PhlepnSouma fC Ser (PICS pr 207-320) 03-1 Coppi 207. Crp Soence Saco fe Pps Alea 3 Nah 2007 MOLECULAR MARKERS FOR IMPROVING SELECTION OF SUGARCANE VARIETIES WITH DOWNY MILDEW RESISTANCE NORVIE L MANIGBAS & LUCILLE VILLEGAS! "Research Associates. Biotechnology Laboratory Philippine Sugar Research Institute, VMC Compound, Victorias City, the Visayas, the Philippines. norviem? yhoo com DNA marker technology has provided new tools in various analyses ranging from phylogenetic analysis, development of high-density maps through PCR-based markers, DNA fingerprinting. cultivar identification, mapping and tagging genes of ‘almost ary trait possible. One of the techniques is the use of molecular markers by tagging genes that are linked (0 wsefil ‘agronomic traits like disease resistance. Ths is done through the application of molecular marker-asssted selection (MAS). These markers can be used to identify disease resistant plants atthe early stages of the breeding cycle, thereby reducing the costs, including labor and time, to produce new varieties of sugarcane. This study was conducted (0 identify reliable ‘molecular markers for downy mildew resistance that can ultimately be incorporated inthe sugarcane breeding program. A ‘mapping population obtained from VMC86-550 x VMC88-354 consisting of 486 progenies were evaluated using 174 ‘microsatellites (SSRs) primers to identify molecular markers for downy mildew resistance. The SSR primers were designed and developed based on the microsatellite-containing sequences of sugarcane by the International Consortium of Sugarcane Biotechnology (CSB). Linkage map was consiructed asing JOINMAP statistical software based on the 147 molecular ‘markers found to be polymorphic. Similarly, phenotypic data were analyzed to determine the association of downy mildew disease resistance to the marker using Fisher's exact test, Chissquare, and Bonferonni correction. A putative marker was ‘identified to be associated with downy mildew resistance. breeding & selection, downy mildew, Joinmap, linkage analysis, LOD scores, marker-assisted selection, microsatellites, Saccharum officinarum, simple sequence repeats, sugarcane A sugarcane fieldINTRODUCTION Molecular techniques are extensively used in improving crop yields and in breeding for resistance against pests and diseases. One of the techniques is the use of molecular markers by tagging genes that are Jinked to useful agronomic traits like disease resistance. This is done through the application of marker-assisted selection (MAS). It involves scoring indirectly for the presence or absence of a desired plant phenotype based on the banding pattems of linked molecular markers (MeCouch & Tanksley 1991). There are a number of ways by which screening efficiency in plant breeding could be increased by using DNA markers, namely: (1) ability to soreen in the seedling stage for traits that are expressed late in the growth of the plant, (2) ability 10 screen for traits that are difficult, expensive, and time- consuming to score phenotypes (eg, resistance 10 pest and diseases and tolerance to abiotic stresses, (3) ability to perform simultaneous marker-assisted selection for several characters at one time. Tight linkage between a marker and a trait has led to the successful transfer of genes through MAS. The challenge is how to capitalize ‘on these novel technologies. The Sugarcane Genome Sugarcane is a complex. aneuploid and autopolyploid. Cultivated sugarcane varieties have 100- 130 chromosomes representing an estimated 10” base pairs and comprising 8-18 copies of a basic x = 8, 2n 40-128 in the persistent weed, Saccharum spontanctn, or x = 10, 2n = 8x = 80 in sugarcane, Saccharum afficinaram (D'Hont et al 1995 & 1998, Ha et al 1999, Irvine 1999). Genomic In Situ Hybridization (GISH) revealed that 80% of chromosomes were inherited from S offcinarun, 10% from S. spontanewn, and 10% were derived from recombinations between the two ancestral species (D’Hont etal 1996), Cultivated sugarcane and its wild relatives are highly heterozygous. Pure inbred do not exist because of difficulty in self-pollination and the random pairing of ‘multiple homologous chromosomes. This random chromosome pairing, which