Molecular techniques for the identification of triploid citrus

Project involves various methods of triploid identification in citrus fruit.. Seedlessness is a desirable horticultural attribute in Citrus and is positively associated with triploidy. The conventional cytological method for triploid identification is a laborious technique as it involves the preparation of foot-tips for chromosomal analysis. Isozymes and digital densitometry, however, offer the facility to distinguish triploid Citrus from large populations of seedlings both quickly and cheaply. Where there are no gene-dosage regulation effects, greater band density, reflecting increased enzyme activity, should be evident in the allozyme contributed by the diploid gamete for a heterozygous locus. To achieve this, appropriate methods of sample preparation, isozyme electrophoresis and digital densitometry were established. The isozymes of four enzymes, malate dehydrogenase, 6-phosphogluconate dehydrogenase, shikimate dehydrogenase, and phosphoglucose isomerase were investigated for band density differences between allozymes. Polyacrylamide gel electrophoresis was employed to study the isozymes of these four enzymes and band density was measured using a digital densitometer. Of the 4 enzymes investigated only allozymes for shikimate dehydrogenase exhibited consistent differences over a wide range of Citrus cultivars. Greater band density was evident in the allozyme contributed by the diploid gamete. The band density ratio between allozymes for triploid Citrus was close to 0.5, while for diploid Citrus band density ratios were close to 1.0. This effect is due to the extra protein coded by the additional gene dose and was not observed in diploids. Shikimate dehydrogenase proved to be an accurate molecular marker for distinguishing between diploid and triploid Citrus.