Chapter 4Chapter 4 Results: Analysis of Autosomal...

Chapter 4Chapter 4Chapter 4Chapter 4 Results: Analysis of Autosomal Results: Analysis of Autosomal Results: Analysis of Autosomal Results: Analysis of Autosomal

MarkersMarkersMarkersMarkers

Analysis of Autosomal Markers

A Genomic Study on the Sub–Structured Chaudhari Tribe of Southern Gujarat 94

ANALYSIS OF AUTOSOMAL MARKERS

4.1. Analysis of Autosomal Markers

The biparently inherited autosomal DNA represents the larger portion of the genome. Of

the estimated 32,000 genes present in the human genome, the majority are located on the

autosomes, while the X chromosome has approximately 1,500 genes, and the Y contains

approximately 78 genes (Venter et al., 2001; Skaletsky et al., 2003). Although these

regions are under functional constraints but the large portion of autosomal DNA contain

neutral variants. Various classes of autosomal markers such as Single Nucleotide

Polymorphism (SNP), Variable Number Tandem Repeats (VNTR), Restriction Fragment

Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP),

Retroelements etc., having characteristic features are known which are widely used to

investigate the human population structure and history. In the present investigation Alu

elements belonging to Short Interspersed Nuclear Element (SINE) family of retroelement

class of autosomal markers and SNPs that happens to be located within restriction sites

were used to determine the Chaudhari population structure and its relation to other Indian

populations. The current section highlights the results obtained from the laboratory analysis

and statistical analysis of autosomal markers in the four study groups. The autosomal

markers screened were seven Alu Insertion Deletion (InDel) polymorphisms and seven

unlinked RFLP loci. The detailed description of the markers employed has been given in

the material and method section. Because of technical reasons, numbers of samples

analyzed were different for different markers. Results have been presented in two parts in

the present chapter. The first section deals with findings from the Chaudhari subpopulations

and the second section deals with their analysis with other population groups.

4.2. Findings from the Four Chaudhari Populations

4.2.1. Genotype and Allele Frequency

4.2.1.1. Alu InDel Polymorphisms

4.2.1.1.1. PV92

Table 4.1 presents the genotype and allele frequency data for PV92 locus in the four

Chaudhari subpopulations. Barring Mota Chaudhari, the frequency of heterozygous

4

A Genomic Study on the Sub–Structured Chaudhari Tribe of Southern Gujarat

genotype was found to be highest

frequency of genotype homozygous for deletion allele.

homozygous for deletion allele genotype, the homozygous for insertion allele was

found in low frequency except in Pavagadhi Chaudhari in which both homozygo

genotypes were observed in similar

similar distribution pattern of alleles with deletion allele

frequency levels than insertion

insertion allele (+) was in Pavagadhi Chaudhari

Chaudhari (0.430). Both Nana and Mota Chaudhari exhibited

of insertion allele. Figure 4.1 displays the

Table 4.1. Genotype and allele frequency distribution at PV92 locus among the Chaudhari

subgroups

Populationa

Genotypeb

Count

VC NC MC PC

+ + 10 07 06

+ – 23 24 18

– – 17 20 26

Total 50 51 50

a VC=Valvi Chaudhari; NC=Nana Chaudhari; MC=Mota Chaudhari; PC=Pavagadhi Chaudharib ++ Homozygous for insertion allele, +– Heterozygous for insertion and deletion alleles, c+ Insertion allele, – Deletion allele

Figure 4.1. Gel photograph showing polymorphism at PV92 locus


Structured Chaudhari Tribe of Southern Gujarat

be highest in all the other Chaudhari subgroups, followed by the

homozygous for deletion allele. In comparison to the


y except in Pavagadhi Chaudhari in which both homozygo

observed in similar frequencies. The study populations exhibited

of alleles with deletion allele (–), displaying higher

n insertion allele (+). It was observed that the highest frequency

insertion allele (+) was in Pavagadhi Chaudhari (0.500), followed by that in

. Both Nana and Mota Chaudhari exhibited almost similar proportion

displays the polymorphism at PV92 locus.

Genotype and allele frequency distribution at PV92 locus among the Chaudhari

Frequency (%) Allelec Frequency

PC VC NC MC PC VC NC

10 20.0 13.7 12.0 24.4 + 0.430 0.373

21 46.0 47.1 36.0 51.2 – 0.570 0.627

10 34.0 39.2 52.0 24.4

41 100.0 100.0 100.0 100.0 1.000 1.000

VC=Valvi Chaudhari; NC=Nana Chaudhari; MC=Mota Chaudhari; PC=Pavagadhi Chaudhari

Heterozygous for insertion and deletion alleles, – – Homozygous for deletion allele

Gel photograph showing polymorphism at PV92 locus

Autosomal Markers

95

, followed by the

In comparison to the


y except in Pavagadhi Chaudhari in which both homozygous

. The study populations exhibited a

displaying higher

the highest frequency of

, followed by that in Valvi

similar proportion

Genotype and allele frequency distribution at PV92 locus among the Chaudhari

Frequency

MC PC

0.300 0.500

0.700 0.500

1.000 1.000

Homozygous for deletion allele



4.2.1.1.2. FXIIIB

Table 4.2 presents the genotype and allele frequency data for FXIIIB locus in the four

Chaudhari subpopulations. In all the groups heterozygous genotype were found in low

frequency compared to the homozygote genotypes. The maximum and minimum

frequency of genotype homozygous for insertion allele was observed in Nana

Chaudhari (51.00%) and Mota Chaudhari (16.00%) respectively. However, the trend

was found to be reversed in the case of genotype homozygous for deletion allele. Its

highest frequency was observed in Mota Chaudhari (72.00%) in contrast to its least

frequency in Nana Chaudhari (25.50%). Frequency of the insertion allele (+) was found

to be higher than the frequency of the deletion allele in all the populations except Mota

Chaudhari. Figure 4.2 displays the polymorphism at FXIIIB locus.

