ASSESSMENT OF GENETIC DIVERSITY AND OUTCROSSING IN SAFFLOWER (Carthamus tinctorius L.) VARIETIES USING SSR MARKERS
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Date
2014
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ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY. RAJENDRANAGAR, HYDERABAD
Abstract
Safflower (Carthamus tinctorius L.) is a member of Asteraceae family originated in the
Middle Eastern region. The cultivated safflower is a diploid with 24 chromosomes and
genome size of about 1.4 Gb. Understanding of genetic diversity in the safflower
germplasm accessions is critical for conservation, maintenance and selection of
appropriate accessions for use in breeding activities. In this study, genetic diversity and
relatedness of 30 Mexican safflower varieties were compared with 30 public bred
Indian cultivars using 50 SSR markers. The genetic diversity measures viz., allele
number, major allele frequency, observed heterozygosity (Ho), expected heterozygosity
(He), polymorphism information content (PIC) and the population parameters viz.,
population structure, genetic admixture, analysis of molecular variance (AMOVA) and
fixation index (Fst) were analyzed across 60 genotypes.
The allele number ranged from 1 to 10 with the mean of 2.7 per locus. The
major allele frequency ranged from 0.386 to 1 with the mean of 0.74. The observed
heterozygosity (Ho) ranged from 0 to 1 with the mean of 0.088. The expected
heterozygosity (He) ranged from 0 to 0.768 with the mean of 0.332. The PIC values for
each SSR primer pair ranged from 0 to 0.751 with the mean of 0.286. The low average
number of alleles, high major allele frequency and low average PIC values across 60
accessions suggested that low level of SSR polymorphism in the safflower cultivars.
Neighbor joining (NJ) tree based on pair-wise simple matching coefficients
clearly placed Indian and Mexican accessions in two distinct clusters. NJ tree also revealed
several subgroups within Mexico and India groups. The model based STRUCTURE
analysis also showed two subpopulations (K=2) clearly thus grouping the Indian and
Mexican cultivars into distinct clusters. However, it was unclear to what extent the two
varietal groups were genetically differentiated. Partitioning of variation through
hierarchical AMOVA showed that about 40% of variation is explained between
populations and 60% between individuals within population. The pair-wise Fst estimate
between Mexico and India groups was 0.39639. These results indicated that both India
and Mexico varietal groups are highly differentiated and a substantial genetic diversity
exists between them, which can be used for generating new variability for improving
agronomic traits in safflower breeding programmes.
In this study, an effort was also made to check outcrossing between nine
safflower genotypes grown under honeybee excluded and protected net condition using
SSR markers. Previously generated genotypic data of 60 genotypes were used to
identify a single or a combination of SSR markers which could uniquely differentiate
one genotype from the other. Five hundred plant progenies of the variety A1 (of the
seeds collected from protected net field) grown in grow-out test (GOT) plot were
genotyped using A1 specific SSR primer pair ct-266. The ct-266 allelic pattern of
progenies was examined to check if any progeny that showed heterozygous allelic
pattern (carrying A1 allele + allele from any parental genotype grown in the outcross
block). It was observed that 496 progenies had the allelic pattern of A1. Only four
progenies had allelic pattern that was different from A1; the pattern was not
heterozygous but was similar to that of two parental genotypes; hence, these plants were
considered as contaminants. Thus, the genotyping results showed that there was no
evidence of outcrossing between A1 and other safflower genotypes under honeybee
excluded protected net condition. Genotyping of 500 A1 progenies (of the seeds
collected from open field) was also done using A1 specific SSR primer pair ct-266. All
the 500 plants showed the allelic pattern of A1 suggesting that outcrossing did not
happen between A1 and other genotypes even under open field conditions. The results
have strengthened the observations of safflower breeders that out-crossing may not
happen in safflower in the absence of honeybees. However, the results are only
indicative and not conclusive because of limited progeny size and genotypes used.
Therefore, the findings need further validation using larger progeny across genotypes.
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Keywords
ASSESSMENT, GENETIC, DIVERSITY, OUTCROSSING, SAFFLOWER, VARIETIES, USING, SSR, MARKERS