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ISSRs: An efficient tool to characterize interspecific F1 hybrids of Brassica species

Kadambari Gupta2, Deepak Prem2, Madan Singh Negi1 and Abha Agnihotri1

1Bioresources and Biotechnology Division, TERI, Darbari Seth Block, IHC Complex, Lodhi Road, New Delhi, 110003,
India.Tel: 91-11-2468 2100 or 2468 2111, Fax: 91-11-24682 144 or 2468 2145. E-mail: abhagni@teri.res.in
2
Center for Bioresources and Biotechnology, TERI School of Advanced Studies, Darbari Seth Block, IHC Complex, Lodhi Road,
New Delhi, 110003, India. Tel.: 91-11-2468 2100 or 2468 2111, Fax: 91-11-2468 2144 or 2468 2145

Abstract

Inter Simple Sequence Repeats (ISSRs) have emerged as an efficient and effective tool for clonal fidelity and genome mapping. In the present study eight ISSR UBC primers were used to characterize a total of 21F1’s involving B. juncea as female and B. campestris and B. carinata as male donors. The size of amplified bands ranged from 400 to 1000 bp resulting into a polymorphism of 92%. Bands specific to female and male parents were present in all hybrids ascertaining the hybrid nature of F1 progeny. The dendrogram constructed using Jaccard’s similarity coefficient grouped parent genotypes and hybrids into four major distinct clusters. All hybrids were first grouped into two clusters based on the presence of male donors, B. campestris and B. carinata, and then further sub- grouped depending upon the presence of female parent in specific crosses. Hybrids generated from cross B. juncea x B. campestris resembled the female and male parents, at a genetic similarity value of 0.71 and 0.45, respectively, while those of cross B. juncea x B. carinata resembled the female and male parent at a genetic similarity value of 0.67 and 0.37, respectively. The genetic similarity values indicated more influence of female parent in the hybrids in both types of crosses. The male parent specific ISSR markers can be converted to co- dominant SCAR or STS markers for hybrid screening. The present study elucidates the utility of ISSR markers as an efficient tool to screen hybrids obtained from similar crosses.

Media Summary

Efficient characterization of F1 brassica hybrids by ISSR markers, in the present study, elucidates their efficacy to be utilized for similar studies to ascertain hybridity.

Key Words

B.juncea, B.carinata, characterization, hybridity, genetic distance

Introduction

Characterization and identification of true hybrids at an early stage is mandatory in studies involving interspecific/ intergeneric hybridization. Biochemical and morphological markers are used to ascertain hybridity in many crop species including Brassicas. While the sensitivity of these markers to environmental and developmental variations limits their applicability, molecular markers can be profitably utilized to screen and identify true hybrids at an early stage. In recent years, ISSRs (Inter Simple Sequence Repeats) have become an efficient tool to study genetic diversity, somaclonal variation, phylogenetic relationships and percent parentage in many crop species (Kantety et al, 1995). Distribution of ISSRs in the eukaryotic genome makes them highly informative (Tauz and Renz, 1984). They are highly polymorphic, reproducible and cost effective, requiring no prior information of the sequence (Bornet et al, 2002).In Brassicas; applications of ISSRs have so far been confined to envisage clonal fidelity, somaclonal variants, and genetic diversity studies (Saal et al, 2001).The present study elucidates the characterization of interspecific Brassica hybrids, involving crosses of B. juncea with B. campestris and B. carinata attempted with a view to generate genetic variability to fungal diseases, through molecular markers using ISSRs.

Materials and Methods

Plant Material

A total of 11 F1 plants established from the cross B. juncea (genotypes: RESJ 830, RESJ 837) x B. campestris (genotypes: RESR 219, RESR 350), and 10 F1 plants from B. juncea (genotypes: RESJ 830, RESJ 837, TERI EP) x B. carinata (genotype: Kiran), were used for characterization. Lyophilized leaf material collected from the putative hybrids was used for DNA extraction by modified CTAB method (Weising et al, 1995).

