Doubled haploid based parental lines are most suitable in predicting heterosis using microsatellites and in development of highly heterotic F1 hybrids in Brassica oleracea

In Brassica oleracea, heterosis is one of the most efficient tools giving impetus to hybrid vegetable industry. In this context, we presented the first report on identifying superior heterotic crosses for yield and commercial traits in cauliflower involving cytoplasmic male sterile (CMS) and doubled haploid (DH) lines as parents. We studied the suitability of SSR and EST-SSRs based genetic distance (GD) and morphological markers based phenotypic distance (PD) in prediction of heterosis when DH based genotypes are used as parents in developing F1 hybrids. Overall 120 F1 hybrids derived from twenty Ogura cybrid CMS lines and six DH based testers were evaluated for 16 phenotypic traits along with their 26 parental lines and 4 commercial standard checks, in 10 × 15 alpha lattice design. The genomic SSR and EST-SSRs based genetic structure analysis grouped 26 parental lines into 4 distinct clusters. The CMS lines Ogu118-6A, Ogu33A, Ogu34-1A were good general combiner for developing short duration hybrids. The SCA effects were significantly associated with heterosis suggesting non-additive gene effects for heterotic response of hybrids. Less than unity value of σ2A/D coupled with σ2gca/σ2scaindicated the predominance of non-additive gene action in the expression of studied traits. The genetic distance estimates among 26 parents ranged from 0.44 to 0.98 and were significantly associated with heterosis for important commercial traits, suggesting the utility of microsatellite based genetic distance in prediction of heterosis in B. oleracea.


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Abstract 24 In Brassica oleracea, heterosis is one of the most efficient tools giving impetus to hybrid 25 vegetable industry. In this context, we presented the first report on identifying superior heterotic 26 crosses for yield and commercial traits in cauliflower involving cytoplasmic male sterile (CMS) 27 and doubled haploid (DH) lines as parents. We studied the suitability of SSR and EST-SSRs 28 based genetic distance (GD) and morphological markers based phenotypic distance (PD) in 29 prediction of heterosis when DH based genotypes are used as parents in developing F 1 hybrids. 30 Overall 120 F 1 hybrids derived from twenty Ogura cybrid CMS lines and six DH based testers  (Table 1). These CMS lines were used as female parent in the 162 breeding programme. The completely homozygous 6 DH inbred lines of snowball cauliflower 163 with abundant pollen production, developed through IMC, were used as testers (Table 1). 164 The CMS and DH lines were selected among the 60 CMS and 24 DH lines developed 165 previously, based on molecular, morphological characterization (data not shown in this 166 publication) and flowering synchronization of lines and testers was also the main consideration 167 in selection of parents. All the recommended package of practices, suggested for raising 168 cauliflower crop at IARI-regional station Baragram farm, were followed to grow a healthy crop  depicting genetic diversity (S1 Table) (Table 3). The variance analysis for combining ability 292 also revealed highly significant differences among 120 testcross progenies for all the 16 traits at 293 0.1% probability, while no significant differences were found among three replications for all the 294 traits except LW, suggesting true presence of inherent variability among all the crosses (Table 3).

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With respect to CL, the highest significant positive SCA effect was observed in the cross   (Fig 1b).  parental CMS and DH lines based on their phylogeny (Fig 2). The PCA revealed that first two  The Euclidean distance (PD) between lines and testers were computed from 16 phenotypic traits 481 (supplement Table S3) and GD was calculated from molecular data based on 87 microsatellite 482 markers (genomic-SSR and EST-SSRs) used for assessment of genetic diversity between parents 483 (S4 Table). The PD was ranged from 2.07 for the cross L16 × T6 (Ogu12A × DH-53-10) to 8.27    Table 7 and Table 8. Among the vegetative traits, for the traits related with 527 earliness, like days to 50% CI and days to 50% CM, the cross combinations Ogu34-1A×DH-53-528 1 and Ogu33A×DH-53-6, showed significantly high MPH in desirable negative direction (Table   529 7). For the days to 50% CI, the testers DH-53-1 and DH-53-9, were involved in 4 crosses 530 individually out of top 10 crosses. For the days to 50% curd maturity, the CMS line Ogu33A as 531 female parent was involved in 6 hybrids for earliness among top 10 hybrids. Ogu33A was also 532 involved as female parent in one of the top 10 cross combinations related to days to 50% CI.

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This line had significantly highest GCA for earliness among all the CMS lines used in the study.

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A short core length is desirable in cauliflower.         The PPMCC of GD, PD with MPH, BPH, SCA and of combining ability with heterosis for ten 592 commercial traits is presented in Table 9 (Figs 4 a to f). The GD and PD exhibited no significant 593 correlation coefficient with SCA for any of the traits (Table 9). SCA showed significantly 594 positive correlation with MPH and BPH for all the traits at P ≤ 0.01. No significant association 595 of GD with MPH and BPH was observed with respect to days to 50% CM, LL, CL and CoL. For 596 the commercial traits viz. PH, GPW, NCW, LW, CD and TMY, significant correlation was observed between GD and MPH in desirable direction for the respective traits (Table 9). The 598 highest value of PPMCC of GD with MPH and BPH in desirable direction was observed for LW.

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Thus, GD exhibited significant correlation with MPH and BPH for six traits out of ten traits 600 studied. However, PD exhibited no significant correlation with heterosis for majority of traits.

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PD showed significant correlation with MPH for only LL in desirable direction (Table 9). PD 602 exhibited significant correlation in undesirable direction for CoL. However, no significant 603 association was observed between parental genetic distances based on phenotypic traits (PD) and 604 molecular data (GD) (r = -0.04) (Fig 5).  reports are available regarding correlations of genetic distances, heterosis and combining ability.

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In the present study, no correlation was observed between two distance measurements, based on 813 morphological data (PD) and molecular data (GD). This is in contrary to the findings of Gupta et In conclusion, our study is the first report on determining heterotic groups based on combining 874 ability for morphological, yield and commercial traits using Ogura cybrid cytoplasm based CMS 875 lines and DH testers. We also presented the first comprehensive report on predicting the