is a characteristic of autopolyploids, makes it necessary to construct linkage maps for each parent of a cross (Ming et al 2001). Studies using molecular markers revealed that single dose restriction fragments (SDRFS) or simplex markers were useful in building genetic maps of sugarcane (Da Silva et al 1995, D'Hont et al 1995, Grivet et al 1996), Despite the complexity of sugarcane genome, the 4 meiosis of sugarcane cultivars involves mainly bivalent pairing (Price 1963, Bumer & Legendre 1993 & 1994) and thus makes it possible to address genetic mapping on the basis of simplex. markers (Wu et al 1992) as mentioned by Raboin et al 2005, The segregating populations used in genetic studies are FI progenies derived from bi-parental crosses (Kang et al 1983, Milligan et al 1990) Microsatellites or simple sequence repeats (SSRs) Microsatellites are composed of tandem repeats of cone to six nucleotides (Litt 1989). These short tandem repeat elements have been found to be both abundant and widely distributed throughout the genomes of many higher plants and animals (Wu et al 1993) and requires only small amount of starting DNA (Powell et al 1996). SSSR analysis is performed by amplification of genomic DNA using pairs of specific primers flanking tandem arrays of microsatellite repeats. Products are typically separated on polyacrylamide gels to visualize DNA bands differing in sizes. Polymorphism is based on the differences in the number of tandem repeats in the amplified regions (Burow & Blake 1998), Any primer or a pair of primers when used to amplify a particular SSR locus in a number of genotypes will reveal SSR polymorphism (Gupta et al 1996). This polymorphism is in the form of differences in length of the amplified product, each length representing an allele at a particular locus. The length differences are attributed to the variation in the number of repeat units at a particular SSR locus. One of the distinguishing, characteristics of these markers is that they can identify polymorphism and inheritance in sugarcane, ‘The objective of this study is to identify refiable molecular markers for downy mildew resistance genes for sugarcane. Downy mildew (Figure 1) is one of the ‘most important diseases of sugarcane in the Philippines. Cane yield reduction due to downy mildew was reported to be 21.7% in a moderately susceptible variety and 465% in a very susceptible variety. Considering sugareane as a 12-month crop, screening and evaluation for disease resistance may take several cropping seasons. Use of MAS presents potential advantages because of its reliable, reproducible and fast way of selecting genotypes resistant to downy mildew. Once a marker is identified, a large number of individuals can be screened in a short period of time. ‘Sugarcane selection for downy mildewMATERIALS & METHODS Mapping population ‘The mapping population consisted of 486 progenies derived from sugarcane cross VMC86-550 x VMC88- 354 was used. VMC86-550 is susceptible to downy mildew and VMC88-354 is moderately resistant. The population comprised unselected seedling clones derived from biparental crosses mentioned above. Crosses were made at the PHILSURIN breeding station in Victorias City, Negros Occidental. Young and old florets of the designated female parent were removed and subjected to hot water treatment (50°C) for 5 min (Nagai 1984, Machado et al 1995, Manigbas et al 2003, Manigas & Villegas 2004). Male and female tassels were combined in an isolated lantem until pollination was completed. Seeds were harvested, dried and sown into seed boxes. Germinated seedlings were transferred separately into seedling cups and maintained in the nursery. Fresh and healthy leaves were sampled for DNA extraction. DNA extraction was done using a modified CTAB method. ‘The extracted DNA was quantified by agarose gel electrophoresis (Figure 2). DNA extraction for PCR amplification ‘Genomic DNA from leaf tissue was extracted from each of the Fl progenies using a modified cetyl-trimethy! ammonium bromide (CTAB) protocol (Muay & Thompson 1980). The extracted DNA was air-dried, resuspended, and incubated in TE buffer with RNAse at 37°C for | hour. DNA concentrations were determined electrophoretically