Indian Journal of Experimental Biology Vol. 43, May 2005, pp. 462-466

Molecular tagging of gene conferring leaf blight resistance using micro satellites in sorghum [Sorghum bicolor (L.) Moench]

Mukta Mitral & K S Boora* Department of B iotechnology and Molecular Biology, CCS Haryana Agricultural University, Hisar 1 25 004 (Haryana), India.

Received 13 lilly 2004; revised 22 November 2004

Resistance to leaf blight in sorghum [Sorghum bicolor (L.) Moench] accession G- 1 1 8 was found to segregate as a single dominant trait in a cross to susceptible cultivar, HC- 136. Molecular marker(s) l inked to the locus for disease resistance was identified using simple sequence repeat (SSR) markers coupled with bulk segregant analysis. Genomic DNA from the parental cultivars and bulks were screened by PCR amplification with 50 simple sequence repeat primer pairs. Out of these, 38 SSR primers produced polymorphism between parents. After screening of these 38 SSRs with resistant and susceptible bulk, one SSR primer, Xtxp 309 produced a unique band of approximately 700 bp only in resistant parent and resistant bulk and a unique band of 450 bp only in susceptible parent and susceptible bulk. Upon screening with individual resistant and susceptible recombinant inbred l ines (RILs), marker Xtxp 309 produced amplification in 23 of the 26 resistant RILs and no amplification was produced in any of the 25 susceptible RILs. The same marker Xtxp 309 produced ampl ification in 2 1 of the susceptible RILs and 3 of the resistant RILs of 450 bp band. This was found to be located at a distance of 3 . 1 2 cM away from the locus governing resistance to leaf blight which was considered to be closely l inked and 7.95 cM away from the locus governing susceptibil i ty to leaf blight. This marker may prove useful in MAS for gene i ntrogression, plant genetic diagnostics and gene pyramiding for resistance via genetic transformation for disease resistance in plants.

Keywords: Gene, Leaf blight resistance, Microsatell i tes, Molecular tagging, Sorghum bicolor

Sorghum ranks fifth among major crops in the world after paddy, wheat, maize and barley, Production of sorghum is adversely affected due to both biotic and abiotic stresses. Leaf blight caused by fungus, ExcerolzilulIl turcicllm, is among the important foliar diseases of the sorghum that causes loss up to 50% (Ref. 1 ) .

In recent years, progress has been made in mapping and tagging the genes of agronomically important traits which form the foundation of marker assisted selection (MAS). Closely linked markers2 has been used successfully for the isolation of a number of important plant genes, including genes for disease resistance3.6. RAPD based DNA markers l inked to anthracnose disease resistant gene have been identified in sorghum? Since RAPD are highly reproducible and dominant markers so simple sequence repeats (SSR)8,9 have been initially employed to detect polymorphism between the parental cultivars. Simple sequence repeats, also known as microsatellites, are based on tandem repeats of short (2-6 bp) DNA sequences. Different alleles

*Correspondent author: E-mail : booraks@hau.ernert . in

can be detected at a locus by the polymerase chain reaction (PCR), using conserved DNA sequences flanking the SSR as primers. The aim of the present study was to identify and map the SSR markers closely linked to the gene for leaf blight resistance in sorghum.

Materials and Methods

Fungal material-Conidia from a specific pathogenic isolate of Exserohilum turcicum, collected from diseased plants leaves from the field, were cultured on casein lactose hydrolysate (CLH) agar medium. Single conidia was picked up from the plate and was again cultured on CLH medium. Conidia were suspended in sterilized distilled water to a final concentration of l x 1 06 conidialml .

Plant materials, inoculation and scoring for disease

resistallce-Sorghum l ines G- 1 1 8 , resistant to leaf blight caused by E. turcicum and HC- 1 36, agronomically superior, but susceptible to this pathogen were selected for the present investigation. Crosses were made between HC- 1 36xG- 1 1 8 and an F2 population consisting of 1 10 pbnts was derived.

MITrAL & BOORA: MOLECULAR TAGGING OF GENE CONFERRING LEAF BLIGHT RESISTANCE 463

These plants along with the parental l ines were raised in the greenhouse following recommended agronomic practices and screened for disease resistance using conidial suspension of a local isolate of E. turcicum as the primary inoculum. F2 genotypes and 20 plants of each of the parent were inoculated with 1 ml conidial suspension ( l x 1 06/ml), into leaf whorl of four week old plants. Data was recorded three times at 1 0 days interval after . inoculation based on symptom expression of the disease. F2 plants were selfed till the F6 progeny, using single seed descent method, to obtain recombinant i nbred lines. Twenty plants of F6 genotype were screened for disease resistance by inoculating the same local isolate of the pathogen, used earlier to screen the F2 genotypes along with parental lines. Plants were scored as resistant recombinant inbred l ines (RILs) when all the plants of F6 progeny showed no disease and susceptible recombinant inbred lines when all the plants had disease symptoms .

