Optimization Of Sampling Size For DNA Based PCR Assay For Hybrid Purity Test In Brinjal and Cauliflower

Abstract

Farmers can harness the full potential of any hybrid only when they get genetically pure seeds of the hybrid. Hence, ensuring the genetic purity of certified seeds of brinjal and cauliflower hybrids is mandatory in India, which is done through field Grow-out Test (GOT) based on the morphological characters of plants grown to maturity. Traditional GOT is costly, tedious, time consuming and environment sensitive. To increase the speed and accuracy of genetic purity testing of hybrids, recent advances in DNA markers have shown promise. newlineTherefore, the present study was undertaken to identify the SSR markers that could be used to test the hybrid purity of three hybrids [viz. 2 brinjal (Arka Anand and Asha), 1 cauliflower (NBH Tania-815)] and to optimize the minimum sample size that can be used for purity assessment of the brinjal and cauliflower hybrids. The experiment was carried out by mixing 95% F1 hybrids with 5% female contaminantss, individually in the sample sets of 100, 200, 300 and 400. For each sample size, PCR-based assay and GOT were carried out to check the hybrid purity. newlineIn GOT, purity evaluation was carried out based on ten morphological traits specified in the published descriptors of brinjal and cauliflower. Plants from four different sample sizes (100, 200, 300 and 400) of brinjal hybrids ( Arka Anand ; Asha ) and cauliflower hybrid ( NBH-Tania 815 ) were studied individually to determine if they were true-to-type for ten morphological characters in the GOT. In case of brinjal hybrid Arka Anand , out of the ten morphological characters analysed; Flower petal no., leaf colour, leaf length, flower petal no., fruit shape and fruit colour exhibited the maximum variation. In case of brinjal hybrid Asha ; flower colour, flower petal no., calyx spininess, fruit shape, fruit colour, calyx colour and presence of fruit stripes exhibited the maximum variation. In case of cauliflower hybrid (NBH Tania-815); plant habit, leaf shape, curd compactness and curd colour exhibited the maximum variation. newlineIn PCR- based assay, a total of 120 and 220 SSR markers were screened for a survey of polymorphism between the parents of brinjal and cauliflower hybrids respectively. 21 SSRs clearly distinguished the parental lines of both the brinjal hybrids and 32 markers showed polymorphism between the parental lines of cauliflower hybrid. Each F1 hybrid were screened with shortlisted polymorphic SSR markers to detect the heterozygosity of hybrids. Polymorphism was looked for, to confirm the co-dominant nature (presence of bands which were found in both the parents) of hybrids. Two SSR markers (eme08D09 and emb01F16) was found to be co-dominant in brinjal; one SSR marker (BrgMS565) was found to be co-dominant in cauliflower. newlineThe percentage of hybrid purity was calculated in GOT assay and in PCR based assay. Hence results indicated that instead of 400 sample size, 100 seedlings are enough to confirm the hybrid purity of brinjal and cauliflower as it showed results comparable with higher sample size. In comparison to that, marker analysis showed the consistent result with GOT. The identified co-dominant markers can be used as referral markers for unambiguous identification, seed purity testing and protection of the hybrids. Hence, it is proposed that molecular marker-based hybrid purity assessment may serve as an effective substitute to traditional GOT. newline newline