using known amounts of DNA as references. Polymerase chain reaction (PCR) was run in a 96-well Biorad thermal cycler. Each 10 uL PCR reaction contained 2 wL of 10x PCR buffer, 15mM of MgCl; ImM_ of dNTP mix (Invitrogen Life Technologies); 5 U/L of DNA tag polymerase, 5 uM of oligonucleotide primer and 25ng/jiL of DNA template The DNA was first denatured for 5 min at 95°C, followed by 35 cycles of 1 min denaturation at 94°C, | min annealing at $5°C and 2 min elongation at 72°C, with a final elongation at 72°C for 7 min, The soaking temperature was 4°C. ‘The PCR products were denatured at 95°C for 5 min and were immediately chilled on ice. Six yl of the PCR product with STR loading buffer was dispensed onto each well of the polyacrylamide gel. Amplification products were resolved on 8% (w/v) polyacrylamide gel at constant voltage (600V) for 3 hours using Bio-Rad ML Manighas & LC Villegas Sequi-Gen GT gel assembly. The gel was stained following Promega DNA silver staining system with some modifications. DNA marker polymorphism screening A total of 174 SSR primers were screened to identify polymorphisms between each pair of parents and among their progenies. The SSR primers used were designed and developed based on the microsatellte- containing sequences of sugarcane by the Intemational Consortium of Sugarcane Biotechnology (ICSB). All progenies were screened from each cross including the parents. PCR was used fo amplify the DNA. The PCR products were resolved through Polyacrylamide Gel Electrophoresis (PAGE) and stained using the Promega protocol of silver staining with slight modifications. Fragment of band scoring and data analysis ‘The SSR banding profiles of the parents and progenies were scored manually for the presence (1) or absence (0) of distinct bands. The banding pattems of the parents and their progenies were compared (Figure 3). RESULTS & DISCUSSIONS A segregation ratio of 249:213 resistant:susceptible was observed in the progenies of a bi-parental cross VMC86-550 x VMC8-354. Twenty four were observed to be moderately resistant. This may indicate the possibility that many genes are responsible for downy mildew resistance. The segregating ratio is skewed but not statistically different from 1:1 (Table 1). This confirms earlier reports by Butterfield & Nuss 2001) that sugarcane bi-parental cross segregates into 1:1 AA total of 174 primers were tested on the parents and their progenies and 147 markers were found 10 be Polymorphic. Microsatellites showing single-band polymorphism (single-dose markers) between the parents and their progenies were used to construct a linkage map (Figure 4). Linkage analysis of all single-dose markers resulted in a very few segregation groups (ie, equivalent to the number of chromosomes in sugarcane genome) in the bi-parental cross used. ‘A total of 486 progenies from the mapping, population were analyzed based of the pseudo test cross strategy. This is usually applied when a cross is between {two heterozygous parents. Progenies from the cross were used t0 construct individual maps of the parents and the cross itself, Single-dose or simplex makers were identified from the 147 polymorphic SSR markers using, 5(PI) VMC86-550_ x (P2) VMC88-354. cross, its reciprocal comprising of 486 progenies. The total number of single-dose markers identified in (PI) VMC86-550 and (P2) VMC88-354 was 12. Single-dose markers were identified in the population based on the segregation ratio of the markers. The results indicated that the proportion of single~ dose markers was 5-8%, which is smaller than was reported by Raboin et al 2005. A linkage map was constructed based on a few single-dose markers and multiplex: markers, but it was inadequate to establish linkage between marker(s) and the resistance gene(s). The association of markers with downy mildew resistance was determined using Fisher's exact test, Chi- square, and Bonferonni correction (Table 1). From the 12 single-dose SSR markers from a cross between VMCB6- 550 and VMC88-354, 8 putative single-dose markers were found with probability of association with downy mildew resistance: 0.05 probability for P1-21, 0.03 for P1_22 (SMC687CS), 0.04 for P6_3, 0.04 for P6_4, 0.02 