PCR analysis using ISSR primers

ISSR’s and PCR were performed following the modified protocol by Kumar et al (2001). A total of 8 UBC (University of British Columbia) ISSR primers (synthesized from Microsynth, Switzerland) were used for DNA amplification. Amplification of DNA was carried out in a reaction mix containing 25 ng of DNA; 10mM of Tris-HCl, 50 mM KCl, 2% formamide, 1.5 mM of MgCl2; 0.25 mM of dNTPs; 0.2 M primers and 1.0 U Taq DNA polymerase per 25 l reaction mix. PCR amplification was performed in Perkin- Elmer thermocycler programmed for 35 cycles as: 1 cycle of 94C for 5 minutes; 35 cycles of 94C for 45 seconds, 50C for 45 seconds, 72C for 1minute 30 seconds followed by 1 cycle of 72C for 7 minutes. The amplification product was visualised on 1.5% agarose gel by staining with Ethidium Bromide.

Data Analysis

For statistical interpretation of DNA fingerprinting results, binary matrix was constructed by scoring the ISSR gels as presence (1) and absence (0) of bands across genotypes. On the basis of binary matrix, similarity matrix was calculated using Jaccard’s coefficient (Jaccard, 1908). The similarity matrix was subjected to Sequential Agglomerative Hierarical Nested Clustering (SAHN) and the phenotypic dendrogram was constructed employing Unweighted Pair Group Method of Arithmetic Averages (UPGMA) by Sneath and Sokal (1973) to group individuals into discrete clusters. The molecular data was analysed using statistical software package NTSYS version 2.0 (Rohlf, 1971).

Results

Eight ISSR UBC primers were employed to ascertain the hybrid nature of a total of 21putative hybrids obtained from crosses of B. juncea with B. campestris and B. carinata genotypes. Out of the eight primers, two primers UBC 840 and UBC 842 did not amplify any band. Rest six primers resulted into a total of 50 bands, out of which 46 bands were polymorphic resulting to 92.0 % polymorphism (Table 1). The size of amplified bands using different primers ranged from 400 bp to 1000 bp. All hybrids showed specific bands to their respective female and male parents, thus ascertaining the hybrid nature of the plantlets. In previous studies, purity of F1 seed in B. oleracea has been determined using male specific bands through RAPD markers (Phillip et al. 2000)]. In our study most of the primers showed one to three male parent specific bands; the number of bands specific to male donor B. carinata was found to be more in comparison to the bands specific to male donor B. campestris.

The dendrogram constructed on the basis of Jaccard’s similarity matrix, followed by UPGMA based clustering analysis (Figure1) showed that the genotypes were grouped into four major clusters viz., cluster BJ (B. juncea genotypes), BC (B. campestris genotypes), BK (B. carinata genotype) and cluster H representing hybrids obtained from them. The three Brassica species used in the present study showed high genetic diversity among themselves with a genetic similarity value of 0.47 between B. juncea and B. campestris, and 0.39 between B. juncea and B. carinata genotypes. In order to study the relationship among the hybrids, cluster H was further grouped into two sub-groups HC (containing hybrids from the cross B. juncea x B. campestris) and HK (containing hybrids from the cross B. juncea x B. carinata), identifying each other at a genetic similarity value of 0.67. Hybrids in the group HC resembled their female and male parents respectively, at a genetic similarity value of 0.71 and 0. 45. Similarly hybrids from group HK resembled their female and male parent at a genetic similarity value of 0.67 and 0.37, respectively. In general all hybrids were intermediate in morphological traits, however, contrary to the molecular genetic similarity showing more closeness to female parent in both crosses, the B.juncea x B.carinata hybrids resembled more closely to the male parent in their morphological characteristics. Based on the presence of female genotype, the cluster HC further sub-grouped the hybrids into two groups, HC1 (RESJ 837) and HC2 (RESJ 830), and the cluster HK divided the hybrids into three groups, HK1 (RESJ 837), HK2 (TERI EP-60) and HK3 (RESJ 830), wherein the female genotype is reflected in the brackets. Grouping of hybrids present in clusters HC and HK into sub groups showed a number of hybrids identical to each other at a genetic similarity value of 1.0 such as hybrids HC1.1, HC1.3 and HC1.4 in sub- group HC1; hybrids HC2.4 and HC2.5; and hybrids HC2.1, HC2.2 and HC2.3 in sub- group HC2. Similarly in sub- group HK2, hybrids HK2.2, HK2.3, HK2.4 and HK2.4 were identical to each other. The remaining hybrids in both the groups also resembled each other closely. Thus all hybrids were first clustered based on the presence of male donors and further sub- grouped depending up on the presence of female parent in specific crosses. Species specific clustering is a common feature in some other plants also (Steiger et al, 2002). This kind of molecular based cluster analysis has been proved efficient to identify and select superior lines and predict progeny performance (Beattie et al, 2003).

