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CROP BREEDING, GENETICS & CYTOLOGY

An Isozyme Marker for Resistance to Root-Knot Nematode in Sugarbeet

^a USDA-ARS-PWA, U.S. Agric. Res. Stn., 1636 East Alisal St., Salinas, CA 93905-3018
^b Harris Moran Seed Co., 100 Breen Rd., San Juan Bautista, CA 95045

* Corresponding author (myu{at}salinas.ars.usda.gov)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Root-knot nematode (Meloidogyne spp.) is a destructive pest of sugarbeet (Beta vulgaris L.) that reduces production in infested areas and is difficult to manage. Identification of nematode-resistant plants is a time-consuming process that is subject to genotype–environment interaction. Development of resistant cultivars/hybrids is the most effective control. This study was conducted to establish a rapid and effective screening technique to detect a large number of sugarbeet genotypes with resistance to Meloidogyne spp. A nematode-resistant sugarbeet germplasm line, Mi-1 Beta, was previously developed using J2 inoculation and screening procedures. Leaf and cotyledon extractions were used in diagnosis. Phosphoglucomutase (PGM) was found to be a potentially useful isozyme marker of resistance in Mi-1 Beta and derived lines in starch gel electrophoresis. Seven banding patterns (four resistant and three susceptible) were produced. All susceptible plants shared the banding pattern of the resistant strains, except for a single PGM band. If demonstrated to be tightly linked to nematode resistance, this novel PGM isozyme marker will accelerate breeding sugarbeet with resistance to root-knot nematode.

Abbreviations: PGM, phosphoglucomutase • PMS, phenazine methosulfate

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
THE ROOT-KNOT NEMATODE, Meloidogyne spp., is an economically important sugarbeet (Beta vulgaris L.) pest that is difficult to control in production fields. Previously, soil fumigation was the most reliable technique. Root-knot nematode is now yield-limiting due to continuing loss and restriction of soil fumigants, thereby preventing farmers from growing sugarbeet crops profitably. Host-plant resistance to root-knot nematode is, therefore, the most environmentally safe means to alleviate sugarbeet production problems and nematicide hazards.

The wild sea beet [B. vulgaris ssp. maritima (L.) Arcang] source of root-knot nematode resistance is effective against multiple species of Meloidogyne spp. (Yu et al., 1999). Introgression of root-knot nematode resistance from wild beet to cultivated sugarbeet has been achieved using second-stage juveniles (J2) inoculation, screening, and pollination procedures in the greenhouse. Improved sugarbeet-breeding materials with resistance to root-knot nematode were further selected for tap-root conformation and root-yield performance.

The process of transferring resistant genes from noncultivated taxa or species to the cultivated genomes, and selecting productive genotypes to become hybrid parents, always has been time-consuming and labor-intensive. A simpler and more accurate screening procedure has been sought for accelerating sugarbeet root-knot nematode-resistance breeding. An isozyme pattern of phosphoglucomutase (PGM) has been shown to be associated with root-knot nematode resistance found in the Mi-1 Beta germplasm lines. The PGM isozyme marker can be employed to easily identify individual Mi-1 Beta genotypes with resistance to Meloidogyne spp.

The objective of this study was to establish a rapid and effective screening procedure to detect a large number of sugarbeet genotypes with resistance to root-knot nematode.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The Beta materials with resistance to Meloidogyne spp. used in this research were the Mi-1 germplasm (PI 593237; Yu, 1997), progeny of its hybrid crosses to sugarbeet, and susceptible controls (sugarbeet 1783 and 4500) available in the USDA-ARS program. Stecklings (seedlings) of the susceptible sugarbeet lines C37, C78, C80, and R876-89-5NB were obtained from R.T. Lewellen, USDA-ARS, Salinas. Crosses to transfer root-knot nematode resistance from wild beet to sugarbeet were conducted in the greenhouse and laboratory. Test plants were examined for root gall formations 35 to 40 d after inoculation of early-stage beet seedlings with 1200 M. incognita J2 per plant (Yu et al., 1999). Individual plants producing none or fewer than 10 small (<1 mm) root galls were classified as resistant. All other plants were classified as susceptible. Seeds were germinated in steam-sterilized sand.

Tissue of cotyledons and expanding young leaves was collected from greenhouse or field (Spence farm in Salinas, CA, sandy loam soil classification) plants, from seedling stage to full maturation for isozyme analysis. A minimum of 55 plants per germplasm line were examined. Samples, about 5 mm² in size, were ground in pre-chilled mortars with 0.1-mL extraction buffer. The extraction buffer consisted of 0.1 M Tris (tris-[hydroxymethyl]aminomethane)–HCl, pH 7.0, 2.45 M of glycerol, 1.25 mM of PVP-40T (polyvinylpyrrolidone), 0.5% (v/v) Triton X-100, and 125 mM 2-mercaptoethanol, the latter added just before use (modified from Aicher and Saunders, 1990). Paper wicks (11 by 2 mm; Northfork Products, Syracuse, IN) were soaked in each sample extract and stored at 4°C.