for P6_18 (mSSCIR2!), 0.02 for P7_3, 0.003 for P7_13, and 0.013 for P7_16 (mSSCIR 12). Bonferonni correction for multiple tests was applied using 0.05 threshold at genome level. Results showed ‘one putative marker, mSSCIRI2-13, had a high association with resistance. However, linkage betwe: marker and resistance gene still need to be established once additional single-dose markers are generated, CONCLUSIONS Application of marker-assisted selection for important traits like downy mildew resistance in ACKNOWI sugarcane may still take 4-5 more years before it can be incorporated in the breeding program. Additional high- throughput markers are required to saturate the map of the complex genome of sugarcane. Confirmatory evaluation of the mapping population in controlled and field conditions is necessary to establish the inheritance of downy mildew in sugarcane, Understanding genetic control for downy mildew resistance requires more bi-parental combinations because of the complexity of the trait. Inheritance of resistance may differ from one population to another (ie, selfed and bi-parental cross) as in the case of rust resistance gene as reported by the Sugar Research and Development Corporation, Australia (2002). They reported that inheritance of rust resistance in the population 1331 is governed by many genes in contrast to the monogenic inheritance reported by CIRAD in the selfed population R570. Recommended follow-up activities include not only SSR markers but also high-throughput markers such as ABLPs to produce more number of markers. he target ‘number of molecular markers is 1,500 for each trait. Current research in identifying molecular markers for downy mildew will continue by generating more molecular markers and further phenotyping the existing population in more controlled conditions (ie, intensive disease pressure) with replications. The work will focus (on improving phenotyping of the existing individuals in the field and in the nursery. Currently, plants are being ‘propagated in the nursery for future screening in different locations. DG ‘The Common Fund for Commodities (CFC), the Intemational Sugar Organization (ISO) and the Philippine Sugar Research Institute Foundation Inc (PHILSURIN) under its Director General and Project Manager, Mr Leon M Arceo, funded this work, The Intemational Consortium for Sugarcane Biotechnology (ICSB), Dr Brigitte Courtois and Dr Angelique D’Hont (CIRAD, France). The mill district development council coordinators (MDDC), Mr Joe! G Ronario (Luzon), Mr Philip Hofilefia, Mr Manuel L Gallego, Mr Herbie R Macapal, Mr Ildefonso V Prietos (Negros), Mr Raul 'V Soquirata (Mindanao), Mr Alfredo P Buayaban (Bogo-Medellin). PHILSURIN-Makati and Bacolod offices, provided administrative and friendly support. LITERATURE CITED Bumer DM & Legendre BL. 1993. Chromosome transmission and meiotic ability stability of sugarcane (Saccharum spp.) hybrid derivatives. Crop Science 33: 600-606 Bumer DM & Legendre BL. 1994. 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SSR marker Sequence Fisher's test Chi-square test Bonferonni SMC687CS-21 (CAGB 0.05 ** 0.03 ** 06S ‘SMC687CS-22 (CAG 0.03 ** 0.02 ** 04s mSSCIR21-3 (GAGG(GA}23 0.04 ** 0.05 ** 10's mSSCIR2I-4 (GA)2GG(GA)23 0.05 ** 0.06" 12's mSSCIR2I-18 (GARGGGALS 0.02 ** 004+ 08" mSSCIRI23 GAs oon oan oa" mSSCIRI2-13 GAS 0.003 * 0.0001 * 0.002* mSSCIRI2-16 (GAS ool* ors 02" * sguscan at 96 ew * significant a $25 owl me not igifiare Figure 1. Downy mildew caused by Peronosclerospora philipinensis NL Manighas & LC Villegaswe Figure 2. Agarose gel (1%) stained with ethidium bromide and treated with RNAse showing the native nucleic acids extracted from the parents. MPIP2PII 2.3.4 5 67 8 9 1011021213 1415 1603 P2P1 267 234 23 32 184 124 104 Figure 3. ‘The SSR marker revealing highly scorable bands in the parents and progenies, PI = VMC86-550, P2= VMC88-354, P3 = PHIL80-13. Lanes I-11 corresponds to the progenies of P2 x PI. Lanes 12-16 corresponds to the progenies of P3 x PI. Lane M- molecular weight marker PBR322 Hae III digest. 10 Sugarcane selection for downy mildewo-prenm gopcpegees opps 1 “4 Je-dose marker-based map between VMC36-550 x VMC88-354 at LOD 5. NL Manighas & LC Villegas u