Table 4.2. Genotype and allele frequency distribution at FXIIIB locus among the

Chaudhari subgroups

Populationa

Genotypeb

Count Frequency (%) Allelec Frequency

VC NC MC PC VC NC MC PC VC NC MC PC

+ + 20 26 08 19 40.0 51.0 16.0 46.3 + 0.550 0.627 0.220 0.573

+ – 15 12 06 09 30.0 23.5 12.0 22.0 – 0.450 0.373 0.780 0.427

– – 15 13 36 13 30.0 25.5 72.0 31.7

Total 50 51 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000

a VC=Valvi Chaudhari; NC=Nana Chaudhari; MC=Mota Chaudhari; PC=Pavagadhi Chaudhari b ++ Homozygous for insertion allele, +– Heterozygous for insertion and deletion alleles, – – Homozygous for deletion allele c+ Insertion allele, – Deletion allele

Figure 4.2. Gel photograph showing polymorphism at FXIIIB locus


4.2.1.1.3. D1

Genotype and allele frequency dist

The frequency of genotype homozygous

proportion in all the four populations. The highest frequency of

for insertion allele was found in Valvi Chaudhari

than frequency observed in Nana Chaudhari

lowest frequency of the genotype

genotype distribution, frequency of the deletion allele (

proportion than insertion allele

polymorphism at D1 locus.

Table 4.3. Genotype and allele frequency distribution at D1

subgroups

Populationa

Genotypeb

Count

VC NC MC PC

+ + 15 15 05

+ – 15 10 21

– – 19 26 24

Total 49 51 50


Figure 4.3. Gel photograph showing polymorphism at D1 locus

650bp 333bp +



allele frequency distribution pattern at D1 locus is presented in Table 4.3.

genotype homozygous for deletion allele was observed in

all the four populations. The highest frequency of genotype homozygous

was found in Valvi Chaudhari (30.60%), which was slightly higher

frequency observed in Nana Chaudhari (29.40%). Mota Chaudhari displayed the

the genotype homozygous for insertion allele. Similar to

frequency of the deletion allele (–) was noticed in higher

proportion than insertion allele (+) in all the populations. Figure 4.3

e frequency distribution at D1 locus among the



07 30.6 29.4 10.0 17.1 + 0.459 0.329

15 30.6 19.6 42.0 36.6 – 0.541 0.608

19 38.8 51.0 48.0 46.3

41 100.0 100.0 100.0 100.0 1.000 1.000

VC=Valvi Chaudhari; NC=Nana Chaudhari; MC=Mota Chaudhari; PC=Pavagadhi Chaudhari

Heterozygous for insertion and deletion alleles, – – Homozygous for deletion allele

Gel photograph showing polymorphism at D1 locus

650bp 333bp + – – – ++

Autosomal Markers

97

sented in Table 4.3.

observed in higher

genotype homozygous

which was slightly higher

Chaudhari displayed the

. Similar to the

) was noticed in higher

Figure 4.3 displays

locus among the Chaudhari

Frequency

MC PC

0.310 0.354

0.690 0.646

1.000 1.000


L



4.2.1.1.4. APO

Table 4.4 presents the genotype and allele frequency data at APO locus in the study

populations. The distribution pattern showed the highest frequency of genotype

homozygous for insertion allele followed by the heterozygous genotype and the

genotype homozygous for deletion allele. However, in Valvi Chaudhari heterozygote

genotypes outnumbered genotype homozygous for insertion allele by 10.00%.

Similarly, the frequency for insertion allele was found in higher proportions in all the

populations as compared to the deletion allele. The frequency of insertion allele was

found to vary between 0.650 among Valvi Chaudhari and 0.775 among Nana

Chaudhari. Figure 4.4 shows polymorphism at APO locus.

Table 4.4. Genotype and allele frequency distribution at APO locus among the Chaudhari

subgroups

Populationa

Genotypeb



+ + 20 30 27 26 40.0 58.5 54.0 63.41 + 0.650 0.775 0.700 0.768

+ – 25 19 16 11 50.0 37.3 32.0 26.83 – 0.350 0.225 0.300 0.232

– – 05 02 07 04 10.0 3.92 14.0 9.76

Total 50 51 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000


Figure 4.4. Gel photograph showing polymorphism at APO locus


4.2.1.1.5. ACE

Table 4.5 presents data pertaining to genotype and allele frequ

populations at ACE locus. Out of the two

genotype homozygous for insertion

genotype homozygous for deletion allele. However, in comparison to

genotype, the frequencies of homozygous genotypes were

frequency distribution showed

allele. Out of the four Chaudhari sub

to be highest in Nana Chaudhari (0.618

Pavagadhi Chaudhari and Valvi Chaudhari which

insertion allele. Figure 4.5 shows polymorphism at ACE locus.

Table 4.5. Genotype and allele frequency distribution

subgroups

Populationa

Genotypeb

Count

VC NC MC PC

+ + 19 20 19

+ – 17 23 20

– – 14 08 11

Total 50 51 50


Figure 4.5. Gel photograph showing polymorphism at ACE locus

190bp 490bp – –



Table 4.5 presents data pertaining to genotype and allele frequency among study

Out of the two homozygous combinations, the frequency of

insertion was observed in higher proportion than that of

for deletion allele. However, in comparison to the heterozygous

homozygous genotypes were found to be less.

frequency distribution showed a higher frequency of insertion allele than the

allele. Out of the four Chaudhari subgroups, the frequency of insertion allele was found

highest in Nana Chaudhari (0.618) followed by that in Mota Chaudhari,

Pavagadhi Chaudhari and Valvi Chaudhari which showed almost similar proportion

shows polymorphism at ACE locus.

