those genetic materials are of little value unless they are evaluated, documented and utilized. Thus, one of our current research emphases has been the systematic evaluation of the entire US collection of sorghum germplasm to identify new sources of greenbug resistance, and to subsequently characterize the newly identified sources to facilitate breeding sorghum with insect resistance.

In this project, these sorghum germplasm accessions from locations around the world were evaluated for their response to greenbug feeding in the greenhouse at the USDA-ARS Plant Science Research Laboratory, Stillwater, Oklahoma. As a result, 21 germplasm accessions were identified that possessed resistance to the greenbug, as shown in Table 1. When compared with susceptible checks, the newly identified accessions had relatively low greenbug damage scores, indicating their inherent resistance to greenbug. Among these resistant sources, four accessions showed high levels of resistance ranging from 1.0 to 2.8 on a 1 to 6 scale (1 = no damage; 6 = 100% damage). The other 17 accessions demonstrated moderate resistance with resistance ratings from 2.9 to 3.8. These sorghum accessions are resistant to greenbug biotype I and offer new resistance sources to sorghum breeding programmes.

Table 1 Greenbug damage scores of the new sources of sorghum germplasm lines

^a Based on greenbug damage to the sorghum seedlings, scores were recorded on a 1–6 scale, where 1 = ≤ 20%, 2 = 20–40%, 3 = 40–60%, 4 = 60–80%, 5 ≥ 80% damage and 6 = plant death.

^b Means with the same letter are not significantly different at probability level of 0.05.

Gene discovery through gene expression profiling

Identifying what genes are expressed in a particular tissue or at a specific time is often very useful to determine their function. Recently, genomics techniques such as DNA microarrays, large-scale gene expression profiling (transcriptome) and associated bioinformatics analysis make it possible to monitor expression of all genes simultaneously in any given tissue or following a specific treatment (Alba et al., Reference Alba, Fei, Payton, Liu, Moore, Debbie, Cohn, D'Ascenzo, Gordon, Rose, Martin, Tanksley, Bouzayen, Jahn and Giovannoni2004).

Using cDNA microarrays, we have recently examined the transcriptional changes in sorghum seedlings and compared the results from parallel systems, greenbug-resistant and -susceptible genotypes, leading to the detection of differentially expressed transcripts during infestation by greenbug biotype I, corresponding to 157 sorghum genes. The experiments showed comprehensive gene activities resulted from up-regulating or activating existing defence pathways in sorghum seedlings in response to greenbug feeding. Of the aphid-induced genes identified in this study, 38 genes exhibited three-fold or higher abundance in their expression and 26 genes were significantly reduced. For further analysis, the genes that showed differential expression were cloned and sequenced. The resulting sequences were then annotated by comparison with GenBank databases using the BLASTX search program. Sequence similarity searches allowed putative functions to be assigned to 16 cDNA clones/genes (Table 2) that were directly or indirectly involved in host defence against greenbug attack. Our detailed studies also suggested that the defence responses against greenbug in sorghum are coordinately modulated by versatile molecular regulators such as salicylic acid, jasmonic acid, abscisic acid and phytohormones (Park et al., Reference Park, Huang and Ayoubi2006).

Table 2 Identification of the defence or defence-related genes that differentially expressed in sorghum seedlings in response to greenbug attack

^a GenBank accession number.

^b BLASTX research was used to determine homologous genes and putative functions.

^c Values of signal intensity ratios showing up- or down-regulation.

Although the application of microarray technology to the analysis of plant defence responses is still a very recent approach (Zhu-Salzman et al., Reference Zhu-Salzman, Salzman, Ahn and Koiwa2004; Park et al., Reference Park, Huang and Ayoubi2006), the initial microarray experiments on studies of plant responses to attack by greenbugs have shown the promise for functional characterization of important processes such as plant defence against aphids. Furthermore, these expression profiling studies completed to date have already identified an amazing number of genes that had not previously been implicated in plant defences against insects. Large sets of informative data were generated that led to the identification of many potential defence-related transcripts. The ability to identify changes in gene expression, particularly in response to disease, insect pest or abiotic stresses is significant since exploration of gene activation in plant defence on a genome-wide scale could be important in discovering novel defence genes or strategies for crop improvement.

Development and application of molecular markers in study of plant defence

The development of DNA markers has facilitated the construction of genetic maps for an economically important plant species. A genetic map depicts the linear arrangement of DNA markers along each chromosome and the genetic distances between adjacent markers. Once the genetic maps are constructed, they can facilitate practical applications in plant breeding such as the identification of important agronomic traits (in many cases QTLs) and marker-assisted selection. Genetic maps are also an important resource for plant gene isolation through map-based cloning and potential for modification without affecting other important traits.

Recently, simple sequence repeats (SSRs) or microsatellites have become the most important DNA marker technology as they proved to be a more dependable, rapid and inexpensive tool for plant genotyping (Yang et al., Reference Yang, de Oliveira, Godwin, Schertz and Bennetzen1996). We have recently constructed a detailed SSR-based genetic map for sorghum (Wu and Huang, Reference Wu and Huang2007). Using the genetic map and SSR markers, we were able to locate one major and a minor QTL on chromosome 9 that are responsible for resistance to greenbug (Wu and Huang, Reference Wu and Huang2008). The major resistance QTL on chromosome 9, designated as QSsgr-09-01, was found to reside in the interval of 7.3 cM between Xtxp289 and Xtxp358, on the basis of linkage and QTL analyses. QSsgr-09-01 accounted for 54.5–80.3% of the phenotypic variation in genetic resistance to greenbug biotype I. The second QTL identified on SBI-09 was flanked by Xtxp67 and Xtxp230, and designated as QSsgr-09-02. QSsgr-09-02 explained 1.3–5.9% of the resistance variation. With the rapidly increasing availability of cDNA clones and expressed sequence tags (ESTs), we took the in silico mining approach to develop EST-SSRs. From the available 25,456 ESTs or cDNA sequences, we have developed 2680 EST-SSRs (Huang, Reference Huang2008). These newly developed sorghum EST-SSR markers represent an additional resource for genetic mapping, comparative genomics and evaluation of co-location between QTLs and functionally associated markers in sorghum. Among these newly identified markers, 200 selected EST-SSR markers were examined for transferability to related cereal crops and the results showed their potential as molecular markers in maize, sugarcane, rice, wheat and barley.