Buffers and the phosphoglucomutase stain were adapted from systems described by Van Geyt and Smed (1984) with a slight modification. The gel buffer consisted of 5 mM of histidinemonohydrochloride, pH 7.0, and the electrode buffer was 0.2 M of trisodium citrate-2 H₂O, pH 7.0. The staining solution for the isozyme PGM consisted of 25 mL of 0.1 M Tris–HCl buffer, pH 8.0; 5 mM of -D-glucose-1-phosphate (glucose-1-P); 7 units glucose-6-phosphate dehydrogenase (G-6-PDH); 5 mM of ß-nicotinamide adenine dinucleotide phosphate (NADP); 2 mL of 0.025 M MgCl₂; 1 mM of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT); and 0.13 mM of phenazine methosulfate (PMS).

The gel was prepared using a 1:2:1 mixture of hydrolyzed starch (Pasteur Merieux Connaught, Charlotte, NC), hydrolyzed potato starch (Starch-Art, Smithville, TX), and potato starch (Sigma-Aldrich, St. Louis, MO) dissolved in gel buffer. The gel solution was heated in a microwave oven until translucent, degassed using a vacuum pump, and poured into trays. After cooling in the refrigerator, the gel was loaded with the sample wicks. The gel tray with loaded samples (including two tracking dye wicks) was placed in a refrigerated, horizontal gel electrophoresis apparatus (Model H4; Gibco-BRL Products, Life Technologies, Grand Island, NY). A constant current of 55 to 65 mA (65 to 85 volts) was applied to the gel. Wicks were removed from the gel 10 to 15 min after the start of the run. Electrophoresis was carried out for 5 1/2 to 6 h, until the tracking dye had moved 5 to 7 cm from the cathodal end. The gel was sliced in 1.5-mm-thick slabs with monofilament thread, and developed in PGM stain with an agar overlay (made up of 2% agar solution in 25 mM of MgCl₂). They were incubated in the dark at 37°C for about 1 h. Isozyme banding patterns were recorded for analysis. Photographs were taken using the Video Copy Processor (P67UA; Ultra-Violet Products, Upland, CA).

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
In addition to phosphoglucomutase, some 20 other enzyme activity stains were investigated with the purpose of discriminating Meloidogyne spp. resistant genotypes in Mi-1 Beta, progeny of its hybrid crosses, and other sugarbeet populations. The tested enzymes were aconitase (ACO), acid phosphatase (ACP), alcohol dehydrogenase (ADH), adenylate kinase (AK), arginine aminopeptidase (AMP), diaphorase (DIA), endopetidase (EP), esterase (EST), fructose-1,6-diphosphatase (FDP), glutamate dehydrogenase (GDH), glutamate oxaloacetate transaminase (GOT), hexokinase (HK), isocitrate dehydrogenase (ICD), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), malic enzyme (ME), peptidase with leucyl-alanine (PEP-LA), 6-phosphogluconate dehydrogenase (PGD), phosphoglucoisomerase (GPI), and shikimate dehydrogenase (SKDH) (Siciliano and Shaw, 1976; Vallejos, 1983; Van Geyt and Smed, 1984; Acquaah, 1992). Among these isozymes, AK and MDH induced isozyme bands in some populations of sugarbeet progeny. However, their banding patterns were inconclusive to detect resistance or susceptibility in the donor plants.

It was determined from the starch gel electrophoresis study that an isozyme of PGM is diagnostic for resistance to Meloidogyne spp. in Mi-1 Beta genotype. Activity of PGM could be shown in two zones on the gel. The first, or faster, zone was polymorphic and consisted of up to three bands, very fast (VF), fast fast (FF), and slow fast (SF) (Fig. 1). The zymogram of Meloidogyne spp.-resistant Mi-1 Beta and its derived progeny comprised four resistant and three susceptible banding types: Type I had only the VF band, Type II had VF and FF bands, Type III had VF and SF bands, and Type IV had all three bands. Among the four banding types, Type II had the highest frequency (Table 1) in the current nematode-resistant sugarbeet population.

View larger version (42K): [in this window] [in a new window]   Fig. 1. A phosphoglucomutase-stained starch gel showing seven banding types induced from root-knot nematode-resistant Mi-1 sugarbeet plants (left to right, two samples each: Types I to IV are resistant, Types VI to VIII are susceptible; VF = very fast, FF = fast fast, SF = slow fast, and S = slower band).

The second (or slower) banding zone consisted of one or two slower (S) bands (Fig. 1) that showed no polymorphism in any of the entries studied. Generally, one S band was present in the slower zone from leaf samples, whereas cotyledons exhibited one or two bands depending on the environment and plant age. All cotyledons from field-grown plants exhibited two bands, while those from the greenhouse showed either one or both bands. Older (yellowed) cotyledons usually exhibited only one band. The S bands did not have apparent diagnostic value to sugarbeet root-knot nematode resistance.