Genotype and allele frequency distribution at ACE locus among the



15 38.0 39.2 38.0 36.6 + 0.550 0.618

16 34.0 45.1 40.0 39.0 – 0.450 0.382

10 28.0 15.7 22.0 24.4

41 100.0 100.0 100.0 100.0 1.000 1.000

VC=Valvi Chaudhari; NC=Nana Chaudhari; MC=Mota Chaudhari; PC=Pavagadhi Chaudhari Heterozygous for insertion and deletion alleles, – – Homozygous for deletion allele

Gel photograph showing polymorphism at ACE locus

– + – + +

Autosomal Markers

99

ency among study

frequency of

observed in higher proportion than that of

the heterozygous

found to be less. The allele

than the deletion

the frequency of insertion allele was found

Mota Chaudhari,

almost similar proportion of

ACE locus among the Chaudhari

Frequency

MC PC

0.580 0.561

0.420 0.439

1.000 1.000


L



4.2.1.1.6. CD4

Genotype and allele frequency distribution for CD4 locus among populations under

study is given in Table 4.6. In all the populations genotype homozygous for insertion

allele was present in overwhelming frequencies than the other genotypes. Genotype

homozygous for deletion allele was found to be absent in Nana and Pavagadhi

Chaudhari populations. The deletion allele (–) in decreasing order of frequency was

observed in Mota Chaudhari, Valvi Chaudhari, Pavagadhi Chaudhari and Nana

Chaudhari. Figure 4.6 shows polymorphism at CD4 locus.

Table 4.6. Genotype and allele frequency distribution at CD4 locus among the Chaudhari

subgroups

Populationa

Genotypeb



+ + 43 46 41 35 86.0 90.2 82.0 85.4 + 0.920 0.951 0.900 0.927

+ – 06 05 08 06 12.0 09.8 16.0 14.6 – 0.080 0.049 0.100 0.073

– – 01 00 01 00 02.0 00.0 02.0 00.0

Total 50 51 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000


Figure 4.6. Gel photograph showing polymorphism at CD4 locus



4.2.1.1.7. PLAT

Data on the genotype and allele frequencies at PLAT locus is given in Table 4.7. It was

observed that heterozygous genotype was most frequent compared to homozygous

genotypes in all the populations. The minimum frequency of (+–) genotype was

observed in Mota Chaudhari (40.00%) and its maximum frequency was seen in Valvi

Chaudhari (60.00%). In case of genotype homozygous for deletion allele, the opposite

trend was seen, with higher frequency in Mota Chaudhari (24.00%) and lowest in Valvi

Chaudhari (6.00%). The allele frequency distribution pattern revealed that Pavagadhi,

Valvi and Nana Chaudhari showed almost similar frequency of insertion allele (0.622-

0.640) which was slightly higher than the frequency observed in Mota Chaudhari

(0.560). Figure 4.7 shows polymorphism at the PLAT locus.

Table 4.7. Genotype and allele frequency distribution at PLAT locus among the Chaudhari

subgroups

Populationa

Genotypeb



+ + 17 20 18 16.0 34.0 40.0 36.0 39.0 + 0.640 0.640 0.560 0.622

+ – 30 24 20 19.0 60.0 48.0 40.0 46.4 – 0.360 0.360 0.440 0.378

– – 03 06 12 06.0 06.0 12.0 24.0 14.6

Total 50 50 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000


Figure 4.7. Gel photograph showing polymorphism at PLAT locus

- - ++ + - L

260bp 570bp

A Genomic Study on the Sub

The overall allele frequency distribution

markers in the study populations is presented in Figure 4.8. The comparison revealed

slight variation in the allele

CD4 and PLAT loci in the Chaudhari subgroups.

Figure 4.8. Frequency distribution of derived (mutant) allele

except CD4} at

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

PV92 FXIII B

Fe

wq

ue

ncy

of

de

riv

ed

all

ele

Analysis of


allele frequency distribution pattern observed at the


slight variation in the allele frequency distribution at the loci examined, except

PLAT loci in the Chaudhari subgroups.

Frequency distribution of derived (mutant) alleles {Insertion (+) for all,

except CD4} at Alu InDel loci among the four study groups

FXIII B DI APO ACE CD4 PLAT

Loci analysed

Valvi Chaudhari

Nana Chaudhari

Mota Chaudhari

Pavagadhi Chaudhari


102

the seven Alu InDel


loci examined, except ACE,

{Insertion (+) for all,

InDel loci among the four study groups

Valvi Chaudhari

Nana Chaudhari

Mota Chaudhari

Pavagadhi Chaudhari



4.2.1.2. Restriction Fragment Length Polymorphisms (RFLPs)

4.2.1.2.1. ESR

Table 4.8 presents the allele and genotype frequency distribution data for ESR locus in

four Chaudhari subpopulations. Barring Mota Chaudhari, all other Chaudhari

subpopulations showed a similar trend of the distribution of genotypes with higher

frequency of genotype heterozygous for the presence and absence of restriction site

followed by that of genotype homozygous for presence of restriction site. Except in

Nana Chaudhari (0.373), the allele for presence of restriction site (+) was observed in

higher frequency than the allele for the absence of restriction site (–) in all the

populations. Figure 4.9 shows polymorphism at ESR locus.