Table 1 DNA amplification of hybrids using six UBC- ISSR primers

Primer#

Primer Sequence*

Amplified bands

Poly-morphic bands

Number of female and male specific bands in hybrids

B.juncea x B.campestris

B.juncea x B carinata

B.juncea

B.campestris

B.juncea

B carinata

UBC 812

5’GA-GA-GA-GA-GA-GA-GA-GA- A-3’

11

10

2

0

2

2

UBC 814

5’CT-CT-CT-CT-CT-CT-CT-CT- A-3’

9

8

3

1

2

2

UBC 818

5’CA-CA-CA-CA-CA-CA-CA-CA-G-3’

11

11

3

2

2

3

UBC 843

5’CT-CT-CT-CT-CT-CT-CT-CT-RA-3’

5

3

3

0

3

0

UBC 848

5’CA-CA-CA-CA-CA-CA-CA-CA-RG-3’

8

8

3

1

2

2

UBC S2

5’CTC-TC-TC-TC-GT-GT-GT-GTG-3’

6

6

1

1

3

2

Total

 

50

46

15

5

14

11

# ISSR primers UBC 840 (5’GA-GA-GA-GA-GA-GA-GA-GA-YT-3’) and UBC 842 (5’GA-GA-GA-GA-GA-GA-GA-GA-YG-3) did not amplify any product
* Y= Pyramidine and R = Purines

Figure 1 UPGMA based clustering analysis of hybrids obtained from crosses involving B. juncea as female and B. campestris and B. carinata as male donors. Dendrogram is divided into four clusters; BJ (B. juncea genotypes), BC (B. campestris genotypes), BK (B. carinata genotypes) and H (All hybrids)

Conclusion

The DNA amplification using ISSRs was highly efficient and characterized the presence of male and female parent specific bands, thus successfully ascertaining the hybridity in F1 progeny. The hybrids showed strong genotypic influence and were thus grouped into different clusters depending upon the involvement of female (B. juncea) genotypes. The genetic similarity values indicated that the hybrids were intermediate in most of their traits but reflected more influence of female parent. The presence of male parent specific bands in all progenies by most primers suggests the possibility of converting these ISSR markers to co- dominant Sequence Characterized Amplified Region (SCAR) or Sequence Tagged Sites (STS) markers. These can be utilized for screening the progenies to identify hybrids obtained from similar kind of crosses. They can also be linked to various economically important traits, such as white rust or alternaria blight, the most economically important fungal diseases of B. juncea in Asian countries, and utilized in marker assisted selection.

Acknowledgement

The authors are thankful to Dr. N.I. Nashaat, Principal Research Scientist, Rothamstad Research, UK, and Prof S. J. Kolte, Department of Plant Pathology, GB Pant University of Agriculture and Technology, India, for providing the seeds of parent lines.

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