The PGM bands (from leaf extractions) detected in the starch gel were stable at all stages of growth tested. The cotyledon was an excellent tissue for isozyme diagnosis. Samples collected from 6 to 29 d after sowing (in greenhouse) showed readable and consistent isozyme bandings, but the bands were clearest when cotyledons were collected between 14 and 21 d after planting. In contrast, if expanding young leaves (at any growing stage of the plant) were used for sample extractions, banding remained clear and readable, that is, the VF and FF bands remained distinct, throughout the whole growth period of the beet plant.

It is interesting to find that the Mi-1 host resistance to multiple species (six species, nine races) of Meloidogyne nematode (Yu et al., 1999) was indexed by the presence of a single VF band (Fig. 1; Table 1). The detection of this PGM isozyme marker, which is an expression of a new putative gene, is a potentially useful and practical means to identify Meloidogyne spp.-resistant Mi-1 genotypes in the sugarbeet root-knot nematode-resistance breeding program. The laboratory test results agreed with those obtained from simultaneous M. incognita J2 inoculation study of the same donor plants (an estimated 2000 entries) in the greenhouse. All test plants that exhibited a VF band in PGM staining were resistant to the root-knot nematode demonstrating that the PGM marker is a reliable indicator of Meloidogyne spp. resistance in Mi-1. This isozyme marker may avoid any uncertainties (if occurring) in the classification of nematode-resistant plants in greenhouse inoculation studies, which is based on gall counts (e.g., zero or <10 galls/seedling) as the criterion for resistance.

The investigation of PGM activities in the genus Beta has generated useful information in plant science. In sugarbeet, Pgm1 was found to segregate independently from loci B (annual growth habit) and R (hypocotyl color) (Aicher and Saunders, 1990). Convincing evidence was obtained on the location of linked pairs of loci Pgm1-Mdh2 and Mdh1-Gdh2 in different B. vulgaris chromosomes (Denisova and Levites, 1999). Genetic deviation in Californian wild beets was found to have two different origins: (i) Beta vulgaris, evolved from subspecies vulgaris and maritima hybridized populations, and (ii) Beta macrocarpa, genetically almost identical to the European accessions (Bartsch and Ellstrand, 1999).

Isozyme markers still are useful as simple, inexpensive means for detection of gene introgression and recombination, for comparative mapping, and for determination of genetic diversity and phylogenetic relationships among plant species (Hart and Langston, 1977; Hoffman, 1999; Horandl et al., 2000). The starch gel electrophoresis employed in the present study demonstrated the utility of PGM as a simple and rapid assay for a putative marker for the selection of Mi-1 Beta individuals resistant to Meloidogyne spp. This marker will facilitate and greatly benefit sugarbeet root-knot nematode-resistance breeding programs in the future if tightly linked to resistance to Meloidogyne spp.

	ACKNOWLEDGMENTS

 
The technical assistance of G.H. Gretz is gratefully acknowledged. This research was supported in part by funds from the California Beet Growers Association, Ltd., 2 West Swain Rd., Stockton, CA 95207.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

Received for publication August 16, 2000.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

• Acquaah, G. 1992. Staining procedures for selected enzymes. p. 97–115. In T.R. Dudley (ed.) Practical protein electrophoresis for genetic research. Dioscorides Press, Portland, OR.
• Aicher, L.D., and J.W. Saunders. 1990. Inheritance studies and clonal fingerprinting with isoenzymes in sugarbeet. Crop Sci. 30:1064– 1072.[Abstract/Free Full Text]
• Bartsch, D., and N.C. Ellstrand. 1999. Genetic evidence for the origin of Californian wild beets (genus Beta). Theor. Appl. Genet. 99: 1120–1130.
• Denisova, F.S., and E.V. Levites. 1999. New approach using the genetic segregation in triploid progeny for the chromosomal localization of genes in higher plants exemplified by Beta vulgaris L. Russ. (English transl.). J. Genet. 35:658–663.
• Hart, G.E., and P.J. Langston. 1977. Chromosomal location and evolution of isozyme structural genes in hexaploid wheat. Heredity 39:263–277.
• Hoffman, D.L. 1999. Inheritance and linkage relationships of morphological and isozyme loci in As-genome diploid oat (Avena spp.). J. Hered. 90:446–452.[Abstract/Free Full Text]
• Horandl, E., J. Greilhuber, and C. Dobes. 2000. Isozyme variation and ploidy levels within the apomictic Ranunculus auricomus complex: Evidence for a sexual progenitor species in southeastern Austria. Plant Biol. 2:53–62.
• Siciliano, M.J., and C.R. Shaw. 1976. Separation and visualization of enzymes on gels. p. 185–209. In I. Smith (ed.) Chromatographic and electrophoretic techniques. Vol. II. 4th ed. William Heinemann Medical Books, London.
• Vallejos, C.E. 1983. Enzyme activity staining. p. 469–516. In S.D. Tanksley and T.J. Orton (ed.) Isozymes in plant genetics and breeding. Part A. Elsevier Science, Amsterdam, the Netherlands.
• Van Geyt, J.P.C.F., and E. Smed. 1984. Polymorphism of some marker enzymes of the sugarbeet (Beta vulgaris L.) investigated by polyacrylamide gel electrophoresis and starch gel electrophoresis. Z. Pflanzenzuchtg. 92:295–308.
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