Table 4.8. Genotype and allele frequency distribution at ESR locus among the Chaudhari

subgroups

Populationa

Genotypeb



+ + 21 16 18 14 42.0 32.0 36.0 34.2 + 0.650 0.373 0.520 0.573

+ – 23 22 16 19 46.0 44.0 32.0 46.3 – 0.350 0.627 0.480 0.427

– – 06 12 16 08 12.0 24.0 32.0 19.5

Total 50 50 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000

a VC=Valvi Chaudhari; NC=Nana Chaudhari; MC=Mota Chaudhari; PC=Pavagadhi Chaudhari b ++ Homozygous for insertion allele, +– Heterozygous for insertion and deletion alleles, – – Homozygous for deletion allele c+ Restriction site present, – Restriction site absent

Figure 4.9. Gel photograph showing polymorphism at ESR locus



4.2.1.2.2. NAT

Table 4.9 presents the genotype and allele frequency data at NAT locus observed in the

study populations. The genotype pattern showed that three out of the four groups had a

higher frequency of (++) genotype, followed by that of (+–) genotype. Genotype

homozygous for the absence of restriction site showed the least frequency in all the

groups. The allele frequency for presence of restriction site in Valvi, Mota and

Pavagadhi Chaudhari was found to range from 0.650–0.695 whereas, it lagged behind

in Nana Chaudhari (0.529). Likewise, the frequency of allele for absence of a restriction

site in Nana Chaudhari was found to stand apart from the other Chaudhari groups which

showed almost the similar proportions of (–) allele. Figure 4.10 shows polymorphism

at NAT locus.

Table 4.9. Genotype and allele frequency distribution at NAT locus among the Chaudhari

subgroups

Populationa

Genotypeb



+ + 23 14 24 20 46.0 27.0 48.0 49.0 + 0.650 0.529 0.650 0.695

+ – 19 26 17 17 38.0 51.0 34.0 41.2 – 0.350 0.471 0.350 0.304

– – 08 11 09 04 16.0 22.0 18.0 09.8

Total 50 51 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000


Figure 4.10. Gel photograph showing polymorphism at NAT locus



4.2.1.2.3. T2

Genotype and allele frequency data for T2 locus is given in Table 4.10. From the table

it is evident that the heterozygous genotype dominated the genotype distribution pattern

in all the four groups. Out of the two homozygous genotypes, all Chaudhari subgroups

showed higher frequency of (++) genotype than (– –) genotype. Similarly, allele for

presence of restriction site was found in higher frequency than the allele for the absence

of a restriction site. Figure 4.11 shows polymorphism at T2 locus.

Table 4.10. Genotype and allele frequency distribution at T2 locus among the Chaudhari

subgroups

Populationa

Genotypeb



+ + 14 20 14 13 28.0 39.2 28.0 31.7 + 0.530 0.647 0.570 0.610

+ – 25 26 29 24 50.0 51.0 58.0 58.5 – 0.470 0.353 0.430 0.390

– – 11 05 07 04.0 22.0 09.8 14.0 09.8

Total 50 51 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000


Figure 4.11. Gel photograph showing polymorphism at T2 locus



4.2.1.2.4. LPL

Table 4.11 illustrates the genotype and allele distribution data at LPL locus in the study

populations. It was observed that the heterozygous genotype occurred with greater

frequency than both homozygous genotypic combinations in all the groups. Where the

frequency of genotype homozygous for presence of restriction site was found to be

more frequent than the genotype homozygous for absence of restriction site in Valvi

and Pavagadhi Chaudhari, it was the other way round in Nana and Mota Chaudhari. In

terms of allele frequency distribution, both Valvi and Nana Chaudhari displayed similar

allele frequencies on one hand and Mota and Pavagadhi Chaudhari on the other. Figure

4.12 shows polymorphism at LPL locus.

Table 4.11. Genotype and allele frequency distribution at LPL locus among the Chaudhari

subgroups

Populationa

Genotypeb



+ + 20 07 15 12 40.8 13.7 30.0 29.3 + 0.643 0.647 0.500 0.561

+ – 23 22 20 22 47.0 43.1 40.0 53.7 – 0.357 0.353 0.500 0.439

– – 06 22 15 07 12.2 43.2 30.0 17.0

Total 49 51 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000


Figure 4.12. Gel photograph showing polymorphism at LPL locus



4.1.1.2.5. PSCR

Data on genotype and allele frequency for PSCR locus in populations under study is

presented in Table 4.12. Genotype with two copies of the allele for presence of

restriction site was present in least frequency in all the groups ranging between 6.00%

in Valvi Chaudhari and 24.00% in Mota Chaudhari. Genotype with both alleles, one for

the presence of restriction site and one for the absence of restriction site, was most

frequent genotype in all the groups. The frequency distribution of the two alleles was

found to be similar in all the groups, with higher frequencies observed for the deletion

allele. Figure 4.13 shows polymorphism at PSCR locus.

Table 4.12. Genotype and allele frequency distribution at PSCR locus among the Chaudhari

subgroups

Populationa

Genotypeb



+ + 03 09 12 04 06.0 17.7 24.0 09.8 + 0.440 0.431 0.490 0.378

+ – 38 26 25 23 76.0 51.0 50.0 56.1 – 0.560 0.569 0.510 0.622

– – 09 16 13 14 18.0 31.3 26.0 34.1

Total 50 51 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000


Figure 4.13. Gel photograph showing polymorphism at PSCR locus



4.2.1.2.6. ADH2

Table 4.13 illustrates the genotype and allele frequency data observed at ADH2 locus in

four Chaudhari subgroups. (++) genotype occurred more frequently in Valvi and Nana

Chaudhari as compared to Mota and Pavagadhi Chaudhari. (+–) genotype frequency

was found to range from 31.40% in Nana Chaudhari to 56.10% in Pavagadhi

Chaudhari. The allele for absence of restriction site was present in higher frequency

than the allele for presence of restriction site in all the groups. Figure 4.14 shows

polymorphism at ADH2 locus.

Table 4.13. Genotype and allele frequency distribution at ADH2 locus among the

Chaudhari subgroups

Populationa

Genotypeb



+ + 08 08 03 04 16.0 15.6 06.0 09.8 + 0.370 0.314 0.240 0.378

+ – 21 16 18 23 42.0 31.4 36.0 56.1 – 0.630 0.686 0.76 0.622

– – 21 27 29 14 42.0 53.0 58.0 34.1

Total 50 51 50 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000


Figure 4.14. Gel photograph showing polymorphism at ADH2 locus



4.2.1.2.7. ALAD

Data on the genotype and allele frequencies at ALAD locus in Chaudhari subgroups is

presented in Table 4.14. Barring Valve Chaudhari, all other Chaudhari subgroups

showed the high frequency of (– –) genotype followed by that of (+ –) genotype. The

frequency of genotype with two copies of the allele for presence of restriction site was

observed to be least in all the groups. The allele frequency distribution pattern showed

that the allele for absence of restriction site was more frequent ranging from 0.633 to

0.847 compared to the allele for presence of restriction site which was found to be

ranging from 0.153 to 0.367. Figure 4.15 shows polymorphism at ALAD locus.