DNA extraction-Leaf tissue was collected· from

3-4 week old seedlings of each of the parental genotypes and F6 genotypes (which include 25 susceptible and 26 resistant RILs) grown in the greenhouse. Midrib was removed from the leaves and laminae were cut into 5- 1 0 cm segments. The samples were freeze dried using lyophilizer. The tissue was ground to fine powder using Willey Mill . The ground tissue of all the recombinant inbred lines and parental genotypes was stored at-20°e.

DNA was isolated from 0.5 g of the ground leaf tissue powder of each genotype using the CT AB methodl O• 1 2. DNA samples were treated with RNase A (50 /.Lg/ml) followed by ethyl alcohol precipitation. The DNA concentration was measured using DNA fluorometer (DNA Quant 200 fluorometer) .

Bulking of DNA samples--Homozygous susceptible bulk was made by mixing DNA (2 /.Lg) from each of 25 susceptible RILs . Resistant bulk was made by combining DNA (2 p.g) from each of 26 resistant RILs \3 .

SSR analysis-A total of 50 SSR primers were screened for polymorphism among the parents HC-1 36 and G- 1 l 8 . PCR was performed in (20 /.Ll ) reaction mixtures containing 50 ng of genomic DNA, Two units of Taq DNA polymerase, 2 /.Ll of 1 0 X Taq

DNA polymerase buffer ( 1 0 mM Tris-HCl, pH 9 .0, 50 rnM KCl, 1 0% Triton X 1 00), 1 00 p.M of each dNTPs, 1 .6 mM MgCh and l /.L l of each forward and reverse primers. Each sample was overlaid with 1 0 /.L l of l ight mineral oil . Amplification reactions were carried out in Biometra T personal and MJ Research PTC 1 00 programmable thermal cycler. peR conditions for microsatellite analysis i ncluded an initial denaturation step of 3 min at 94°C and following 30 cy'cles of amplification with denaturation at 94°C for 1 min, annealing at 50°C for 1 min and extension at noc for 2 min. Final extension was carried out at noc for 1 0 min. PCR amplified DNA fragments were resolved in 2% (w/v) agarose gels by electrophoresis in 1 X TBE buffer l4. Amplified DNA fragments were stained with ethidium bromide and photographed using UV gel documentation system (Pharmacia-Biotech).

Bulked segregant analysis-Primers showing polymorphism were screened to find out markers l inked to the target locus using resistant and susceptible bulks along with parental DNA. PCR amplification conditions were maintained similar to those used during screening of primers to detect polymorphism.

Linkage analysis-Each allele was scored with bulks and the marker which showed l inkage with the alleles in the bulks was analysed with individual RIL. Marker Xtxp 309 amplified an allele in the inbred lines similar to resistant parent except in 3 l ines but not in any of inbred lines which were similar to susceptible parent. The same marker amplified another allele in inbred lines similar to susceptible parent except in 4 lines and in 3 of the inbred lines which were similar to resistant parent. Each locus was scored for parental alleles to generate the database. The database consisted of columns for each locus and l ines for each inbred. A FORTRAN program was written that compared each column one by one. In a pair of columns the absolute difference was recorded for each inbred that differed in allele assignments. No differences were recorded when a 0 was present in a given inbred at one of the two loci. The sum of absolute differences was divided by the number of functional (nonzero) comparisons; this fraction, R, represents the proportion of recombinant l ines. R is related to the proportion of recombinants in a single meiosis, r, by the equation , r = R/(2-2R), where r is map distance in Morgans I5 , 16 .

-464 INDIAN J EXP BIOL, MAY 2005

Results and Discussion Genetic analysis of inheritance of gene for

resistance to leaf blight-F, of a HC 1 36xG- 1 1 8 cross was resistant to leaf blight local biotype indicating that leaf blight resistance is controlled by a dominant gene. Out of total 1 03 F2 plants screened, 77 plants were found to be resistant and 26 were susceptible to leaf blight. Hence, there was a Mendelian ratio of 3 : 1 segregation for resistant and susceptible genotype of F2 population . This confirmed that a single gene controlled the disease resistance. These genotypes were further grown to create RILs which were produced by single seed descent method and screening for disease resistance was done in F6