Table 4.14. Genotype and allele frequency distribution at ALAD locus among the

Chaudhari subgroups

Populationa

Genotypeb



+ + 03 03 01 02 06.2 05.9 2.04 4.88 + 0.367 0.206 0.153 0.171

+ – 30 15 13 10 61.2 29.4 26.53 24.39 – 0.633 0.794 0.847 0.829

– – 16 33 35 29 32.6 64.7 71.43 70.73

Total 49 51 49 41 100.0 100.0 100.0 100.0 1.000 1.000 1.000 1.000


Figure 4.15. Gel photograph showing polymorphism at ALAD locus

A Genomic Study on the Sub

Figure 4.16 presents the comparison of allele frequencies observed

loci in the four study groups

distribution at almost all the loci among the

Figure 4.16. Frequency distribution of allele

RFLP loci among the four study groups

4.2.2. Hardy–Weinberg

The allele frequency estimates

goodness–of–fit test to determine if the observed genotype frequencies in the study

populations deviate from the Hardy

statistical tests were being performed

correction, an adjustment made to

After applying Bonferroni’s correction for multiple

data, the observed genotype frequencies were found to be in

expected Hardy–Weinberg proportions for all the loci except

Mota and Pavagadhi Chaudha

Valvi Chaudhari. The overall pattern

the Hardy–Weinberg expectations.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

ESR NAT

Fre

qu

en

cy a

f a

lle

le f

or

the

pre

sen

ce o

f

Re

stri

ctio

n s

ite

Analysis of


Figure 4.16 presents the comparison of allele frequencies observed

loci in the four study groups. The figure indicated the differences in allele frequency

almost all the loci among the four Chaudhari subgroup.

Frequency distribution of alleles for presence of restriction site at the

RFLP loci among the four study groups

Weinberg Equilibrium

The allele frequency estimates at the examined loci were subjected to chi

fit test to determine if the observed genotype frequencies in the study

populations deviate from the Hardy–Weinberg proportions (Table 4.15)

statistical tests were being performed simultaneously on each population

an adjustment made to p value, was applied to avoid false positive results

applying Bonferroni’s correction for multiple comparisons on

data, the observed genotype frequencies were found to be in agreement with their

Weinberg proportions for all the loci except at FXIII

and Pavagadhi Chaudhari, at D1 locus in Nana Chaudhari and at PSCR

he overall pattern of genotype distribution was in accordance with

Weinberg expectations.

NAT T2 LPL PSCR ADH2 ALAD

Loci analysed

Valvi Chaudhari

Nana Chaudhari

Mota Chaudhari

Pavagadhi Chaudhari


110

at the seven RFLP

differences in allele frequency

subgroup.

for presence of restriction site at the

at the examined loci were subjected to chi–square

fit test to determine if the observed genotype frequencies in the study

(Table 4.15). Since multiple

population, Bonferroni’s

to avoid false positive results.

on Alu and RFLP loci

agreement with their

at FXIIIB locus in Nana,

and at PSCR locus in

of genotype distribution was in accordance with

Valvi Chaudhari

Nana Chaudhari

Mota Chaudhari

Pavagadhi Chaudhari



Table 4.15. Chi square (χ2) test for estimation of Hardy–Weinberg Equilibrium at Alu

InDel and RFLP loci among the four subdivisions of Chaudhari population

Locus χ2

Estimates

Valvi

Chaudhari

Nana

Chaudhari

Mota

Chaudhari

Pavagadhi

Chaudhari

PV92 0.189 0.002 1.021 0.024

FXIIIB 7.759 12.582* 21.147* 12.464*

D1 7.211 17.675* 0.016 1.635

APO 0.489 0.226 2.834 2.490

ACE 4.902 0.104 1.601 1.769

CD4 1.707 0.135 0.617 0.255

PLAT 4.562 0.086 1.773 0.008

ESR 0.006 0.653 6.443 0.114

NAT 1.358 0.027 3.194 0.019

PSCR 14.699* 0.078 0.000 1.525

ALAD 4.930 0.515 0.026 0.788

T2 0.001 0.688 1.677 2.168

LPL 0.024 0.157 2.000 0.327

ADH2 0.491 3.757 0.008 1.525

* χ2 statistically significant at pcorrected <0.0035, df=1, after applying Bonferroni’s correction.

4.2.3. Heterozygosity

Table 4.16 illustrates the heterozygosity levels observed at 14 studied loci in thr four

Chaudhari subgroups. The heterozygosity analysis revealed overall high heterozygosity

level in all the four groups, ranging from 0.421 in Mota Chaudhari to 0.457 in Valvi

Chaudhari. Examination of heterozygosity at individual loci revealed variation between

the study groups. Minimum heterozygosity level was observed at CD4 locus in all the

populations. Whereas, maximum heterozygosity value (0.500) was seen at various loci

in the study populations. Loci FXIIIB, D1, ACE and T2 exhibited highest

heterozygosity in Valvi Chaudhari. Nana Chaudhari showed similar values at ESR and

NAT loci. Although, Mota Chaudhari had the lowest average heterozygosity compared

to the other subgroups, three loci namely, ESR, PSCR and LPL were found to harbor

the maximum heterozygosity in Mota Chaudhari. Only one locus namely, PV92 was

observed to show highest heterozygosity value in Pavagadhi Chaudhari.



Table 4.16. Heterozygosity estimates and their Standard Errors for Alu InDel and RFLP

among the four subdivisions of Chaudhari population

Locus Heterozygosity ± Standard Error

Valvi

Chaudhari

Nana

Chaudhari

Mota

Chaudhari

Pavagadhi

Chaudhari

PV92 0.495±0.015 0.473±0.025 0.424±0.037 0.500±0.009

FXIIIB 0.500±0.012 0.473±0.025 0.347±0.047 0.495±0.018

D1 0.500±0.011 0.481±0.022 0.432±0.036 0.463±0.032

APO 0.460±0.029 0.353±0.046 0.424±0.037 0.360±0.050

ACE 0.500±0.012 0.477±0.024 0.492±0.017 0.499±0.016

CD4 0.149±0.045 0.094±0.039 0.182±0.048 0.137±0.049

PLAT 0.465±0.027 0.465±0.028 0.498±0.014 0.476±0.027

ESR 0.460±0.029 0.500±0.011 0.500±0.008 0.495±0.018

NAT 0.460±0.029 0.500±0.009 0.460±0.029 0.429±0.040

PSCR 0.498±0.014 0.495±0.015 0.500±0.007 0.476±0.027

ALAD 0.469±0.026 0.330±0.047 0.262±0.051 0.287±0.055

T2 0.500±0.009 0.461±0.029 0.495±0.016 0.482±0.025

LPL 0.464±0.028 0.461±0.029 0.500±0.007 0.499±0.016

ADH2 0.471±0.026 0.435±0.035 0.368±0.045 0.476±0.027

Average

Heterozygosity 0.457±0.023 0.429±0.027 0.421±0.028 0.434±0.029

4.2.4. Genetic Differentiation among Populations

4.2.4.1. Gene Diversity Estimate

Table 4.17 presents the gene diversity estimates based on examined loci in Chaudhari

subgroups. Gene diversity analysis in the four subgroups revealed an overall high levels

of heterozygosity (HT=0.441). It was observed that on an average each subgroup

harbored significant amount of heterozygosity (HS=0.431). Estimate of inter population

difference (GST) was found to be 2.30%. The four populations seemed to differ most at

FXIIIB locus (GST=10.20%) and least at ACE locus (0.30%).



Table 4.17. Gene diversity estimates at the studied Alu InDel and RFLP loci among the

four subdivisions of Chaudhari population

Locus HT HS GST

PV92 0.480 0.469 0.023

FXIIIB 0.500 0.449 0.102

D1 0.471 0.465 0.013

APO 0.400 0.395 0.013

ACE 0.488 0.487 0.003

CD4 0.140 0.139 0.005

PLAT 0.473 0.471 0.005

ESR 0.490 0.485 0.010

NAT 0.466 0.458 0.016

PSCR 0.491 0.488 0.006

ALAD 0.348 0.334 0.041

T2 0.484 0.480 0.008

LPL 0.500 0.477 0.045

ADH2 0.439 0.433 0.014

All Loci 0.441 0.431 0.023

4.2.4.2. Genetic Differentiation using Wright’s F–Statistics

Table 4.18 presents results of fixation–index–based measures of departure from expected

heterozygosity, commonly used to quantify population structure. At the first level, FIS

was estimated which compared average observed heterozygosity of individuals in each

subpopulation and the average Hardy–Weinberg expected heterozygosity for all

subpopulations. From the table it was observed that the subpopulations on average had

7.92% deficiency of heterozygotes as expected if there was nonrandom mating taking

place. The next level in the hierarchy was the average expected heterozygosity for

subpopulations compared with expected heterozygosity for the total population i.e. FST.

The result indicated that there was 2.29% less heterozygosity on average for the four

subpopulations compared with the heterozygosity expected in case of panmixia. The

final level in the hierarchy was FIT. The Level of FIT indicated that the homozygosity was

10.04% greater than would be expected in a randomly mating, panmictic population with

the same allele frequencies.



Table 4.18. Estimates of F–Statistics at the studied Alu InDel and RFLP loci among the

four subdivisions of Chaudhari population

Locus FIS FST FIT

PV92 0.0399 0.0225 0.0615

FXIIIB 0.5126 0.1023 0.5625

D1 0.3069 0.0128 0.3157

APO 0.0756 0.0132 0.0878

ACE 0.1879 0.0027 0.1901

CD4 0.0570 0.0048 0.0615

PLAT –0.0358 0.0044 –0.0312

ESR 0.1324 0.0100 0.1411

NAT 0.1024 0.0163 0.1170

PSCR –0.1932 0.0064 –0.1855

ALAD –0.0468 0.0462 0.0016

T2 –0.1323 0.0079 –0.1233

LPL 0.0373 0.045 0.0806

ADH2 0.0446 0.0139 0.0578

All loci 0.0792 0.0229 0.1004

4.2.4.3. Analysis of Molecular Variance (AMOVA)

The analysis of molecular variance across the four populations on the basis of allele

frequency data on 14 autosomal loci showed 98.39% of variability due to within

population differences and 1.61% (p<0.05) due to differences among the Chaudhari

subpopulations. The results have been presented with other AMOVA results in

Table 4.23 at the end of the chapter.

4.2.4.4. Genetic Distance and Neighbor Joining Tree

To find out the genetic relation among the study groups, genetic distances were

computed based on the allele frequencies observed at 14 autosomal loci. From the Table

4.19, it is evident that the highest genetic proximity i.e least genetic distance was

between Nana Chaudhari and Mota Chaudhari (0.003) followed by that between

Pavagadhi and Nana Chaudhari (0.005). Mota Chaudhari showed the highest genetic

distance with Valvi Chaudhari. The dendrogram (Figure 4.17) generated using the data

indicated the genetic proximity between Nana and Mota Chaudhari by clustering the

two populations in one group.