Locus

Table I-Microsatell ite primer pairs producing polymorphic bands

Bands scored Polymorphic Monomorphic Total

Pep C Xtxp 47 Xtxp 6 1 Xtxp 67 Xtxp 69 Xtxp 84 Xtxp 97 Xtxp 1 00 Xtxp 1 05 Xtxp 1 14 Xtxp 1 15 Xtxp 1 59 Xtxp 1 83 Xtxp 201 Xtxp 225 Xtxp 23 1 Xtxp 262 Xtxp 266 Xtxp 273 Xtxp 274 Xtxp 279 Xtxp 286 Xtxp 287 Xtxp 292 Xtxp 309 Xtxp 3 1 9 Xtxp 32 1 Xtxp 339 Xtxp 348 Xtxp 350 Xtxp 354 Xtxp 357 Xtxp 37 Xtxp 25 Xtxp 4 Xtxp 3 Xtxp 296 Xtxp 302

5 9 6 1 2 5 1 4 5 5 3 7 2 6 3 1 1 1 1 4 1 7 5 2 1 1 5 2 7 2

I 2 2 2 I 3 2 1 3 2 2

0 5 1 1 0 0 6 0 1 2 4 9 4 5 2 6 4 9 0 5 0 3 0 7 0 2 3 9 2 5 2 1 3 3 14 6 1 0 1 2 3 1 0 2 7 1 3 0 1 1 3 8 2 4 0 7 0 2 0 I I 2 0 2 3 5 3 5 I 2 2 5 4 6 3 4 I 4 2 4 3 5

generation. Upon screening of F6 progeny, it was found that 25 genotypes were susceptible and 26 genotypes were resistant to the disease.

SSR analysis-out of 50 SSR primer pairs used, 46 primer pairs (92%) produced amplification in the parental genotypes (HC 1 3 6 and G 1 1 8). A total of 245 discrete bands were produced with an average uf 6.4 bands per primer. Four primers did not produce any amplification, whereas 5 primers did not amplify the parent G- 1 1 8, and HC- 1 36 was not amplified by 2 primers. 38 primers (76%) produced 1 63 distin-:t polymorphic bands between the parents. Primer Xtxp 84, Xtxp 266, Xtxp 32 1 , Xtxp 339, Xtxp 357 and Xtxp 4 produced only one polymorphic band, whereas primer Xtxp 67 produced maximum number of polymorphic bands i :e . 1 2 (Table 1 ) .

Marker Xtxp 309 generated a product that differed in two parents. This primer produced a DNA fragment of approximately 700 bp in resistant parent and resistant bulk only and another unique band of approximately 450 bp in susceptible parent and susceptible bulk only (Fig. I ). Marker Xtxp 309 was screened with individual susceptible and resistant RILs in order to check the closeness of the marker to the gene for leaf blight resistance. This marker Xtxp 309 was found to be located at a distance of 3 . 1 2 cM away from locus governing resistance to leaf blight

Fig I-Lane 1 Lambda DNAlEcoRI + Hind III double digest Marker, Lane 2, 6 & 1 0 HC- 1 36, Lane 4, 8 & 12 G- 1 18, Lane 3, 7 & 1 1 Susceptible bulk, Lane5, 9 & 1 3 Resistant bulk.

MITrAL & 1300RA: MOLECULAR TAGGING OF GENE CONFERRING LEAF I3 LIGHT RESISTANCE 465

(Fig. 2 A-C) and 7.95 cM away from locus governing susceptibility to leaf blight (Fig. 3 A-C).

SSR marker Xtxp 309 was mapped on sorghum linkage group G (LGG) of the sorghum RFLP and SSR linkage map produced by Texas A & M University, College Station, Texas, USA.

Acknowledgement We are thankful to Rockefeller Foundation, New

York, USA for financial support.

Fig. 2.(A}-Lane 1 Lambda DNA/EeoR 1 + Hind I I I double digest maker, Lane 2 0- 1 1 8, Lane 3-9 Resistant R ILs ( 1 -7) ; (13)-Lane I Lambda DNA/EeoR I + Hind II I double digest maker. Lanc 2 0-I 1 8, Lane 3-9 Resistant RILs (8- 1 4); and (C)-Lanc I Lambda DNAlEcoR I + Hind III double digest maker, Lane 2- 1 3 Resistant RILs ( 15-26)

Fig. 3 (A}-Lane I Lambda DNA/EcoR I + Hind 1 I I double digest maker, Lane 2 HC- 1 36, Lane 3- 1 3 Susceptible R I Ls ( I - I I ); (8)­Lane I Lambda DNA/EcoR I + Hind I I I double digest maker. Lane 2-7 Susceptible RILs ( 1 2- 1 7) ; and (C)-Lane I Lambda DNA/EcoR I + Hind I I I double digest maker, Lane 2 HC- 1 36, Lane 3- 1 0 Susceptible RILs ( 1 8-25)

466 INDIAN J EXP BIOL, MAY 2005

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