Table 4.19. Genetic distance matrixa based on 14 autosomal loci among the four

Chaudhari subgroups

Valvi

Chaudhari

Nana

Chaudhari

Mota

Chaudhari

Pavagadhi

Chaudhari

Valvi Chaudhari 0 0.0106 0.0137 0.0047

Nana Chaudhari 0.0161 0 0.0204 0.0060

Mota Chaudhari 0.0254 0.0030 0 0.0157

Pavagadhi Chaudhari 0.0237 0.0050 0.0190 0

aStandard genetic distance values are in 10¯

1 codon difference per locus along with standard error above

the diagonal.

Figure 4.17. Neighbor–Joining tree showing genetic relation between the Chaudhari

subdivisions

4.3. Comparison of Study Populations with Other Population Groups

The following section presents the result obtained from the analysis of the study

populations along with other Indian populations. Based on the ethno-historical

information and availability of comparable data from the secondary sources two data

sets were created. Dataset 1 included allele frequency data at autosomal loci among the

nine Indo–European speaking tribes of Gujarat, including the study populations. The

allele frequency data used for the analysis was compiled from Kshatriya et al. (2011).

Dataset 2 included allele frequency data at autosomal loci among Indo–European

speaking groups of Gujarat and other populations of India, compiled from various

published studies. These datasets were subjected to diverse analytical tools to examine

firstly, the genetic structure of Indo–European speaking tribes of Gujarat and secondly,

to understand the genetic affinities of these Indo–European speaking tribal populations

of Gujarat with other Indian populations.



4.3.1. Dataset 1: Genetic Relation between Neighbouring Indo–European (IE)

Speaking Tribal Populations of Gujarat

The first comparison was based on the allele frequency data of 14 biallelic autosomal

markers in the four study groups and five neighbouring Indo–European speaking tribal

groups namely, Dhodia, Dubla, Konkana, Vasava and Gamit of Gujarat. The allele

frequency data used for the analysis was compiled from Kshatriya et al. (2011). Allele

frequency data used for this analysis is given in Appendix XIII.

4.3.1.1. Heterozygosity

Table 4.20 presents the heterozygosity estimates among the nine Indo–European

speaking tribal populations of Gujarat based on the allele frequencies of 14 autosomal

markers. Although, the heterozygosity values of the majority of the loci were found to

be high in most of the populations, certain loci such as APO, CD4, ALAD and ADH2

showed low heterozygosity in some of the populations. Overall, the average

heterozygosity was found to be substantially high in all the groups, which varied from

0.404 in Konkana to 0.457 in Valvi Chaudhari.

Table 4.20. Heterozygosity estimates at the studied 14 autosomal loci among the nine

Indo–European speaking tribal groups of Gujarat

Locus Valvi

Chaudhari

Nana

Chaudhari

Mota

Chaudhari

Pavagadhi

Chaudhari

Dhodia Dubla Konkana Vasava Gamit

PV92 0.495 0.473 0.424 0.500 0.499 0.500 0.500 0.498 0.485

FXIIIB 0.500 0.473 0.347 0.495 0.484 0.484 0.495 0.497 0.481

D1 0.500 0.481 0.432 0.463 0.456 0.433 0.437 0.431 0.423

APO 0.460 0.353 0.424 0.360 0.280 0.305 0.339 0.318 0.394

ACE 0.500 0.477 0.492 0.499 0.462 0.400 0.498 0.375 0.428

CD4 0.149 0.094 0.182 0.137 0.091 0.116 0.204 0.208 0.095

PLAT 0.465 0.465 0.498 0.476 0.456 0.479 0.496 0.451 0.495

ESR 0.460 0.500 0.500 0.495 0.480 0.476 0.495 0.497 0.492

NAT 0.460 0.500 0.460 0.429 0.456 0.466 0.461 0.460 0.431

PSCR 0.498 0.495 0.500 0.476 0.500 0.492 0.478 0.497 0.496

ALAD 0.469 0.330 0.262 0.287 0.353 0.229 0.309 0.436 0.196

T2 0.500 0.461 0.495 0.482 0.500 0.487 0.485 0.455 0.485

LPL 0.464 0.461 0.500 0.499 0.497 0.499 0.463 0.500 0.487

ADH2 0.471 0.435 0.368 0.476 0.458 0.416 0.000 0.436 0.438

All loci 0.457 0.429 0.421 0.434 0.427 0.413 0.404 0.433 0.416



4.3.1.2. Genetic Differentiation among Populations

4.3.1.2.1. Gene Diversity Estimate

Table 4.21 presents data on genomic diversity analysis for 14 loci among the Indo-

European speaking tribal populations from Gujarat. From the table it was observed that

the total average heterozygosity among the tribes of Gujarat was high (0.433). Most of

this genomic diversity could be attributed to individual variations within the population as

reflected by high HS value (0.423). Whereas, 2.20% of the total genetic diversity

accounted for variation between the populations (GST). Maximum genetic differentiation

between the populations was observed with respect to FXIIIB and ADH2 loci (5.80%).

At loci ESR and PSCR minimum differentiation between the populations was seen.

Table 4.21. Gene diversity analysis at the studied 14 autosomal loci among the nine Indo–

European speaking tribal groups of Gujarat

4.3.1.2.2. Analysis of Molecular Variance (AMOVA)

Considering all the Indo–European speaking tribes of Gujarat as one group, the analysis

of molecular variance across the populations on the basis of allele frequency data on 14

autosomal loci showed 99.21% of variability due to within population differences and

HT HS GST

PV92 0.494 0.484 0.020

FXIIIB 0.500 0.471 0.058

D1 0.453 0.449 0.010

APO 0.363 0.357 0.017

ACE 0.471 0.457 0.031

CD4 0.143 0.141 0.010

PLAT 0.477 0.473 0.007

ESR 0.490 0.487 0.006

NAT 0.460 0.456 0.009

PSCR 0.494 0.491 0.006

ALAD 0.331 0.317 0.039

T2 0.486 0.481 0.010

LPL 0.497 0.484 0.027

ADH2 0.411 0.387 0.058

All loci 0.433 0.423 0.022



0.79% (p<0.05) due to differences among Gujarat populations. The results have been

presented with other AMOVA results in Table 4.23 at the end of the chapter.

4.3.1.3. Population Structure and Gene Flow

The Harpending and Ward (1982) model for population structure and gene flow was

employed to understand the population structure and the level of gene flow among the

Indo–European speaking tribal groups of Gujarat. The model is based on regression of

heterozygosity on genetic distance from the gene frequency centroid. Data on average

heterozygosity (Hi) and the genetic distances from the centroid (rii) in the studied

groups is given in Table 4.22. It was seen that average heterozygosity in the pooled

population (0.432) did not differ significantly from the regression coefficient (0.434),

signifying that these populations have received a similar proportion of gene flow. The

plot (Figure 4.18) made between observed heterozygosity (Hi) and the distance from the

gene frequency centroid (rii) made it possible to understand the population structure of

the populations in a better way. The grouping of majority of populations near the

theoretical line of regression coupled with moderate distance from the centroid signifies

that Indo–European speaking tribal groups of Gujarat have neither received a large

amount of gene flow nor experienced absolute isolation.

Table 4.22. Genetic heterozygosity (Hi) and genetic distances from the centroid (rii)

among the nine Indo–European speaking tribal groups of Gujarat

rii±SE Hi±SE

Valvi Chaudhari (VC) 0.032±0.012 0.452±0.024

Nana Chaudhari (NC) 0.025±0.011 0.425±0.028

Mota Chaudhari (MC) 0.036±0.020 0.417±0.026

Pavagadhi Chaudhari (PC) 0.011±0.003 0.429±0.027

Vasava (Vas) 0.019±0.006 0.433±0.022

Gamit (Gam) 0.012±0.004 0.416±0.032

Konkana (Kon) 0.047±0.030 0.404±0.039

Dhodia (Dho) 0.009±0.003 0.427±0.030

Dubla (Kon) 0.010±0.004 0.413±0.031

Regression analysis: Hi = b (1 − rii); Hi plotted against 1 − rii through the origins has t = 0.804,

7 df, p > 0.05. Regression coefficient through origin: b = 0.434 ± 0.014; H = 0.432 ± 0.0270


Figure 4.18. Regression of genetic heterozygosity (

among the nine Indo

4.3.2. Dataset 2: Genetic Affinities o

Populations of Gujarat

Dataset 2 included the allele frequency data on 14

population groups of India. The allele frequency data for the

populations considered for analysis of molecular variance

XIV. The analysis was carried out between different combinations of populations,

grouped on the basis of language and

considered in different categories were

4.3.2.1. Analysis of Molecular

The results of the analysis have been presented in T

results of previous sections.

influence in determining the population clustering as

was observed between the groups classified on the basis of their lingui

Least among groups variance was seen between the Indo

linguistic groups of India (0.1

population groups (0.51%). Indo

Gam

Dho

Dub

PC

0.30

0.32

0.34

0.36

0.38

0.40

0.42

0.44

0.46

0.48

0.50

0.00 0.01



Regression of genetic heterozygosity (Hi) on distance from centroid (

Indo–European speaking tribal groups of Gujarat

Genetic Affinities of Indo–European (IE) Speaking Tribal

e frequency data on 14 autosomal loci among different

. The allele frequency data for the autosomal loci and the

for analysis of molecular variance has been given in

The analysis was carried out between different combinations of populations,

anguage and ethnicity. Number of populations and markers

dered in different categories were different, depending on the availability of data.

olecular Variance (AMOVA)

ave been presented in Table 4.23 along with

. In comparison to ethnicity, language showed more

influence in determining the population clustering as 1.71% of among group variance

was observed between the groups classified on the basis of their linguistic affiliation.

Least among groups variance was seen between the Indo–European and Dravidian

linguistic groups of India (0.11%) followed by that between the castes an

%). Indo–European speaking tribes of Gujarat showed

Vas

Kon

VC

NCMC

0.02 0.03 0.04 0.05 0.06

Autosomal Markers

119

) on distance from centroid (rii)

European speaking tribal groups of Gujarat

European (IE) Speaking Tribal

l loci among different

autosomal loci and the

been given in Appendix

The analysis was carried out between different combinations of populations,

. Number of populations and markers

different, depending on the availability of data.

along with AMOVA

In comparison to ethnicity, language showed more

% of among group variance

stic affiliation.

European and Dravidian

%) followed by that between the castes and tribal

European speaking tribes of Gujarat showed

0.07



minimum between group variance with Dravidian speaking groups of India (1.44%),

followed by higher variance with other Indo–European speaking groups of India

(2.83%). In all comparisons, within the population component of variance was found to

explain the major component of variance. All the comparison values were found to be

statistically significant.

Table 4.23. Extent of genetic differentiation estimated by AMOVA among the Indo–

European speaking tribes of Gujarat and other Indian populations on the

basis of autosomal loci allele frequencies

Category

Among groups

variance

(In %)b

Among

population

within groups

variance

(In %)b

Within

population

variance

(In %)b

Chaudhari subgroups as one group 1.61 98.39

IE speaking tribes of Gujarat as one

group 0.79 99.21

Castes and Tribal groups of India 0.51 5.02 94.48

4 linguistic groups of Indiaa 1.71 3.69 94.6

IE and DR linguistic groups of India

0.11 3.60 96.29

IE speaking tribes of Gujarat and DR

speaking groups of India 1.44 2.65 95.91

IE speaking tribes of Gujarat and IE

speaking groups of India 2.83 1.63 95.54

a IE:Indo–European; DR:Dravidian; AA:Austro–Asiatic; TB:Tibeto–Burman

b All the values are significant, p < 0.05

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